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https://openalex.org/W1963929162
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https://zenodo.org/records/2172247/files/article.pdf
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French
| null |
Recherches Sur La Nutrition Des Thallophytes A L'Aide Des Amides
|
Bulletin de la Société botanique de France
| 1,901
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public-domain
| 3,388
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Bulletin de la Société Botanique de France ISSN: 0037-8941 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tabg17 Bulletin de la Société Botanique de France Recherches Sur La Nutrition Des Thallophytes A
L'Aide Des Amides M. L. Lutz Date: 04 June 2016, At: 15:34 Download by: [189.219.133.207] (1) G. Ville, Définir par la végétation l'etat moleculaire des corps (C.
R., t. LVII, 1863, p. -t6!1).
(2) Hampe, Landw. Ve1·suchs-Stationen, 1866.
(3) 1\ente, Landw. Versuchs-Stationen, 1~1>6, p. 463.
(4) Pfelfcr, Unters. über Proteinkorper. (Jahrb. {ü1· w. Bflf., t. VIH, 1872,
p. 4'29).
(5) Ca pus, Sur le rôle physiol. dr. l'aspa1·a!}'ine (Ann. agron., t. Y, i879,
p. 578).
(6) Lœw et Bokorny, Chem.-physiol. Unters. üb. Algen (lourn. {ül' prakt.
Chemie.-Neue Folge, Bd 36, 1887). M. L. Lutz To cite this article: M. L. Lutz (1901) Recherches Sur La Nutrition Des Thallophytes
A L'Aide Des Amides, Bulletin de la Société Botanique de France, 48:4, 325-334, DOI:
10.1080/00378941.1901.10831849 Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=tabg17
Download by: [189.219.133.207]
Date: 04 June 2016, At: 15:34 Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=tabg17 Download by: [189.219.133.207] Download by: [189.219.133.207] 325 LUTZ. -
NUTIUTION DES 'J'IIALI.OPIIYTES A L' AID:" DJ.:S Alli lES. les Champig·nons supérieurs, et notre C'h.~l' et hono: é confrère,
M. Brevière, s'occupe avec ardeur du 'a~tc grour e de~ Champi-
gnons infél'ieurs. Enfin, de notre eàt ·., nous es érons pouvoir
publier à bt·er ddai le résultat de nos rechert"hes sur les Lichens
d'Auvergne. M. Lutz fait à la Société ta communication suivante: p
(2) Hampe, Landw. Ve1·suchs-Stationen, 1866. ( )
p
(3) 1\ente, Landw. Versuchs-Stationen, 1~1>6, p. 463. (5) Ca pus, Sur le rôle physiol. dr. l'aspa1·a!}'ine (Ann. agron., t. Y, i87
p. 578). (6) Lœw et Bokorny, Chem.-physiol. Unters. üb. Algen (lourn. {ül' pra
Chemie.-Neue Folge, Bd 36, 1887). p
(4) Pfelfcr, Unters. über Proteinkorper. (Jahrb. {ü1· w. Bflf., t. VIH, 1872,
4'29). (1) G. Ville, Définir par la végétation l'etat moleculaire des corps (C.
, t. LVII, 1863, p. -t6!1). (1) Schimper, Z11r Frage der ,\ssimilation der Mineralsalze durch die
grit11e Pflunze (Flora, t. LXXIII, lleft. III, p. 2U7).
(2) l:mmerling, Beitr. z. Kenntn. der chem. Vorgiinge in der Pflanze.
(Laradw. Versuclls-Statione11, t. XXX, 18lH, p. 109).
(3) Lœw, Centratbl. (ür Agrikultur-Chemie, Hl96, p. 4Ut.
(4) Mt'unier. Etudes sur l'asparagine (Ann. agron., t88U, p. 27:i).
(5) Borodine, lht role pliysiol. et de la rtipartitùm de l'asparagine dans
le regne végétal (Ann. agron., t. V, 1880, p . .i87).
(6) Frank, LQ, statistique de l'azote en agricult~tre (Landw. Jahrb., 1888,
p. 4!1. Trad. in Ann. ag1·on., 1888, 2, p. 2!).
(7) Kinoshita, Coll. agr. de Tokio (Bull. vol. Il, n" 4., p. 196).
(8) O'Muller, Jnal. Amer. Chem. Soc., t. XVII. 1895, no 2, p. 118.
(9) Miyachi, Coll. agr. Tokio, Bull., vol. Il, p. <1.87.
(10) :"akamura, Coll. agr. Tokio, Bull., vol. Il, n• 7. p. 465.
(11) Schulze, De l'influence des hydrates de carbone su1· la formation des
albumi11oïdes dans les plantes (Landw. Jahrb., 1898, p. 516).
(12) Lœw et Bokorny, Chein. physiol. Studien über Algen (Jnal f. Prakt.
Chem., neue Folge, Bd 36, 1887).
·
(13) Frank, La statistique de l'azote en agric~tlture (Landw. Jahrb., 1888,
p. 421). Downloaded by [189.219.133.207] at 15:34 04 June 2016 Depuis longtemps déjà l'attention des expérimentateurs a été
attirée sut· l'emploi de certaines ainides comme source d'azote
pout· les végétaux. C'est ainsi que G. Ville (1) admet que \( l'urée
produit sut· la végétation une influence favorable des plus actives»
et que « son effet ulil·~ est juste égal à celui des sels ammonia-
caux >l. Cette opinion est également émise par Hampe (2) en ·!866;
enfin Bcnte (:1), dès 186;), considère l'acétamide comme assimi-
lable. Pendant plusiems années la qttcslion est ensuite délaissée, mais
le tt·avail mag·istr·al de Pfelle1' (4) ,:ur la formation et le rôle de
l'aspar;tginc la remet à l'ordre du jour. Dès lors, les observations
se multiplient, spécialement en Allema!!'ne et en Russie; ne pou-
vant les passer toutes en revue, je renverrai à leur sujet au Mémoire
publié en 1879 par Capus (5) daus les Annales ag1'onomiques. Depuis celte date, un g:rand nombre d'auteurs ont continué à
s'occuper de l'asparagine. C'est ainsi que Lœw et Bokorny (6) ont
pu nourrir des Spirogym à l'aide de cette substance, que Schim- :SÉAt";CE liU ~6 JUILLET 1\lOJ. 32li per (1) ct Emmerling- (2) la considèrent comme lenne de migra-
tion de l'azote dans les tissus végt\laux, que Lœw (3) observe une
relation entre l'asparagine et
le~ l1ydrales de carbone dans la
plante ct constate le rôle important joué par l'ammoniaque dans
ces transformations. Parmi les autres travaux se rapportant à l'as-
similation de l'asparagine, on peut citer encore ceux de Meunier( 4),
de Borodine (5), de Frank (6), de I\inoshita(7), de IJ'~Iullcr (8), de
Miyachi (9), de Nakamura ('10), de Schulze (11), etc. La résultante de ces di\·ers travaux est que l'asparagine se con-
duit non seulement comme une substance assimilable, mais qu'elle
joue un rôle des plus importants dans les phénomènes intimes de
la nutrition des végétaux. ownloaded by [189.219.133.207] at 15:34 04 June 2016 Downloaded by [189.219.133.207] at 15:34 04 Jun En dcho1·s de l'asparagine, peu de travaux sont à signaler con-
cemantles amides: Lœw et Uokorny ( 12) constatent que les Algues
vivent mal dans l'urée, tandis que Frank (1:3), essayant l'urée,
l'acide urique, el l'acide hippurique admet que ces substances
sont assimilables. Downloaded by [189.219.133.207] at Il m'a paru de quelque intérêt de continuer sur les amides la
série d'expériences déjà effectuées sur les amines, les alcaloïdes ct
les nitriles, et de rechercher comment ces composés se conduisent
vis-à-vis des végétaux. p
(
p
)
(12) Lœw et Bokorny, Chein. physiol. Studien über Algen (Jnal f. Prakt.
Chem., neue Folge, Bd 36, 1887).
· (13) Frank, La statistique de l'azote en agric~tlture (Landw. Jahrb., 1888,
p. 421). Downloaded by [189.219.133.207] at 15:34 04 June 2016 La présente Note est relative à la nutrition
des Algues et des Champignons. ( )
y
g
p
(10) :"akamura, Coll. agr. Tokio, Bull., vol. Il, n• 7. p. 465. (11) Schulze, De l'influence des hydrates de carbone su1· la formation des
lbumi11oïdes dans les plantes (Landw. Jahrb., 1898, p. 516). f p
(
p
)
(12) Lœw et Bokorny, Chein. physiol. Studien über Algen (Jnal f. Prakt. Chem., neue Folge, Bd 36, 1887). · g
(13) Frank, La statistique de l'azote en agric~tlture (Landw. Jahrb., 1888,
p. 421). 1
~ 1
~
.,
"
"
~
~
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~
~
=
-::l
-~
~
,_
1 ·; 0
c
'ë
3
1
'ë. '"
SURSTANCJ<.:s
.S' :a
'"
0
~
.... "
~
..;:
~
.,
t1. Eau distillée ...................... jwoo lwoo 1 woo lwoo !woo 11
Azotate de potasse ............... +· ~uo 1 o 1 --o -r ~
~- o 1
Amide ........................... ·l o jo.OH9J0.11 ~-F-~ 1 ~~·!0.1/ll jo
Phosphate de potasse ..... :.~.~-~-~~;~oo lo-~wo jo.:wo Jo.:wo Jo.:wo 10
Sulfate de magnésie ............... ,0.200 Jo.200 10.200 Jo.~oo 10.200 Jo
Sulfate de chaux ............. : ... .jo.200 jo 200 10.200 10.200 ,0.200 Jo
-Sulfate de fer .•.................. ·1 traces 1 traces 1 traces 1 traces 1
traces 1 t
q. s.j q. s.,.q. s. SÉANCE DU 26 Jl'ILLET 1901. SÉANCE DU 26 Jl'ILLET 1901. ::128 I. -
Nutrition des ,ligues. De même que dans toutes les observations précédentes, le
liquide nutritif servant de milieu de culture type est le liquide
de Molisch. Pour chacune des amides étudiées, il a été préparé
une solution différant de la première par la substitution aux 09',200
d'azotate de pota~se de la quantité d'amide renfermant le même
poids d'azote. La diminution du potassium est compensée par
l'addition de 09',200 de sulfate de potasse. Voici les formules de ces divers milieux, résumées en tableau. nloaded by [189.219.133.207] at 15:34 04 June 2016 Ces liquides ont été répartis dans des fioles d'Erlenmeyer bou-
chées au coton et stérilisées par tyndalisation (chauffes de 20 mi-
nutes à GO•, à 2-1. heures d'intervalle). Des expériente!l_ préliminaires
ont montré que l'on peut, par ce procédé, stériliser parfaitement
des solutions neutt·es d'amides sans entrainerde décomposition de
ees substanees. Downloaded by [189.219.133.207] at 15:34 04 Jun D'autre part, à l'aide de cultures répétées dans du liquide de
~iolisch, j'ai obtenu les deux espèces suivantes d'Algues à l'état de
pureté et hien exemptes de miero-organismes : 1• Plenrococcus miniatus; 2• Raphidium polymorphum. Le 15 juillet 1899, les dive1·s milieux, préparés comme il vient
d'ètre dit, ont été ensemencés avec une trace d'une de ces deux
espèces, puis ils ont été placés près d'une fenêtre, à la température
ordinaire. Au bout d'une dizaine de jours un début manifeste de
végétation pouvait se note1· dans la plupart des flacons. Seuls ceux
qui renfermaient la benzamide, la salicylamide et l'acétanilide ne
présentaient aucune tl'ace de multiplication des cellules déposées
comme semence. Au bout d'un mois, les Algues apparaissaient
dans la benzamide. Enfin, le 20 septembre, lors de l'arrêt de l'ex-
périence, toutes les fioles avaient cultivé, sauf celles qui conte-
naient la salicylamide et l'acétanilide. Les divers liquides de culture ont alors été examinés au double
point de vue de la présence de bactéries et de celle de composés
ammoniacaux. Dans aucun cas je n'ai trouvé la moindre trace de
micro-org·anismes, pas plus que de sels ammoniacaux. On peut
donc admettre que les amides qui ont servi à la nutrition des
Alg·ues ont été absorbées sans avoir subi de fermentation préalable. ~
1 1 Sans azOIP ...... ; ....... 1 HiOO
1
70.000
1 6. 50
1
0
1 Il. 60
1 0. 4
~- ~q Rau~;l~difi:.;-: ......... ,15001 __ 70.000 1 6Ji0 j 4~50 f{Ï.60 1 0.4
31 F'ot·miami<le ............. I. -
Nutrition des ,ligues. ltil?O f
66.79'~ j 6.rlo 1
il
1 ~1.60 1 0.4
41 Acétamide .............. ,1tJOO ! 63.a87 1 6.50 \
o 1 0.60 j 0.4
51 Propiouamide ........... ,1500 / 60.381
1 6.50 ! 0 , 0.60 [ 0.4
. 61 Ilutyrami•le ............. l1500 1 57.17516.50 1
0 ! 0.60 / O. 7J Benzamide .............. ,1500 1 47.556-1 6.50 r
0 ( 0 60 1 0.4
tl' Salicylamide ............ ,1500 j
'~7.556 j 6.50 1
0
1 0.60 1 0.4
91 Asparagine ............. ,1500 j 63.587j6.50 /
0
j 0.60 1 0.4
10
Urée ................... ,1500 1 69.397 \ 6.50 1
0
/ 0.60 1 0.4
tt
Oxamide ................ ft500 1 fl6. 79t. 1 6.50 1
0 j 0.60 [ 0.4
121 Succinamide ............ ,1500 J
63:~R7 j 6.50
1
0
1 0.60 j 0.4
131 Acétanilide ............ ·11500 1 63.587 / 6.50 j
0 / 0.60 1 0.4 330 SEANCE DU 2ü JUILLET 1901. Plusieurs autres essais, effectués dans les mêmes conditions ont
conduit ù des résultats analogues. If.- Nutrition des t::bampigoous. Parlant toujours du même principe, j'ai choisi pour faire ces
recherches une série de modifications du liquide de Haulin calcu-
lées de manière à présenter une composition élémentaire cons-
tante, la nature du composé azoté offert comme aliment variant
seule. Ces solutions nutritives devant être parfaitement neutres,
par suite de la facile décomposition des composés amidés, l'acide
tartrique a, en outre, été remplacé par une quantité convenable de
tartrate neutre de potasse. Downloaded by [189.219.133.207] at 15:34 04 June 20 Les formules des différents milieux sont résumées ci-après : Tous ces liquides ont été introduits dans des fioles d'EI'lenmayer,
à raison de 100. cent. cubes pat· fiole, puis ils ont été stérilisés par
tyndalisation à 60', et finalement ensemencés a l'aide de chacune
des trois espèces suivantes: Downloaded by [189.219.133.207] at 1 1 • Aspergillus niger ; 2• Aspergillus repens; 3• Penicillium glancum. Une première expérience a été effectuée avec de l'Aspergillus
niger, elle n'a été que qualitative. L'ensemencement a eu lieu le
6 mai 1899. Les divers milieux ont été alors disposés dans une
étuve à .38• et abandonnés jusqu'au 8 juin. J'ai pu ainsi constater que Je Ch~1mpignon, outre le liquide type,
s'était développé dans les flacons renfermant la formiamide, l'acé-
tamide, la pmpionamide, la butyramide, l'asparag·ine, l'urée, et
très légèrement dans la succinamide et I'oxamide. Rien n'avait
poussé en présence de la benzamide, de la saJicylamide et de
l'acétanilide. Les autres expériences ont été quantitatives. Deux ont été faites
avec de l'Aspergillus repens, la dernière avec du Penicillium glatt-
cum. Dans les deux premiers cas, les flacons de cultùre ont été mis
à l'étuve à 38•; dans celui 1iu Penicillium, ils ont été maintenus à
la température ordinaire. A la fin de chaque expérience, les champignons ont été recueillis
snr des filtres tarés, puis lavés, séchés à l'étuve et pesés. ~
3
4 6 EXPÉRIENCES
LIQUIDES A BASE DE
~
1 = '<>
1 ~
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=
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Poid~dechampignon., 0.032 2.07'21 1.5:17,1.R:2:-.JI.lm/t.8R
t•• EXPÉRIENCE •
(en grammes)
Aspergillus repens. l;ai_n ~ar rapport au'
» 12.0!01 f.56511.ill:~~; 1.90Uji.R4
'(!!juillet 1899_
hqmde sans azote. 5 août 189~). Pc~te _par rappnrl au'
» 1
)
1
» 1
»
1
»
1
,. hqmde sans azole. . If.- Nutrition des t::bampigoous. Poidsdechampignon., 0.021 t.4fi111.502 1.7ü911.G8~
2' EXPERIENCE :)
Aspergillus repens
Gai_n par rapport au
» 11. 14011 . '~8111 . 7 481
1
1 . Gr.gl 0. 99
(8 . . i"OO
hqmde sans azote. Janvier "
-
21janvier 1900). Pe1:te _par rapport aul
»
1
» 1
»
/. ))
1 » 1
»
hqmde sans azote. . 1
Poids de champi~non. 3' EXPERIENCE :
r;~~~~::~~g!:um -~-;a-fi11-q
1
-,f-d~-~ ~--:;-,~~:-:'~-~:.-.~.-~~-~.~~~--» --!-\-~-.f.-l9-9+-j-~-.2-6-tf-[-o-_:{--6--'ol:-1-.-o-6
2 février1900).1 Per_te par rapport au'
» 1
»
1
» 1
,
1. » 1
,
hqmde san> azote. . 332 S~:ANCE Dll 26 JUILLET 1 !)0 J. Pour plus de simpficilé, je vais réunir lous les résullats en un
seul tableau. En examinant les résultats consignés dans ce tableau, on peut
constater f)lle les amides de la série gTassc ont t'Lé assimilées,
tandis que celles de la série aromatique n'ont donné lieu à aucun
développement de Champignon. D'autre part, on p·cut également rema1·quer CJUe l'assimilabilité
des divet'ses amides n'est pas soumise à une loi de gradation ana-
logue à celle qui a été observée avec les amides. L'oxamide a bien
donné un rendement plus faible que les amides inférieurl's de la
série, mais les poids de Champignons constatés dans les liquides
à base de f'ormjamide, de propionamide. d'acétamide, de hutyra-
mide, par exemple, ne sc présentent pas en échelle décroissante
marquée, et souvent mt\me sont si peu différents qu'il est difficile
d'y voir autre chose que des Yariations d'ordre purement expéri-
mentnl. ownloaded by [189.219.133.207] at 15:34 04 June 2016 On peut noter nussi combien sont importants les chiffres obte-
nus avec l'asparag-ine et l'urée: le liquide de Raulin servant de
type ne donne pas de meilleurs rendements, et les idées de G. Ville
rapportées au déhut de cette Note recoivent une confirmation
absolue. Une mention spéciale est due aux essais de pureté des liquides
nutritifs après culture. Ayant constaté dans quelrpJes milieux une
légère production d'ammoniaque, production d'ailleurs incons-
tante, se rencontrant, par exemple, dans une fiole ensemencée
avec de !'Aspergillus niger et ne s'observant pas avec la mème
amide en présenl:e d'Aspergill-us repens ou de Penicillium glau-
cum, j'ai fait un grand nombre d'ensemencements en Yariant les
conditions expérimentales de manière à élucider cette question. (1) Travail fait au laboratoire des Hautes-Études (Botanique) de l'École de
pharmacie de Paris.
(2) Czapek, Zur Kenntniss der Slickstoffversorgung und Eiweissbildung
bei Aspergillus niger IBer. d. d. Bot. Gesell., pp. 130-139, 1901);- lbid.,
Untersuchungen über die Stickstoffgewinnung und Eiweiss bildung der Pflan-
zen (Hofmeister's Beitriige z. chem. Pltysiol. und Pathol., Bd 1, Heft 10-12,
PP- 538-560, 1902). If.- Nutrition des t::bampigoous. p
q
Ces ensemeneements ont été faits soit directement avec le
Champignon après tyndalisation du liquide nutritif, soit après
addition de divers antiseptiques peu puissants: thymol, chlo-
roforme, fluorure de sodium. D'autres essais ont été effectués en
mélangeant aux solutions d'amides des solutions des ferments
solubles sécrétés par les Champignons en expérience, soit en sou-
mettant le tout à la filtration à la bougie, soit après addilion
d'antiseptiques : thymol, chloroforme, fluorure de sodium. Enfin
l'examen bactériologique des milieux a été opéré (cultures sur
bouillon peptone). 333 LUTZ. -
NUTfiiTION DES TIIALLOI'HYTES A L'AIDE DES AMIDES. LUTZ. -
NUTfiiTION DES TIIALLOI'HYTES A L'AIDE DES AMIDES. Les résultats de ces observations peuvent se résumer ainsi qu'il
suit : Toutes les fois qu'une production d'ammoniaque est constatée
dans les cultures, c'est qu'elles ont été le siège de fermentations
secondaires mic1·obiennes. En l'absence absolue de micro-organismes, le Champignon se
développe fort bien sans qu'il y ait formation d'ammoniaque aux
dépens des amides en présence. Les ferments solubles sécrétés pal' l'Aspet·yillus nigP'/'1 1'.4sper-
gilltts repens et le Penicillium glaucnm ne produisent pas d'hy-
dratation des amides capable de les transformer en sels ammo-
niacaux. UN CAREX NOUVEAU POUR LA FLOHE FRAN(IAISE;
par M. Ernest IIALINVAUD •·t l<'rère HJ';~RIBAUit .JO!!iiEPH {l) 1. line heureuse rrucoutre. 1. line heureuse rrucoutre. l\1. G:tbt·iel Vialon, habitant Monaco, herborisait fe ~7 mai 190'1,
sm· les bords du Donaréou, atllnent de la ri,·e gauche du Var,
dans lequel il se jette à un kilomètre et demi au nord de la petite
g·are de Colomars; un Carex, nouveau pour notre confrère, attira
son attention. Les caractères du fruit, si importants dans le genre Carex,
n'étant pas suffisamment marqués à la fin de mai, notre zélé con-
frère revint le 16 juin et récolla la plante en meilleur état. Ne la
trouvant pas décrite dans les ouvrages qu'il possédait, il s'empressa
de la cor11muniquer il son ancien maître de Clermont-Fenand (2),
et celui-ci, n'y reconnaissant aucune des espèces françaises qui lui
sont familières envoya les échantillons à Paris pour être confrontés
avec les types que renferment les gTands herbiers. (1) L'ordre de mérite, d'accord ici avec l'alphahHilple, Higeait que le nom
de mon tl'op modeste collal:oratem· et ami pi·I·céMtt Ir mien; son affectueuse
insistance ne l'a point permis. (Ern. Maliuvaud.) (2) M. Gabriel Vialon, natif de Clermont-Fe!Taml, est un ancien élève du
Pensionnat des F'ri~res de cette vilte. Downloaded by [189.219.133.207] at 15:34 04 June 2016 On peut donc admettre que les Champignons sont susceptibles
d'assimiler l'azote des amides, sans lui faire subir de modification
préalable. Il est vrai que cette condition est rarement réalisée
dans la pratique, pal' suite de l'extrême altérabilité de ces corps
sous l'action des micl'o-organismes, par exemple. Néanmoins s'il
était permis d'étendœ aux Phanérog·ames les conclusions de cette
étude, la propriété de l'assi111ilabilité directe des amides pourrait
être appliquée aux phénomènes de migTation des substances qua-
ternaires dans le corps des plantes, migrations dans lesquelles
l'asparagine paraît jusqu'ici jouet· un rôle important. Dans un prochain travail, j'étudierai l'action particulière exer-
cée par les composés organiques à noyau aromatique sur la végé-
tation des Cham pignons ( 1 ). [Note ajoutée pendant l'impression. -
Dans l'intervalle qui s'est
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n'aient pas été conçues dans le même esprit que les miennes, il a été
néanmoins amené à examiner l'action sur le développement de l'Asper-
gillus niger de plusieurs amides et acides amidés fJUC j'avais également
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Role of Sox9 in BPD and its effects on the Wnt/β-catenin pathway and AEC-II differentiation
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Version of Record: A version of this preprint was published at Cell Death Discovery on January 11th,
2024. See the published version at https://doi.org/10.1038/s41420-023-01795-2. Article Keywords: Sox9, lncRNA GAS5, Wnt/β-catenin, AEC-II, differentiation
Posted Date: August 16th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3066052/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Additional Declarations: (Not answered) Version of Record: A version of this preprint was published at Cell Death Discovery on January 11th,
2024. See the published version at https://doi.org/10.1038/s41420-023-01795-2. Page 1/30 Abstract The excessive activation of the Wnt/β-catenin signaling pathway is an important regulatory mechanism
that underlies the excessive proliferation and impaired differentiation of type 2 alveolar epithelial cells
(AEC-II) in bronchopulmonary dysplasia (BPD). Sox9 has been shown to be an important repressor of the
Wnt/β-catenin signaling pathway and plays an important regulatory role in various pathophysiological
processes. We found that the increased expression of Sox9 in the early stages of BPD could
downregulate the expression of β-catenin and promote the differentiation of AEC-II cells into AEC-I,
thereby alleviating the pathological changes in BPD. The expression of Sox9 in BPD is regulated by long
noncoding RNA growth arrest-specific 5. These findings may provide new targets for the early intervention
of BPD. Introduction Pulmonary organogenesis requires precise timing and coordination to influence the spatial organization
and function of parenchymal cells. Owing to exposure to hyperoxia and inflammation, the development
of the lungs in preterm infants born during the cystic phase of lung development may be permanently
altered, resulting in persistent structural damage and lifelong respiratory impairment, which can lead to
bronchopulmonary dysplasia (BPD)[1],[2]. Hyperoxia can induce the BPD phenotype and alter the
composition of cellular compartments, especially alveolar epithelia, stromal fibroblasts, capillary
endothelia, and the macrophage population[3]. The abnormal ability of type 2 alveolar epithelial cells
(AEC-II) to proliferate and differentiate into AEC-I is an important factor in this pathological change. Animal experiments have shown that in the pathogenesis of BPD, various signaling factors, such as
transforming growth factor β, connective tissue growth factor, fibroblast growth factor 10, vascular
endothelial growth factor, caveolin-1, wingless and int-1 (WNT)/ß-catenin, and elastin, are abnormally
regulated[4]. The WNT signaling pathway is one of the key signaling pathways involved in the regulation
of the stem and progenitor cells of various organs. In addition to its role in embryonic and fetal lung
development, the Wnt signaling pathway is crucial to lung homeostasis and regeneration[5]. Our previous
studies on the canonical and non-canonical pathways mediated by Wnt3a and Wnt5a have shown that
both alveolar epithelial cell damage repair and alveolarization are regulated in hyperoxia-induced lung
injury[6] and that Wnt5a is involved in the pathological process of neonatal hyperoxia-induced injury[7]. The activity of the Wnt pathway is regulated by various means, such as the acylation of the Wnt protein,
stability of the Wnt receptor, and abundance of the β-catenin protein. During embryonic development,
Sox9 plays an important role in the regulation of sex determination[8], cartilage development[9], and
development of digestive organs[10] through mutual antagonism with the Wnt signaling pathway. During
lung development, Sox9 regulates normal branch morphogenesis and lung epithelial development
through various signaling pathways, inhibits the premature initiation of alveolarization, and is expressed
in adult airway distal progenitor cells and maintains their normal function[11, 12]. The activation of Sox9 is Page 2/30 Page 2/30 Page 2/30 crucial for the recovery of lung function after acute lung injury[13]. In this study, we preliminarily verified
the effects of Sox9 in alveolar dysplasia in BPD and its relationship with the Wnt signaling pathway. Introduction Long noncoding RNAs (lncRNAs) are long-chain RNA molecules that play important roles in many
biological functions, including cell growth and developmental processes, by regulating cellular signaling
pathways, such as the JAK/STAT, PI3K/Akt, and Wnt signaling pathways[14, 15].High expression of lncRNA
Growth arrest-specific transcript 5 (GAS5) can inhibit cell cycle progression, whereas the downregulation
of GAS5 can inhibit cell apoptosis and promote cell division[16]. GAS5 is downregulated in childhood
pneumonia, acute lung injury, and asthma, and its overexpression can cause the inhibition of
inflammation and epithelial cell apoptosis[17–19]. In our previous genomic study on lipopolysaccharide-
induced acute lung injury, the expression of GAS5 was significantly altered[20], but its expression pattern
in BPD was unclear. Through a bioinformatics analysis, we found several microRNAs (miRNAs) that
coexisted with GAS5 and Sox9, which may constitute a competing endogenous RNA regulatory pathway. In this study, we explored whether there is a regulatory relationship between GAS5 and Sox9. Changes in
Sox9 expression were found to affect the key factors involved in the Wnt/β-catenin signaling pathway
that regulates the process of alveolarization. Results In BPD, the alveolar structure is simplified, alveolarization is impaired, and expression of Sox9 is
increased in the early stage of the disease In BPD, the alveolar structure is simplified, alveolarization is impaired, and expression of Sox9 is
increased in the early stage of the disease The hematoxylin and eosin staining of the lung tissue (Fig. 1A) showed that as the rats aged, the
hyperoxic model group (H) started to show inflammatory cell infiltration from day 3 and changed
significantly from day 7 onward. The alveoli became enlarged, number of alveoli decreased, alveolar wall
thickened, and alveolar crest and secondary septum became less visible or even disappeared. From days
14 to 21, the size of the alveolar cavity increased significantly, alveolar fusion appeared, the number of
alveoli was significantly reduced, and the alveolar structure was simplified. The RAC ,which is positively correlated with lung development ,refers to the number of alveoli on the
vertical line from the center of the respiratory bronchioles to the nearest fibrous septum (or pleura) and is
an important indicator for evaluating the maturity of developing lungs.In Fig. 1B, the RAC of the control
group gradually increased, indicating that the alveoli undergoing alveolarization were gradually maturing. The RAC value of the model group began to decrease significantly on day 7, and the difference was more
significant from days 14 to 21 (P < 0.001). This indicated that disordered alveolarization is present in
BPD. Then we detected the expression of the AEC-II surface marker SPC and the AEC-I surface marker AQP5 in
animal models. The expression of SPC was increased while AQP5 was decreased. The changes in their Page 3/30 Page 3/30 expression were significant after 7 days of exposure to hyperoxia (Fig. 1C, 1D, 1E), which confirmed the
hyperproliferation of AEC-II cells in BPD and their impaired conversion to AEC-I. expression were significant after 7 days of exposure to hyperoxia (Fig. 1C, 1D, 1E), which confirmed the
hyperproliferation of AEC-II cells in BPD and their impaired conversion to AEC-I. The results of the PCR and western blotting analyses showed that in the early stage of BPD the
expression of Sox9 increased (Fig. 1F, 1G), exhibited a downward trend, and was lower than that in the
control group after 7 days. The immunofluorescence staining of the tissue sections (Fig. Results 1H, 1I) showed that with prolonged
hyperoxic exposure, the immunofluorescence intensities of Sox9 and AQP5 gradually weakened, whereas
the fluorescence intensity of SPC gradually increased, suggesting that Sox9 may be involved in the
regulation of AEC-II cell proliferation and differentiation. The PCR and western blot results further showed that the expression of SPC and Sox9 increased in the in
vitro cell model (Fig. 1J, 1K, 1L), which was consistent with the early manifestations in the BPD animal
model. The distribution of Sox9 in the nuclei and cytoplasm of cells changes during the proliferation and
transformation of AEC-II cells. The cytoplasmic expression of Sox9 is increased and nuclear expression
of AEC-II cells is decreased in hyperoxic culture. The results of the immunofluorescent double staining of the primary AEC-II cells showed that the
expression and fluorescence intensity of SPC gradually decreased during the culture process (Fig. 2A). By
contrast, the expression and fluorescence intensity of AQP5 increased (Fig. 2B), suggesting that the
primary AEC-II cells had differentiated into AEC-I. Sox9 was expressed in the nuclei of the primary AEC-II
cells, and its expression gradually weakened over time. The expression of Sox9 increased in the hyperoxic
culture, especially in the cytoplasm; however, the change in its expression in the nucleus was not
significant. The nucleocytoplasmic protein was extracted in vitro for a western blot analysis. The results showed that
the nuclear expression of the Sox9 protein in the cells of the model group decreased, whereas its
expression in the plasma increased (Fig. 2C, 2D). Exogenous Sox9 can improve alveolarization in animal models of BPD by promoting AEC-II cell
proliferation and differentiation into AEC-I We administered exogenous Sox9 to animal models of BPD. In the model group, exogenous
overexpressed plasmid was injected into the trachea every other day from days 5 to 14.The staining of
the lung tissue sections showed that the development of the alveoli in the BPD Sox9-overexpression
group was improved compared with that in the BPD model group (Fig. 3A). The number of alveoli
increased, their structure became more complete, their size was more uniform, and the RAC value
increased (Fig. 3B), which indicated that exogenous Sox9 can improve the degree of alveolarization in
BPD lung tissue. The immunofluorescence double staining (Fig. 3C, 3D) also showed that the number of
SPC-labeled cells decreased compared to that in the model group, while the fluorescent expression of Page 4/30 AQP5 was enhanced. This confirmed that exogenous Sox9 can promote the proliferation and
differentiation of AEC-II cells into AEC-I. AQP5 was enhanced. This confirmed that exogenous Sox9 can promote the proliferation and
differentiation of AEC-II cells into AEC-I. We further detected the changes in SPC expression after administering Sox9 in an in vitro cell model. The
expression of SPC decreased after the knockdown of Sox9 under hyperoxic conditions and increased
after the overexpression of Sox9 (Fig. 4A, 4B, 4C). Sox9, therefore, promotes the proliferation of AEC-II
cells in BPD. To verify the effects of Sox9 on AEC-II cell differentiation, we overexpressed Sox9 in primary cells and
analyzed the degree of AEC-II cell differentiation using flow cytometry. The results showed that the
number of AEC-II cells increased after the overexpression of Sox9 in the control group (Fig. 4D),while in
the model group the number of AEC-I cells increased. Double immunofluorescence staining showed that
in the model group, SPC expression was increased after the knockdown of Sox9 in the AEC-II cells and
decreased after Sox9 had been overexpressed. In addition, the AEC-II cells with overexpressed Sox9 in the
model group tended to grow from the typical round and oval-island shape to the divergent polygonal
shape of the AEC-I cells (Fig. 4E, 4F). Compared to the control, in the model group, the AQP5 fluorescence
signal of the AEC-I cells overexpressing Sox9 was enhanced, and the morphologies of the cell
membranes were clearer and more continuous (Fig. 4G, 4H). The overexpression of Sox9 may, therefore,
promote the rapid differentiation of AEC-II cells into AEC-I under hyperoxia. Exogenous Sox9 can improve alveolarization in animal models of BPD by promoting AEC-II cell
proliferation and differentiation into AEC-I The Wnt/β-catenin pathway is overactivated in BPD, and Sox9 can downregulate the expression of β-
catenin and affect its nucleoplasmic distribution Sox9 expression in BPD is regulated by the lncRNA GAS5 but not through the miR-1912-3p pathway The PCR results of the animal model tissues showed that the expression of GAS5 in the model group was
significantly increased and gradually decreased,that was positively correlated with the expression of
Sox9.The expression of miR-1912-3p showed an overall upward trend that was negatively correlated with
Sox9 (Fig. 7A). In vitro cell models, GAS expression increased and miR-1912-3p expression decreased,
which was consistent with the early expression of animal models (Fig. 7B). We intervened with GAS5 in an in vitro cell model and detected the changes in the expression of Sox9
using PCR and western blotting (Fig. 7C, 7D, 7E). The results showed that the knockdown and
overexpression of GAS5 could decrease or increase the expression of Sox9, respectively. The extracted primary cells were subjected to the knockdown or overexpression of GAS5. The
immunofluorescence double staining showed that the fluorescent intensity of Sox9 was weakened or
enhanced, respectively (Fig. 7F, 7G, 7H, 7I). The results of the dual-luciferase reporter gene assay showed that rno-miR-1912-3p could regulate the
expression of luciferase with Gas5 (Fig. 7J, 7K). The overexpression of miR-1912-3p further reduced the
luciferase activity of SOX9 WT-3ʹUTR. Additionally, the regulatory activity of Sox9 was restored after the
predicted site mutation but that of GAS5 was not significantly restored, indicating that rno miR-1912-3p
mimics may not have significant interactions with the predicted site of GAS5 but can bind to Sox9
through the predicted site. The Wnt/β-catenin pathway is overactivated in BPD, and Sox9 can downregulate the expression of β-
catenin and affect its nucleoplasmic distribution Our previous studies showed that the Wnt/β-catenin pathway is overactivated in BPD. In this study, as our
previous results, β-catenin exhibited an overall upward trend in the model group compared with that in the
control group. Its expression further decreased on days 1–3 and increased after day 7 (Fig. 5A, 5B, 5C). We further detected the changes in β-catenin expression after the Sox9 intervention in an in vitro cell
model. The results showed that the expression of β-catenin increased after knockdown of Sox9 and
decreased after overexpression of Sox9 (Fig. 5D, 5E, 5F). The nuclear and cytoplasmic distribution of β-catenin in the in vitro cell model was examined using
western blots. The expression of the total and plasma β-catenin proteins in the cells of the model group
decreased, but the nuclear expression of β-catenin increased (Fig. 6A, 6B). The total protein expression
level (in both the nucleus and plasma) increased after overexpression of Sox9 (Fig. 6C), with the increase
in the plasma protein expression being more significant. However, the total protein expression of β-
catenin decreased, its nuclear protein expression decreased, and its plasma protein expression increased
(Fig. 6D, 6E), which confirmed that the overexpression of Sox9 can reduce the expression of β-catenin in
the nucleus. Next, we intervened Sox9 in primary AEC-II cells and further performed β-catenin + SPC
immunofluorescence double staining. The results (Fig. 6F) showed that the fluorescent s Next, we intervened Sox9 in primary AEC-II cells and further performed β-catenin + SPC
immunofluorescence double staining. The results (Fig. 6F) showed that the fluorescent signal of β- Page 5/30 Page 5/30 catenin was significantly enhanced after the knockdown of Sox9, while it was weakened after the
overexpression of Sox9. Compared with that in the control group, the expression of β-catenin in the
nucleus increased after the knockdown of Sox9 in the model group. These results suggest that the
overexpression of Sox9 can not only downregulate the overall expression level of β-catenin but also
affect the nuclear expression of β-catenin, thereby regulating the activation of the Wnt pathway. Discussion The main pathological changes in BPD include alveolar simplification and pulmonary vascular dysplasia,
which are the results of a complex interaction between changes in alveolar and vascular development,
prenatal and postnatal pathogenic factors, and pulmonary repair processes[21]. In BPD, the excessive
proliferation of AEC-II cells and their failure to differentiate into AEC-I cells are important reasons for
insufficient damage repair. The Wnt/β-catenin signaling pathway has been found to play an important
regulatory role in lung development, lung injury repair and regeneration, and lung disease
progression[22],including non-small-cell lung cancer, chronic obstructive pulmonary disease, and
idiopathic pulmonary fibrosis[23–26]. The canonical Wnt/β-catenin pathway is overactivated in BPD. Inhibiting the Wnt/β-catenin signaling pathway can reduce the proliferation of AEC-II cells, promote their
differentiation into AEC-I cells[27], and also attenuate hyperoxia-induced lung injury in neonates[28]. Therefore, it is important to identify the factors in the relative signaling network that directly affect the key Page 6/30 Page 6/30 transcription factor β-catenin. As a member of the Sox family, Sox9 participates in biological processes
through various signaling pathways, such as Notch, PI3K/AKT, Hippo, and Wnt, and has been found to
exhibit tissue and spatiotemporal specificity[29–31]. Crosstalk between these different signaling pathways
forms a complex signal-regulatory network[32, 33]. Our study explored the protective role of Sox9 in BPD
development and its effect on the Wnt pathway. We found that the increased expression of Sox9 in the
early stages of BPD could downregulate the expression of β-catenin and promote the differentiation of
AEC-II cells into AEC-I, thereby alleviating the pathological changes in BPD. The lncRNA GAS5 was further
found to regulate the expression of Sox9 and may serve as a target for the early diagnosis or intervention
of BPD. These findings contribute important nodes to the formation of the Wnt signaling pathway
network. Our study found that the expression level of Sox9 was increased in the early stage of BPD. Additionally,
exogenous Sox9 improved alveolarization in animal models of BPD. This was achieved by promoting the
proliferation of AEC-II cells and differentiation of AEC-I cells, which highlighted these cells as potential
targets for the early intervention of BPD. Sox9 belongs to the SoxE subgroup and is located in the 3 Mb
region of chromosome 17, which lacks other protein-coding genes. Sox9 is a dynamically expressed
transcription factor and maintains stem cell function during lung development[34]. Rockich et al. Discussion showed
that the expression of Sox9 during lung development maintains the undifferentiated state of distal lung
progenitors, is down-regulated starting at embryonic day 16.5, concurrent with the onset of terminal
differentiation of type 1 and type 2 alveolar cells. The removal of Sox9 leads to premature alveolar
differentiation, whereas the addition of Sox9 prevents differentiation and inhibits the epithelial transition
from columnar to squamous epithelium that allows the adult alveoli to form[12]. Another study on
cultured lung organoids confirmed that the inactivation of Sox9 did not affect the differentiation of
mouse alveolar cells or ability of Sox9 to promote the recovery of damaged lung function[35]. In our study,
the tissue sections of the animal models transfected with Sox9 showed improved alveolarization, an
increased number of alveoli, a more complete structure, more uniform-sized alveoli, and increased RAC
values. The results of the immunofluorescence double staining also showed that the number of SPC-
labeled undifferentiated AEC-II cells was decreased compared with that in the model group, while the
fluorescent expression of AQP5 was enhanced. This indicates that exogenous Sox9 can promote the
proliferation and differentiation of AEC-II cells into AEC-I. These conclusions seemingly contradicted each
other, but additional experiments revealed that the expression of Sox9 in BPD increased and that its
localization changed significantly. We further found that the expression of Sox9 decreased in the nucleus and increased in the cytoplasm of
the primary AEC-II cells induced with hyperoxia; therefore, we speculated that the overexpression of Sox9
in BPD may promote the differentiation of AEC-II cells through its nucleocytoplasmic translocation. The
mechanisms by which the downregulation of the nuclear expression of Sox9 promotes AEC-II cell
differentiation may be similar to those involved in the development of embryonic alveoli. The HMG
domain of Sox9 contains two nuclear localization signal sequences and one leucine-rich nuclear export
signal sequence and exhibits a high translocation rate between the nucleus and cytoplasm. Numerous Page 7/30 Page 7/30 studies have confirmed that the regulation of organogenesis by the Sox9 protein is not static but is a
highly dynamic and complex process caused by the nucleocytoplasmic shuttling of Sox9. The
nucleocytoplasmic translocation of Sox9 determines its function. During sex development, the balance
between the nuclear import and export of Sox9 determines the level of transcriptionally active Sox9 in the
nucleus. Discussion Its nuclear transfer further shifts the fate of undifferentiated somatic cells in primitive gonads
away from the ovariogenesis pathway to testicular differentiation[36]. During kidney development, Sox9 is
activated by nucleocytoplasmic translocation and promotes the proliferation and differentiation of renal
tubular epithelial cells[37]. During corneal development and injury repair, Sox9 is synthesized in the
cytoplasm of limbal stem or progenitor cells. When it translocates into the nucleus, Sox9 triggers the
transformation of limbal epithelial progenitor cells into proliferating, transiently expanding cells and
differentiation into the correct lineage. Regenerative potential is acquired through this process, which is
achieved through the cooperation of Sox9 and Wnt/β-catenin signaling in a mutually inhibitory
interaction[38]. The expression of Sox9 is increased during the formation of BPD, and the process of
nuclear transfer occurs during the differentiation of AEC-II cells, which reflects that the transcriptional
regulation of Sox9 plays an important role in the molecular mechanisms underlying BPD formation. However, the specific mechanisms underlying the changes in the nuclear and cytoplasmic distribution of
β-catenin remain to be further elucidated. Members of the canonical WNT signaling pathway play an important role in regulating the development
of mammalian lung organs. The deletion of Wnt2/Wnt2b can lead to a reduction in the number of
tracheal branches in the lungs and severe lung hypoplasia[39]. Wnt7b stimulates embryonic lung growth
by coordinating increased epithelial and mesenchymal cell replication[8]. In mouse lung epithelial cells,
the differentiation of alveolar epithelial cells with knocked-out β-catenin is hindered, and the lungs remain
in the initial stage of tracheal formation[9]. In the process of lung development and injury repair, there is
crosstalk between the Wnt pathway and other signaling pathways. During the pseudoglandular stage of
lung development, the fibroblast growth factor and WNT signaling pathways work together to support
mesenchymal growth and coordinate epithelial morphogenesis[40]. Our previous studies also confirmed
that the YAP pathway can regulate the expression of β-catenin in a manner that is independent of Wnt3a
in BPD[41]. The SOX family of transcription factors has further emerged as a regulator of canonical Wnt/
β-catenin signaling. SOX proteins can regulate β-catenin activity through various ways, including protein–
protein interactions, the binding of SOX factors to the promoters of Wnt target genes, recruitment of co-
repressors or co-activators, regulation of protein stability, and nuclear translocation[42]. Discussion Our study also
revealed that not only can the overexpression of Sox9 in BPD reduce the overall expression level of β-
catenin but also that the nucleocytoplasmic translocation of Sox9 can change the nucleocytoplasmic
distribution of β-catenin by reducing its nuclear expression to inhibit the activation of the Wnt/β-catenin
signaling pathway. β-Catenin is a membrane-bound protein that acts as a central mediator of
transcriptional co-activators in canonical Wnt/β-catenin signaling[43]. β-Catenin is localized in the
cytoplasm during lung development and is usually localized in the nuclei of undifferentiated primitive
epithelial, differentiated alveolar epithelial, and adjacent stromal cells[44]. β-Catenin accumulates in the Page 8/30 Page 8/30 cytoplasm and translocates to the nucleus, thereby triggering Wnt target genes through T-cell factor and
lymphoid enhancer factor proteins[43], which is the key mechanism in activating the canonical Wnt
pathway. Our experiments demonstrate that the overexpression of Sox9 in BPD reduces the nuclear
expression of β-catenin. Sox9 regulates β-catenin expression in many ways. Several studies have
confirmed that Sox9 can directly bind to β-catenin, thereby interfering with the formation of the β-
catenin/T-cell factor complex. Sox9 can also directly bind to β-catenin through the C-terminus. This
results in the degradation of both proteins by the proteasome[45]. It has also been found that the N-
terminus of Sox9, including the HMG domain, promotes the degradation of β-catenin, while the C-terminal
transactivation domain inhibits the transcriptional activity of β-catenin without affecting its stability. Sox9 can also bind to components of the β-catenin destruction complex and relocate them to the
nucleus. The nuclear localization of Sox9 is necessary and sufficient to promote the phosphorylation of
β-catenin in the nucleus and its subsequent degradation[46]. A recent study confirmed that Sox9 can
affect the stability of β-catenin and promote its degradation through the transcriptional activation of
mastermind-like transcriptional activator 2[39]. Sox9 overexpression in BPD further reduces the nuclear
expression of β-catenin. This may be caused by the reduction of cytoplasmic β-catenin levels in the
nucleus or because the nuclear expression of Sox9 promotes the increased degradation of β-catenin in
the nucleus. The specific mechanisms underlying these actions remain to be further studied. However,
these processes do not seem to promote the increased phosphorylation or degradation of β-catenin in the
cytoplasm as the expression level of the β-catenin protein in the cytoplasm is increased. The role of noncoding RNAs in development and disease has become a topic of interest in recent years. Conclusion The increased nuclear entry of Sox9 in AEC-II cells in hyperoxia-exposed lung injury downregulates the
expression of β-catenin, enhances the ability of AEC-II cells to differentiate into AEC-I cells, and
antagonizes disordered alveolarization caused by the excessive activation of the Wnt pathway. This can
effectively improve the coordination and centrality of the network regulation of the cell damage repair
signaling pathway. Although GAS5 regulates Sox9 in this process, it does not act through the predicted
miR-1912-3p, and its specific mechanisms of action in this process require further study. Discussion Many lncRNAs, including MALAT1, CASC2, H19, and NEF, have been confirmed to be associated with
BPD. Our study confirmed that the expression of the lncRNA GAS5 is increased in BPD and can regulate
the expression of Sox9. LncRNAs are RNAs whose transcripts exceed 200 nucleotides and are not
translated into proteins. GAS5 plays multiple roles in lung diseases. For example, its expression is
reduced in non-small-cell lung cancer, and its upregulation can inhibit tumor proliferation, invasion, and
migration. GAS5, therefore, acts as a tumor suppressor by regulating various miRNAs, inhibiting
epithelial–mesenchymal transition, activating signaling pathways, and inhibiting cell cycle progression
and other pathways[47–51]. In acute lung injury, asthma, childhood pneumonia, and other diseases, GAS5
can affect the pyroptosis of bronchial epithelial cells, inhibit inflammatory responses and apoptosis, and
promote the proliferation of airway smooth muscle cells[52]. GAS5 present in exosomes from patients
with non-small-cell lung cancer may serve as an ideal serum-based non-invasive marker for identifying
early-stage non-small-cell lung cancer[53]. The expression of GAS5 is increased in BPD and can regulate
the expression of Sox9, thereby affecting AEC-II cell differentiation. We envisage that GAS5 may be used
as an evaluation index in children with BPD to assess the severity and prognosis of the disease by
collecting sputum, bronchoalveolar lavage fluid, or peripheral blood. The mechanisms by which lncRNAs regulate gene expression are complex. They can directly bind to DNA
or transcription factors to regulate gene expression at the transcriptional level. LncRNAs can also target
mRNAs, miRNAs, or proteins and regulate their activity and stability to exert post-transcriptional effects or Page 9/30 Page 9/30 interfere with epigenetic cytoplasmic complexes, thereby inhibiting or activating gene expression[31, 54, 55]. We screened miRNAs that may have binding sites with GAS5 and Sox9 using the TargetScan database
and other biological information networks. Eleven miRNAs, including miR-18a-5p, miR-200b-3p, miR-
450b-5p, and miR-1912-3p, were identified. Unfortunately, the results showed that miR-1912-3p could not
participate in the regulation of Sox9 by GAS5 through our predicted binding site; therefore, the
mechanisms underlying the regulation of Sox9 by GAS5 require further study. Animals Forty adult Sprague–Dawley (SD) rats (8–10 weeks of age, weighing 200–250 g; Changsheng
Biotechnology Co., Ltd., Liaoning, China) were raised in specific-pathogen-free animal houses and mated
in cages with a male-to-female ratio of 4:1. Pregnant female SD rats were fed independently for 21–23
days and delivered naturally. Construction of the in vivo BPD model A total of 200 newborn SD rats were randomly divided into a normal oxygen control group (21% O2) and
hyperoxygen model group (85% O2) within 12h after birth. Twenty young rats were randomly selected on
days 1, 3, 7, 14, and 21 of the study. Lung tissues were collected for the subsequent experiments. Cell culture and construction of the in vitro BPD model Cell transfection The siRNAs (si-GAS5 and si-Sox9), overexpression plasmids (oe-GAS5 and oe-Sox9), and corresponding
negative controls (si-nc and oe-nc) of GAS5 and Sox9 were obtained from SyngenTech (Beijing, China). The cells were seeded in 6- or 24-well plates for 12–24 h before the experiment. Transfection was
performed using the Polyplus jetPRIME transfection kit (Polyplus Transfectio, France) according to the
manufacturer's instructions. MiR-1912-3p mimics and inhibitors and the corresponding negative controls were purchased from
Guangzhou RiboBio Co., Ltd. The recombinant plasmids were transfected into AEC-II cells using the
RiboFECTTMCP transfection reagent (RiboBio Co. Ltd., Guangzhou, China) according to the
manufacturer’s protocol. Primary cell model in vitro Primary cell model in vitro Primary AEC-II cells were extracted from newborn SD rats within 48h. The extracted cells were inoculated
in Petri dishes, the control group was placed in ordinary incubators (21% O2, 5% CO2), and the model
group was incubated in a high-oxygen incubator (85% O2, 5% CO2). The cells were collected after 24, 48,
or 72 h for subsequent experiments. Cell culture and construction of the in vitro BPD model The rat AEC-II cell line RLE-6TN (ATCC, Manassas, VA, USA) was purchased and cultured in RPMI-1640
medium (Gibco, Grand Island, NY, USA) at 37°C with 5% CO2. The medium was supplemented with 10%
fetal bovine serum (Gibco) and 1% penicillin/streptomycin (HyClone, South Logan, UT, USA). The cells
were inoculated in a six-well plate and incubated for 24 h. The cells incubated in an ordinary incubator
(21% O2, 5% CO2) for 48 h were used as the control group, and those incubated in a high-oxygen
environment (85% O2, 5% CO2) for 48 h were used as the hyperoxia model group. Page 10/30
Isolation of primary AEC-II cells Isolation of primary AEC-II cells Page 10/30 Page 10/30 AEC-II cells were isolated, purified, and cultured in the neonatal rats. After cardiopulmonary lavage, the
excised lung tissues were minced on ice and predigested with 0.25% trypsin-EDTA (Gibco, Thermo Fisher
Scientific) for 10 min. The same volume of DMEM/F12 medium (Gibco, Thermo Fisher Scientific) with
10% fetal bovine serum (Gibco, Thermo Fisher Scientific) was then added to terminate the predigestion,
after which the mixture was discarded. Trypsin-EDTA (0.25%) was added to the lung tissue to digest for
20 min, and an equal volume of DMEM/F12 medium was added to terminate the digestion and collect
the digested liquid. Afterward, 0.1% collagenase type I (Gibco, Thermo Fisher Scientific) was added to the
lung tissue to digest the cells for 30 min, and an equal volume of DMEM/F12 medium was added to
terminate the digestion and collect the digested liquid. The alternate digestion process was repeated three
times. The harvested cell suspension was filtered through a 200-mesh cell strainer and centrifuged (4°C, 5
min, and 200 x g). The cell pellet was resuspended in DMEM/F12 medium with 10% fetal bovine serum
and 1% penicillin-streptomycin (HyClone) and incubated in a 5% CO2 incubator at 37°C. The fibroblasts
were removed and purified using the differential attachment method (repeated four times for 40, 40, 40,
and 120 min). After 12 h of culture, the cells were found to be round, oval, or polygonal in shape under a
light microscope and showed island-like adherent growth. The particles with clear contrast in the
cytoplasm were AEC-II cells, and the cell purity was > 90%, as detected by surfactant protein C (SPC)
immunofluorescence staining. RNA extraction and quantitative real-time PCR RNA extraction and quantitative real-time PCR Total RNA was extracted from the cells (107 cells) and tissues (100 mg) using TransZol reagent
(TransGen, Beijing, China) according to the manufacturer’s instructions. PerfectStart Green qPCR
SuperMix (TransGen, Beijing, China) was used for the real-time PCR analysis to test the expression levels
of GAS5, miR-1912-3p, Sox9, β-catenin, SPC, and aquaporin 5 (AQP5). The primer sequences were GAS5
forward 5′-GCA AGC TCC ACA CAA GGT CCT TC-3′ and reverse 5′-TGT TCA AGC ATC CAT CCA GTC ACC-
3′;Sox9 forward 5′-ACT CCG GCT CCT ACT ACA GC-3′ and reverse 5′-CTG CGC TGG GTT CAT GTA GG-3′;β-
catenin forward 5′-AGG ACA AGC CAC AGG ACT AC-3′ and reverse 5′-CAG CAG TCT CAT TCC AAG CC-
3′;SPC forward 5′-CCC AGG AGC CAG TTT CGC ATT C-3′ and reverse 5′-GAC GAC AAG GAC TAC CAC CAC
AAC-3′;AQP5 forward 5′-CCA TGA ACC CAG CCC GAT CTT TC-3′and reverse 5′-CCC TAC CCA GAA GAC
CCA GTG AG-3′;β-actin forward 5′-GGA GAT TAC TGC CCT GGC TCC TA-3′and reverse 5′-GAC TCA TCG
TAC TCC TGC TTG CTG-3′༛U6 forward 5′-CTC GCT TCG GCA GCA CA-3′ and reverse 5′-AAC GCT TCA CGA
ATT TGC GT-3′; miR-1912-3p primer was synthesized by Guangzhou RiboBio Co., Ltd, and the sequence
was not provided due to technical patents involved. The reaction was performed in a 20 µl mixed reaction
system according to the kit instructions. The amplification procedures were performed in an Applied
Biosystems 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA), and the PCR
procedure was as follows: 94 ℃ for 30 s for pre-degeneration, followed by 94 ℃ for 5 s and 60 ℃ for 30
s for 40 cycles. The results were analyzed following the dissociation protocol. The expression levels of
the target genes were calculated using the 2−ΔΔCt method. In vivo transfection For in vivo transfection, rats were suspended at 45 degrees by the upper teeth on a rodent dosing board
and the trachea was visualized using a fiber optic stylet connected to an endotracheal tube (Biolite small Page 11/30 animal intubation system, Kent Scientific Corp, USA). The trachea was intubated and Sox9-
overexpression plasmid(1µg/g)༈SyngenTech, Beijing, China༉ in 50 µl saline was delivered followed by
200 µl of air. The rats were injected once every other day from the fifth day of high oxygen until the 14th
day, and the lungs were taken from the rats and their lungs were harvested and subsequently frozen in a
-80° deep-frozen refrigerator for later analysis of tissue sections. Luciferase reporter assay Wild-type (WT) and mutant target fragments of GAS5 and Sox9 were constructed, and their
corresponding vectors (Wt-GAS5, Mut-GAS5, Wt-Sox9, and Mut-Sox9) were established. miR-1912-3p and
miR-NC were co-transfected into HEK293T cells. After transfection for 48 h, luciferase activity was
determined using a dual-luciferase reporter assay system (E1960, Promega, Madison, WI, USA,) according
to the manufacturer’s instructions. Western blot analysis The expression of SOX9, β-catenin, SPC, and AQP5 in the tissues and AEC-II cells was detected using a
western blot analysis. Protein samples were extracted from the cells using radioimmunoprecipitation
assay buffer with 1% phenylmethylsulfonyl fluoride. The protein concentration was determined using
bicinchoninic acid protein quantification. The proteins (20–30 µg/lane) were separated using 10%
sodium dodecyl-sulfate polyacrylamide gel electrophoresis and subsequently transferred onto
polyvinylidene fluoride membranes. The membranes were blocked with protein-free rapid sealing fluid for
15 min and then probed at 4°C overnight with the following antibodies: anti-SOX9 (1:500, Santa Cruz,
Santa Clara, CA, USA), anti-β-catenin (1:10 000, Proteintech, Shanghai,China), anti-SPC (1:500,
Proteintech), anti-AQP5 (1:500, Santa, USA), and anti-β-actin (1:20 000, Proteintech). The membranes
were then incubated with horse radish peroxidase-conjugated secondary antibody (1:5 000). An enhanced
chemiluminescence substrate (Millipore, Billerica, MA, USA) was used to visualize the signals. β-Actin
was used as a loading control. Page 12/30 Page 12/30 Pathological assessment Histopathological changes in the lung tissue were observed using hematoxylin and eosin stains. Under a
microscope (Nikon Corporation), a perpendicular line was drawn from the center of the most peripheral
bronchiole to the pleura or nearest interlobular septum. The number of alveoli transected by this line was
defined as the radial alveolar count (RAC), which is an important index that is used to evaluate the stages
of lung development. Histopathological changes in the lung tissue were observed using hematoxylin and eosin stains. Under a
microscope (Nikon Corporation), a perpendicular line was drawn from the center of the most peripheral
bronchiole to the pleura or nearest interlobular septum. The number of alveoli transected by this line was
defined as the radial alveolar count (RAC), which is an important index that is used to evaluate the stages
of lung development. Immunofluorescence staining The positive expression of SOX9, β-catenin, SPC, and AQP5 in the tissues and cells was observed using
immunofluorescence staining. The paraffin-embedded sections or primary cell slides were sealed with
10% goat serum at room temperature (22–24 °C) for 1 h and then incubated at 4°C overnight with the
following antibodies: anti-sox9 (1:100, Santa Cruz), anti-β-catenin (1:200, Proteintech), anti-SPC (1:100,
Proteintech), and anti-AQP5 (1:100, Proteintech). The sections were then incubated with Coralite-594 or
Coralite-488 (Proteintech) at room temperature for 2 h in the dark for fluorescent secondary antibody
labeling. DAPI was incubated at room temperature in the dark for 5 min for nuclear staining. The film was
sealed with Antifade Mounting Medium (Seven, Beijing, China) and observed under a fluorescence
microscope (E800, Nikon, Japan) at a field of vision of ×400. Detection of AEC-II cell differentiation After transfection, the primary cells were cultured in normoxic (21% O2, 5% CO2) and hyperoxic (85% O2,
5% CO2) incubators for 48 h. The collected cells were sealed with 5% bovine serum albumin solution at
room temperature for 1 h. The AEC-I cells were fluorescently labeled using the AQP5 antibody
(Biorbyt,Cambridge, England), and the AEC-II cells were fluorescently labeled with anti-rabbit IgG (H + L)
F(AB')2 Fragment (PE Conjµgate) (CST, KS, USA) and Furin AB (Novus Biologicals, CO, USA). The
fluorescently labeled cell suspensions were assessed using flow cytometry (BD, Franklin Lake, NJ, USA). Statistical analysis Data are presented as the mean ± standard deviation of three independent experiments and were
processed using GraphPad Prism 7 statistical software. The differences in the data were analyzed using
a Student’s t-test or two-way analysis of variance. P < 0.05 indicated statistically significant results. Page 13/30 Page 13/30 Availability of data and materials Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author
on reasonable request The datasets used and/or analyzed during the current study are available from the corresponding author
on reasonable request Authors' contributions WX and DW designed the study. DW wrote and revised the manuscript. DW performed the research. DB
and MY helped to raise the animals and analyze the data. BW provided the preliminary experimental data
of the research group. All authors read and approved the final manuscript. Ethics approval and consent to participate Ethics approval and consent to participate All animal experiments were examined and approved by the Experimental Animal Ethics Committee of
China Medical University. All animal experiments were examined and approved by the Experimental Animal Ethics Committee of
China Medical University. Patient consent for publication Not applicable. Funding The present study was supported by grants from the National Natural Science Foundation of China (no. 81771621), the Key Research and Development Guidance Plan of Liaoning Province (no. 2019JH8/10300023). The present study was supported by grants from the National Natural Science Foundation of China (no. 81771621), the Key Research and Development Guidance Plan of Liaoning Province (no. ) Declarations Acknowledgements The author is grateful to Shengjing Hospital Benxi Experimental Base and laboratory teachers for their
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Pathological changes in lung tissue and changes in SPC, AQP5, and Sox9 expression levels in BPD. 1A Lung histomorphological changes (×200) In the control group the lung septum became thinner Figure 1 Figure 1 Figure 1 Figure 1 1J:
qRT-PCR was used to detect the mRNA expression levels of SPC and Sox9 in the in vitro cell model. 1K:
The protein expression levels of SPC and Sox9 in the in vitrocell model were detected using western blots
(N: normoxic control group, H: hyperoxic model group). 1L: The intensity of the target band was
normalized to β-actin. (This blot is the cropped image ) (***P < 0.005, and ****P< 0.001 compared with
the control group). The expression of SPC and Sox9 increased in the in vitro cell model. cavity size increased significantly, number of alveoli decreased, and alveolar structure was simplified. N,
normoxic control group; H, hyperoxic model group. Scale bar: 50 μm. 1B. Comparison of RAC values in
lung tissues (N, normoxic control group; H, hyperoxic model group; ****P< 0.001) compared with the
control group. The RAC of the model group was significantly decreased from day 7 onwards. 1C: qRT-PCR
was used to detect the mRNA expression levels of SPC and AQP5 in the model and control groups at
different time points. 1D: Western blots were used to detect the protein expression levels of SPC and
AQP5 in the model and control groups at different time points. (N, normoxic control group; H, hyperoxic
model group). 1E: Target band intensity was normalized to β-actin. (**P < 0.01, ***P< 0.005, and ****P <
0.001 compared with the control group). Compared with the control group, the expression of SPC in the
model group was significantly increased and the expression of AQP5 was significantly decreased after 7
days of hyperoxia. 1F: mRNA and protein expression levels of Sox9 in the model and control groups at
different time points were detected using qRT-PCR and western blots. (N, normoxic control group; H,
hyperoxic model group). 1G: Target band intensity was normalized to β-actin. (**P < 0.01, ***P < 0.005,
and ****P < 0.001 compared with the control group). 1H: Paraffin-embedded tissue sections with Sox9
(red) + SPC (green) immunofluorescence double staining (×400). 1I: Paraffin-embedded tissue sections
were double stained with Sox9 (red) + AQP5 (green) immunofluorescence (×400). N, normoxic control
group; H, hyperoxic model group. Scale bar: 50 μm. Compared with the control group, the expression of
SPC in the model group increased significantly after 7 days, and the fluorescence signal was enhanced,
while the expression of AQP5 and Sox9 was decreased, and the fluorescence signal was weakened. Figure 1 Pathological changes in lung tissue and changes in SPC, AQP5, and Sox9 expression levels in BPD. Pathological changes in lung tissue and changes in SPC, AQP5, and Sox9 expression levels in BPD. 1A. Lung histomorphological changes (×200). In the control group, the lung septum became thinner,
alveolar crest appeared, alveolar cavity was uniform, and number of alveoli increased. In the model group,
as the days passed, the alveolar septum thickened, alveolar crest was atypical or disappeared, alveolar Page 19/30 Page 19/30 cavity size increased significantly, number of alveoli decreased, and alveolar structure was simplified. N,
normoxic control group; H, hyperoxic model group. Scale bar: 50 μm. 1B. Comparison of RAC values in
lung tissues (N, normoxic control group; H, hyperoxic model group; ****P< 0.001) compared with the
control group. The RAC of the model group was significantly decreased from day 7 onwards. 1C: qRT-PCR
was used to detect the mRNA expression levels of SPC and AQP5 in the model and control groups at
different time points. 1D: Western blots were used to detect the protein expression levels of SPC and
AQP5 in the model and control groups at different time points. (N, normoxic control group; H, hyperoxic
model group). 1E: Target band intensity was normalized to β-actin. (**P < 0.01, ***P< 0.005, and ****P <
0.001 compared with the control group). Compared with the control group, the expression of SPC in the
model group was significantly increased and the expression of AQP5 was significantly decreased after 7
days of hyperoxia. 1F: mRNA and protein expression levels of Sox9 in the model and control groups at
different time points were detected using qRT-PCR and western blots. (N, normoxic control group; H,
hyperoxic model group). 1G: Target band intensity was normalized to β-actin. (**P < 0.01, ***P < 0.005,
and ****P < 0.001 compared with the control group). 1H: Paraffin-embedded tissue sections with Sox9
(red) + SPC (green) immunofluorescence double staining (×400). 1I: Paraffin-embedded tissue sections
were double stained with Sox9 (red) + AQP5 (green) immunofluorescence (×400). N, normoxic control
group; H, hyperoxic model group. Scale bar: 50 μm. Compared with the control group, the expression of
SPC in the model group increased significantly after 7 days, and the fluorescence signal was enhanced,
while the expression of AQP5 and Sox9 was decreased, and the fluorescence signal was weakened. Figure 1 1J:
qRT-PCR was used to detect the mRNA expression levels of SPC and Sox9 in the in vitro cell model. 1K:
The protein expression levels of SPC and Sox9 in the in vitrocell model were detected using western blots. (N: normoxic control group, H: hyperoxic model group). 1L: The intensity of the target band was
normalized to β-actin. (This blot is the cropped image ) (***P < 0.005, and ****P< 0.001 compared with
the control group). The expression of SPC and Sox9 increased in the in vitro cell model. Page 20/30 Figure 2 Figure 2 Figure 2
Changes in the expression of Sox9 during primary AEC-II culture and differentiation (×400). 2A: Sox9 and SPC immunofluorescence double staining. 2B: Sox9 and AQP5 immunofluorescence
double staining. N is the control group; H is the model group. During the culture of the primary AEC-I
the expression of SPC gradually decreased and the fluorescence signal gradually weakened with cu Figure 2 Changes in the expression of Sox9 during primary AEC-II culture and differentiation (×400). 2A: Sox9 and SPC immunofluorescence double staining. 2B: Sox9 and AQP5 immunofluorescence
double staining. N is the control group; H is the model group. During the culture of the primary AEC-II cells,
the expression of SPC gradually decreased and the fluorescence signal gradually weakened with culture Page 21/30 Page 21/30 time, while the expression of AQP5 gradually increased and the fluorescence signal gradually enhanced. In the control group, Sox9 showed nuclear expression in the primary AEC-II cells. Its expression increased
in the hyperoxia model and showed plasma expression. 2C: Changes in Sox9 protein expression in an in
vitro cell model. 2D: Total protein and plasma protein target bands were normalized to β-actin, and
nucleoprotein target band intensity was normalized to PCNA. (N, control group; H, hyperoxia model
group). Total protein expression of Sox9 in the hyperoxia cell model was increased, its nuclear protein
expression was decreased, and its plasma protein expression was increased.(This blot is the cropped
image )(****P < 0.001 compared with the control group). Page 22/30 Figure 3
Effect of exogenous Sox9 on the pathological changes in lung tissue Figure 3
Effect of exogenous Sox9 on the pathological changes in lung tissue. Figure 3 Effect of exogenous Sox9 on the pathological changes in lung tissue. 3A: Lung tissue section staining showed that, compared with the BPD model group, the BPD Sox9-
overexpression group showed improved alveolar development, increased number of alveoli, more
complete structure, and more uniform size. N: normoxic control group, H: hyperoxic model group, H OE- Page 23/30 Page 23/30 Sox9: hyperoxic Sox9-overexpression group. Scale bar: 50 μm. 3B: Comparison of RAC values in lung
tissues (N, normoxic control group; H, hyperoxic model group; H OE-Sox9: hyperoxic Sox9-overexpression
group (****P < 0.001). RAC increased in the overexpression group compared with that in the model group. 3C: Paraffin-embedded tissue sections with Sox9 (red) + SPC (green) immunofluorescence double
staining. 3D: Paraffin-embedded tissue sections were double stained with Sox9 (red) + AQP5 (green)
immunofluorescence. Compared with the model group, in the overexpression group, the number of SPC-
labeled cells was decreased, while the fluorescent expression of AQP5 was enhanced. Sox9: hyperoxic Sox9-overexpression group. Scale bar: 50 μm. 3B: Comparison of RAC values in lung
tissues (N, normoxic control group; H, hyperoxic model group; H OE-Sox9: hyperoxic Sox9-overexpression
group (****P < 0.001). Figure 2 RAC increased in the overexpression group compared with that in the model group. 3C: Paraffin-embedded tissue sections with Sox9 (red) + SPC (green) immunofluorescence double
staining. 3D: Paraffin-embedded tissue sections were double stained with Sox9 (red) + AQP5 (green)
immunofluorescence. Compared with the model group, in the overexpression group, the number of SPC-
labeled cells was decreased, while the fluorescent expression of AQP5 was enhanced. Sox9: hyperoxic Sox9-overexpression group. Scale bar: 50 μm. 3B: Comparison of RAC values in lung
tissues (N, normoxic control group; H, hyperoxic model group; H OE-Sox9: hyperoxic Sox9-overexpression
group (****P < 0.001). RAC increased in the overexpression group compared with that in the model group. 3C: Paraffin-embedded tissue sections with Sox9 (red) + SPC (green) immunofluorescence double
staining. 3D: Paraffin-embedded tissue sections were double stained with Sox9 (red) + AQP5 (green)
immunofluorescence. Compared with the model group, in the overexpression group, the number of SPC-
labeled cells was decreased, while the fluorescent expression of AQP5 was enhanced. Page 24/30 Page 24/30 Figure 4 Effect of Sox9 on the proliferation and differentiation of AEC-II cells. Effect of Sox9 on the proliferation and differentiation of AEC-II cells. Effect of Sox9 on the proliferation and differentiation of AEC-II cells. 4A: SPC mRNA expression level changes after Sox9 intervention. 4B: SPC protein expression level
changes after Sox9 intervention. 4C: Target band intensity is normalized to β-actin. (Intervention group,
NC, negative control). SPC expression decreased after Sox9 knockdown and increased after Sox9
overexpression. (This blot is the cropped image ) (*P < 0.05 and ****P < 0.001 compared with the NC). 4D:
Flow cytometry analysis of primary cells (N, control group; H, model group; C, control group; si-Sox9,
siRNA knockdown Sox9; oe-Sox9, overexpression of Sox9) showing increased number of AQP5-labeled
cells in the model group. 4E: Sox9 (red) + SPC (green) immunofluorescence double staining in the control
group. 4F: Sox9 (red) + SPC (green) immunofluorescence double staining in the model group. 4G: Sox9
(red) + AQP5 (green) immunofluorescence double staining in the control group. 4H: Sox9 (red) + AQP5
(green) immunofluorescence double staining in the model group. Compared with the control group, in the
model group, the overexpression of Sox9 decreased the expression of SPC and increased the expression
of AQP5. Page 25/30 Figure 5 Changes in the expression of β-catenin. 5A: Changes in β-catenin mRNA expression levels in the model and control groups at different time
points, as identified by qRT-PCR. 5B: Western blots were used to detect the expression level of the β-
catenin protein in the model and control groups at different time points. 5C: Target band intensity was Page 26/30 Page 26/30 normalized to β-actin. 5D: qRT-PCR was used to detect the changes in β-catenin mRNA expression after
Sox9 intervention. 5E: Protein expression level of β-catenin after Sox9 intervention was detected by
western blots. 5F: Target band intensity was normalized to β-actin. (N, normoxia control group; H,
hyperoxia model group). (This blot is the cropped image ) (*P < 0.05, *** P < 0.005, and **** P < 0.001
compared with the control group). normalized to β-actin. 5D: qRT-PCR was used to detect the changes in β-catenin mRNA expression after
Sox9 intervention. 5E: Protein expression level of β-catenin after Sox9 intervention was detected by
western blots. 5F: Target band intensity was normalized to β-actin. (N, normoxia control group; H,
hyperoxia model group). (This blot is the cropped image ) (*P < 0.05, *** P < 0.005, and **** P < 0.001
compared with the control group). normalized to β-actin. 5D: qRT-PCR was used to detect the changes in β-catenin mRNA expression after
Sox9 intervention. 5E: Protein expression level of β-catenin after Sox9 intervention was detected by
western blots. 5F: Target band intensity was normalized to β-actin. (N, normoxia control group; H,
hyperoxia model group). (This blot is the cropped image ) (*P < 0.05, *** P < 0.005, and **** P < 0.001
compared with the control group). Page 27/30
Figure 6 Figure 6 Page 27/30 Page 27/30 Changes in the distribution of β-catenin in the nucleus and plasma. 6A: The expression level of the β-catenin protein was detected by western blots. 6B: The total protein and
plasma protein target band intensity was standardized using β-actin, and the nuclear protein target band
intensity was standardized using PCNA. (N, normoxia group; H, hyperoxia group). Based on the decreased
total protein expression, the nuclear expression of the β-catenin protein increased, and its plasma
expression decreased in the model group(This blot is the cropped image ). 6C: Changes in the Sox9
protein nucleoplasmic expression level after the overexpression of Sox9 in the hyperoxic cell model. Figure 5 6D: 6A: The expression level of the β-catenin protein was detected by western blots. 6B: The total protein and
plasma protein target band intensity was standardized using β-actin, and the nuclear protein target band
intensity was standardized using PCNA. (N, normoxia group; H, hyperoxia group). Based on the decreased
total protein expression, the nuclear expression of the β-catenin protein increased, and its plasma
expression decreased in the model group(This blot is the cropped image ). 6C: Changes in the Sox9
protein nucleoplasmic expression level after the overexpression of Sox9 in the hyperoxic cell model. 6D:
Changes in the β-catenin protein nucleoplasmic expression level after the overexpression of Sox9 in the
hyperoxia cell model. 6E: Total protein and plasma protein target band intensity was normalized to β-
actin, and the nuclear protein target band intensity was normalized to PCNA. (oe, overexpression; nc,
negative control). After the overexpression of Sox9, the total protein and nuclear plasma expression of
Sox9 increased, total protein of β-catenin decreased, nuclear expression decreased, and plasma
expression increased. (This blot is the cropped image ) (*P < 0.05 and ****P < 0.001 compared with the
control group). 6F: β-Catenin (red) + SPC (green) immunofluorescence double staining in the primary AEC-
II cells. (N, normoxia group; H, hyperoxia group; C, blank control; si, small interfering RNA knockdown; oe,
plasmid overexpression; nc, negative control). The knockdown of Sox9 increased the expression of β-
catenin, and the overexpression of Sox9 decreased the expression of β-catenin. Page 28/30 Page 29/30
Figure 7
Changes in the expression of GAS5 and miR-1912-3p. 7A: Expression level of GAS5 and miR-1912-3p in lung tissue. With the prolongation of hyperoxia
exposure, the expression of the lncRNA GAS5 in the model group gradually decreased, but its overall level
was still higher than that in the control group. The expression of miR-1912-3p in the model group Figure 7 Changes in the expression of GAS5 and miR-1912-3p. Changes in the expression of GAS5 and miR-1912-3p. Changes in the expression of GAS5 and miR-1912-3p. 7A: Expression level of GAS5 and miR-1912-3p in lung tissue. With the prolongation of hyperoxia
exposure, the expression of the lncRNA GAS5 in the model group gradually decreased, but its overall level
was still higher than that in the control group. The expression of miR-1912-3p in the model group Page 29/30 Page 29/30 increased gradually, which was lower than that in the control group on days 1–3 and higher than that in
the control group on day 7. 7B: Expression level of GAS5 and miR-1912-3p in the in vitro cell model. The
expression of GAS5 was increased and miR-1912-3p was decreased in the model group.7C: qRT-PCR was
used to detect the change in the Sox9 mRNA expression level after the GAS5 intervention. 7D: Expression
level of Sox9 protein after GAS5 intervention was detected using a western blot. 7E: Target band intensity
was normalized to β-actin. N, normoxic group; H, hyperoxic group(This blot is the cropped image ) (***P <
0.005 and **** P < 0.001 compared with the control group). 7F: Sox9 (red) + SPC (green)
immunofluorescence double staining in the control group. 7G: Sox9 (red) + SPC (green)
immunofluorescence double staining in the model group. 7H: Sox9 (red) + AQP5 (green)
immunofluorescence double staining in the control group. 7I: Sox9 (red) + AQP5 (green)
immunofluorescence double staining in the model group. In each group, the knockdown of GAS5
weakened the fluorescence of Sox9, and the overexpression of GAS5 enhanced the fluorescence of Sox9. 7J: Double luciferase report of GAS5 and miR-1912-3p. After co-transformation with rno-miR-1912-3p, the
reporter fluorescence expression of r-Gas5-MUT did not increase significantly compared with that of r-
Gas5-WT. 7K: Double luciferase report of Sox9 and miR-1912-3p. After co-transmutation with rno-miR-
1912-3p, the reporter fluorescence expression of r-Sox9-MUT increased compared with that of r-Sox9-WT. increased gradually, which was lower than that in the control group on days 1–3 and higher than that in
the control group on day 7. 7B: Expression level of GAS5 and miR-1912-3p in the in vitro cell model. The
expression of GAS5 was increased and miR-1912-3p was decreased in the model group.7C: qRT-PCR was
used to detect the change in the Sox9 mRNA expression level after the GAS5 intervention. Figure 7 7D: Expression
level of Sox9 protein after GAS5 intervention was detected using a western blot. 7E: Target band intensity
was normalized to β-actin. N, normoxic group; H, hyperoxic group(This blot is the cropped image ) (***P <
0.005 and **** P < 0.001 compared with the control group). 7F: Sox9 (red) + SPC (green)
immunofluorescence double staining in the control group. 7G: Sox9 (red) + SPC (green)
immunofluorescence double staining in the model group. 7H: Sox9 (red) + AQP5 (green)
immunofluorescence double staining in the control group. 7I: Sox9 (red) + AQP5 (green)
immunofluorescence double staining in the model group. In each group, the knockdown of GAS5
weakened the fluorescence of Sox9, and the overexpression of GAS5 enhanced the fluorescence of Sox9
7J: Double luciferase report of GAS5 and miR-1912-3p. After co-transformation with rno-miR-1912-3p, the
reporter fluorescence expression of r-Gas5-MUT did not increase significantly compared with that of r-
Gas5-WT. 7K: Double luciferase report of Sox9 and miR-1912-3p. After co-transmutation with rno-miR-
1912-3p, the reporter fluorescence expression of r-Sox9-MUT increased compared with that of r-Sox9-WT. double staining in the model group. In each group, the knockdown of GAS5 weakened the fluorescence of Sox9, and the overexpression of GAS5 enhanced the fluorescence of Sox9. 7J: Double luciferase report of GAS5 and miR-1912-3p. After co-transformation with rno-miR-1912-3p, the
reporter fluorescence expression of r-Gas5-MUT did not increase significantly compared with that of r-
Gas5-WT. 7K: Double luciferase report of Sox9 and miR-1912-3p. After co-transmutation with rno-miR-
1912-3p, the reporter fluorescence expression of r-Sox9-MUT increased compared with that of r-Sox9-WT. Page 30/30 Page 30/30
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https://openalex.org/W4240417333
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http://ejournal.unp.ac.id/students/index.php/pek/article/download/7328/3408
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Indonesian
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Pengaruh Persepsi Mahasiswa Tentang Profesi Guru dan Lingkungan Keluarga Terhadap Miinat Menjadi Guru
|
Jurnal Ecogen
| 2,019
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cc-by
| 5,583
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PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN
KELUARGA TERHADAP MIINAT MENJADI GURU Shinta Haryawan, Bustari Muchtar, Rita Syofyan
Jurusan Pendidikan Ekonomi Fakultas Ekonomi
Universitas Negeri Padang
Jl. Prof. Dr. Hamka Kampus UNP Air Tawar Padang
Email : shintaharyawanfe11@gmail.com Abstract: The purpose of this research was to determine 1) The influence students perception about teacher
profession of interest to become a teacher. 2) The influence family environment of interest become a teacher. 3) The
influnce of students perception about teacher profession and family environment of interest to become a teacher. This type of research is descriptive and associative research. The population of this study is all students from
economic education padang state university in 2014, amount of the population is 88 people. The sampling
technique of data collection is done by Total Sampling with amount of sample is 82 people because the 7 people
have finished their studies. The data used are primary and secondary data. This research had two categories of
variable The first was the independent variable, the paerceptions of students about the teaching prefession . The
first was the independent variable, the perceptions of students about the teaching profession and family
environment. The second dependent variable, namely interests become a teacher. The analytical method used is
multiple regression analysis. The result show that. 1) Student perception about teacher profession significant effect
to interest become a teacher. 2) Family environment are significant effect to interest become a teacher. 3)
Perception of students about the profession teacher and family environment jointly had a significant effect to
interest become a teacher. Keyword: Perception, The Profession Of Teacher, Family Environment, Interset 218 218 EcoGen
Volume 2, Nomor 3, 5 September 2019 Volume 2, Nomor 3, 5 September 2019 Menurut Slameto (2010:180) minat adalah suatu rasa lebih suka dan rasa ketertarikan pada suatu hal atau
aktivitas, tanpa ada yang menyuruh. Minat pada dasrnya merupakan penerimaan akan suatu hubungan antara diri
sendiri dengan sesuatu yang ada di luar diri. Semakin kuat dan dekat hubungan tersebut, maka akan semakin besar
minat yang akan ditimbulkan. Begitu juga dengan minat menjadi guru apabila seseorang mengatakan ia berminat
menjadi guru maka ia akan bersungguh-sungguh mencari tau informasi tentang profesi guru, adanya perhatian yang
lebih terhadap profesi guru dan adanya keinginan untuk mempelajari lebih dalam lagi sehingga keinginan untuk
menjadi guru pada mahasiswa tercapai. Minat menjadi guru adalah ketertarikan seseorang terhadap profesi guru
yang ditunjukkan dengan adanya perasaan suka, senang, tertarik dan ingin menjadi guru. Berdasarkan hasil observasi awal yang penulis lakukan terhadap 30 responden yang merupakan mahasiswa
Jurusan Pendidikan Ekonomi Fakultas Ekonomi Universitas Negeri padang yang terdaftar pada tahun 2014 yang
dilakukan pada tanggal 17 April 2017. Pernyataannya satu soal berbentuk pertanyaan yaitu apakah anda berminat
menjadi guru? Berikan alasannya!. Berikut hasil observasinya: Tabel 1. Hasil Observasi Awal Variabel Minat Menjadi Guru Tabel 1. Hasil Observasi Awal Variabel Minat Menjadi Guru
Konsentrasi
Jumlah
Keterangan
Tidak
Berminat
%
Kurang
Berminat
%
Sangat
Berminat
%
ADP
8
0
0
4
13
4
13
Akun
7
1
3
2
7
4
13
Ekop
8
1
3
4
13
3
10
TTN
7
1
3
4
13
2
7
Total
30
3
10
14
47
13
43
Sumber : Data Primer Sumber : Data Primer Berdasarkan tabel 1 diatas, diperoleh gambaran bahwa rata-rata dari keseluruhan minat meenjadi guru pada
mahasiswa tergolong masih rendah. Dapat dilihat dari persentase mahasiswa yang memilih tidak berminat menjadi
guru, yaitu sebesar 10%, sedangkan persentase mahasiswa yang memilih kurang berminat menjadi guru, yaitu
sebesar 47%, dan persentase mahasiswa yang memilih sangat berminat menjadi guru, yaitu sebesar 43%. Alasan
yang dikemukakan oleh responden yang tidak berminat menjadi guru yaitu pertama, mahasiswa merasa kemampuan
keguruan pada diri mahasiswa masih kurang. Kedua, dilihat dari segi fnansial guru jauh dari yang diharapkan. Ketiga, jurusan pendidikan ekonomi merupakan pilihan terakhir bagi mahasiswa untuk memasuki perkuliahan di
UNP. Keempat, mahasiswa memang tidak berminat menjadi guru melainkan diluar bidang kegguruan. Sedangkan
mahasiswa yang kurang berminat menjadi guru dikarenakan pertama, mahasiswa menganggap profesi guru masih
kalah gengsi dengan profesi lainnya seperti profesi dosen, pegawai swasta dan wirausaha dilihat dari segi gaji,
tempat kerja dan lain-lain. PENDAHULUAN Pendidikan adalah investasi jangka panjang dan menjadi kunci untuk masa depan yang lebih baik dalam
kehidupan berbagsa dan bernegara. Tanpa adanya pendidikan yang memadai dan berkualitas, maka bangsa
indonesia akan semakin tertinggal. Jalur pendidkan dapat ditempuh di berbagai lembaga pendidikan formal dan
informal. Perguruan tinggi merupakan salah satu lembaga pendidikan formal yang diserahi tugas dan tanggungjawab
untuk mempersiapkan mahasiswa sesuai dengan tujuan sinstem pendidikan nasional. Universitas Negeri Padang
merupakan salah satu lembaga Perguruan Tinggi Negeri di Sumatera Barat yang menghasilkan para lulusan tenaga
pengajar dan pendidik yang berkualitas serta menghasilkan tenaga ahli dibidangnya masing-masing. Jurusan pendidikan ekonomi merupakan satu-satunya jurusan kependidikan di fakultas ekonomi universitas
negeri padang yang akan menghasilkan calon-calon guru profesional nantinya. Sebagai sarjana pendidikan,
mahasiswa pendidikan ekonomi juga dibekali dengan berbagai keahlian. Keahlian tersebut akan membantu mereka
untuk dapat bekerja dibidang pendidikan dan non-kependidikan. Pada jurusan pendidikan ekonomi terdapar empat
konsentrasi keahlian yaitu akuntansi, administrasi perkantoran, tata niaga dan ekonomi koperasi. Masing-masing
dari konsentrasi memiliki kelebihan tersendiri sehingga akan menghasilkan calon guru ekonomi yang berkualitas. Menurut Undang-undang No.14 Tahun 2005 tentang Guru dan Dosen Pasal 1 (2005:2) guru adalah pendidik
profesional dengan tugas utama mendidik, mengajar membimbing, mengarahkan, melatih, menilai, dan
mengevaluasi peserta didik pada pendidikan anak usia dini jalur pendidikan formal, pendidikan dasar, dan
pendidikan menengah. Menurut Sardiman (2012:125) guru adalah salah satu komponen mnusiawi dalam proses
pembelajaran, yang ikut berperan dalam usaha membantu sumber daya manusia yang potensial di bidang
pembangunan. Artinya guru merupakan salah satu komponen mikro dari sistem pendidikan yang sangat strategis
dan banyak mengambil peran penting dalam proses pendidikan secara luas khususnya dalam pendidikan di sekolah
dan subjek yang sangat sentral bagi terselenggaranya mutu pendidikan yang berkualitas. PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU 219 PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU Volume 2, Nomor 3, 5 September 2019 Volume 2, Nomor 3, 5 September 2019 dewasa nanti dalam memilih profesi pekerjaan. Karena keluarga merupakan pusat pendidikan yang pertama dan
utama sekali oleh karena itu disinilah kepribadian dan pola pikir anak terbentuk. Jadi keluarga mempunyai
pengaruh yang besar terhadap perkembangan kepribadian anak dan minatnya untuk menjadi seorang guru. dewasa nanti dalam memilih profesi pekerjaan. Karena keluarga merupakan pusat pendidikan yang pertama dan
utama sekali oleh karena itu disinilah kepribadian dan pola pikir anak terbentuk. Jadi keluarga mempunyai
pengaruh yang besar terhadap perkembangan kepribadian anak dan minatnya untuk menjadi seorang guru. l Observasi Kedua Variabel Persepsi Tentang Profesi Guru dan Lingkungan Keluarga. Tabel 2. Hasil Observasi Kedua Variabel Persepsi Tentang Profesi Guru dan Lingkungan Keluarga. No. Pernyataan
Ya
Tidak
1. Apakah profesi guru merupakan profesi yang menyenagkan
30
-
2. Apakah profesi guru merupakan keinginan dari dalam diri sendiri
15
15
3. Apakah profesi guru merupakan keinginan dari kedua orang tua
13
17
4. Apakah ada di lingkungan keluarga anda yang berprofesi sebagai guru
17
13
5. Apabila orang tua anda berprofesi sebagai guru apakah anda ingin
mengikuti profesi dari kedua orang tua
9
21
6. Apabila orang tua anda berprofesi sebagai pengusaha apakah anda
ingin mengikuti profesi dari kedua orang tua
20
10
Sumber : Data Olahan 2017 Berdasarkan hasil observasi kedua yang dilakukan pada tanggal 13 November 2017 digambarkan pada tabel 2,
maka dapat dilihat bahwa persepsi mahasiswa menyukai profesi guru karena profesi yang menyenangkan. Guru
merupakan profesi yang mulia dan mencerdaskan anak-anak bangsa. hal ini sangatlah bagus untuk mahasiswa yang
memiliki keinginan yang tinggi dan kesadaran akan panggilan jiwa dalam dirinya untuk berkeinginan menjadi guru
karena keinginan tersebut datang dalam dirinya sendiri bukan dari orang lain. Namun ada juga mahasiswa yang
tidak berkeinginan menjadi guru karena mahasiswa lebih tertarik untuk melanjutkan pendidikan S2 menjadi dosen,
ingin bekerja dikantor dan ada juga yang tertarik menjadi pengusaha muda yang sesuai dengan kemampuan yang
mereka miliki. Adapun pengaruh dari lingkungan keluarga dapat dilihat dari adanya lingkungan keluarga yang berprofesi
sebagai guru baik itu orang tua, paman, bibi, dan kakak. Hal ini menandakan banyak atau sedikitnya informasi yang
diperoleh oleh mahasiswa mengenai profesi guru baik positif maupun negatif akan sangat berpengaruh bagi
mahasiswa dalam memilih karir nantinya sebagai guru karena seorang anak akan memilih profesi yang hampir sama
dengan orang tuanya ataupun lebih karena mereka dapat melihat dan merasakan banyaknya keuntungan yang
diperoleh dari profesi tersebut. Volume 2, Nomor 3, 5 September 2019 Kedua, pengangkatan guru PNS semakin lama semakin sulit. Ketiga, kepribadian dan
kewibawaan keguruan mahasiswa masih kurang. Sedangkan mahasiswa yang memilih sangat berminat menjadi guru
yaitu karena profesi guru yang mulia dapat mencerdaskan anak bangsa dan penerus bangsa indonesia. Selain itu
mahasiswa terpanggil jiwanya untuk menjadi guru, karena ingin berbagi ilmu pengetahuan yang didapat mahasiswa
selama mengikuti perkuliahanserta ingin menambah wawasan keguruan dan ingin mewujudkan cita-citanya menjadi
guru. Berdasarkan hasil wawancara dan observasi yang telah penulis lakukan kepada mahasiswa terdapat beberapa
faktor yang mempengaruhi minat menjadi guru pada mahasiswa yaitu faktor internal dan eksternal. Faktor internal
adalah faktor dari dalam diri mahasiswa yang dapat mempengaruhi minat mahasiswa untuk menjadi guru secara
langsung seperti persepsi mahasiswa mengenai profesi guru itu sendiri dilihat dari pengalaman sewaktu sekolah,
diperkuliahan, pengalaman di waktu PPLK disekolah, dan masyarakat sekitar. Positif atau negatifnya persepsi yang
dihasilkan terhadap profesi guru akan sangat berpengaruh terhadap minat mahasiswa untuk menjadi seorang guru
nantinya. Sedangkan faktor eksterenal yaitu faktor luar diri mahasiswa yang dapat mempengaruhi dan
menumbuhkan minat mahasiswa untuk menjadi guru, seperti lingkungan keluarga dan lingkungan masyarakat. Lingkungan keluarga juga berperan penting dalam memberikan pengarahan dan pengalaman kepada anaknya untuk
menentukan minatnya menjadi seorang guru. Khususnya bagi orang tua mahasiswa yang berprofesi sebagai guru
dapat memberikan informasi penting dan positif mengenai profesi guru dan menjadi panutan bagi anaknya setelah PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU 220 220 PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU Volume 2, Nomor 3, 5 September 2019 Volume 2, Nomor 3, 5 September 2019 kemauan, keinginan dan rasa senang. Faktor internal ini dapat memberikan pengaruh yang lebih besar dan langsung
dibandingkan faktor eksternal terhadap minat mahasiswa menjadi guru. Berdasarkan kenyataan yang ada mahasiswa tidak berminat menjadi guru dikarenakan profesi guru merupakan
profesi yang sulit seperti pengangkatan untuk menjadi guru PNS semakin lama semakin sulit, untuk menjadi guru
lulusan S1 kependidikan harus mengikuti pendidikan profesi guru (PPG), tidak sesuai dengan kepribadian
mahasiswa, dilihat dari segi pendapata guru tidak sesuai dengan yang diharapkan dan mahasiswa merasa
kemampuan keguruan dalam diri mahasiswa belum maksimal, selain itu masih banyak profesi lain yang lebih
menjanjikan menurut mahasiswa yang sesuai dengan keahlian yang mereka miliki. Berdasarkan kenyataan yang ada mahasiswa tidak berminat menjadi guru dikarenakan profesi guru merupakan
profesi yang pengangkatan untuk menjadi guru PNS semakin lama semakin sulit, untuk menjadi guru lulusan S1
kependidikan harus mengikuti pendidikan profesi guru (PPG), tidak sesuai dengan kepribadian mahasiswa, dilihat
dari segi pendapata guru tidak sesuai dengan yang diharapkan dan mahasiswa merasa kemampuan keguruan dalam
diri mahasiswa belum maksimal, selain itu masih banyak profesi lain yang lebih menjanjikan menurut mahasiswa
yang sesuai dengan keahlian yang mereka miliki. Dalam menjalankan profesi sebagai guru, sebaiknya didasarkan pada hasrat dan keinginan yang kuat terhadap
profesi tersebut. Dengan adanya hasrat yang kuat dimiliki dalam diri mahasiswa maka akan terbentuknya minat
mahasiswa untuk menjadi guru dengan sendirinya. Minat yang tinggi akan menimbulkan perasaan senang, suka,
tertarik, dan ingin menjadi guru yang profesional. Sehingga membuat seseorang menampakkan keinginan yang
tinggi untuk berprestasi dalam bidang keguruan tanpa mengenal rasa bosan dan lelah dalam menjalaninya. Adanya
persepsi mahasiswa yang positif maupun negatif tentang profesi guru turut mempengaruhi minat mahasiswa menjadi
guru. Jika mahasiswa memiliki persepsi yang positif terhadap profesi guru, maka akan meningkatkan minatnya
menjadi guru. Sebaliknya jika mahasiswa memiliki persepsi negatif terhadap profesi guru maka minat menjadi guru
juga akan menurun. Oleh karena itu diperlukan minat yang kuat untuk menjadi guru agar memberikan dorongan
positif untuk mencapai cita-cita yang diinginkan. Berdasarkan uraian dan fenomena-fenomena dari permasalahan yang ada diatas, maka penulis tertarik untuk
melakukan penelitian yang berjudul “Pengaruh Persepsi Mahasiswa Tentang Profesi Guru dan Lingkungan Keluarga
Terhadap Minat Menjadi Guru pada Mahasiswa Jurusan Pendidikan Ekonomi FE UNP”. Dimana penelitian ini
bertujuan untuk mengunggkapkan : (1). Pengaruh persepsi mahasiswa tentang profesi guru terhadap minat menjadi
guru. (2) Pengaruh lingkungan keluarga terhadap minat menjadi guru. Volume 2, Nomor 3, 5 September 2019 Selain itu seorang anak di didik, dibesarkan, dan diajarka bertanggung jawab,
bersikap dan berperilaku tidak jauh dari latar belakang orang tua dan keluarganya. Berdasarkan penelitian yang telah dilakukan oleh Alim, Arif Firman 2014 hasil penelitian menunjukkan bahwa
faktor-faktor yang mempengaruhi minat profesi guru mahasiswa terdiri atas lima yaitu emosional yang memiliki
persentase sebesar 17,23%, sedangkan persepsi sebesar 19,42%, motivasi sebesar 16,54%, bakat sebesar 25,41%,
dan penguasaan ilmu pengetahuan sebesar 21,40%. Penelitian yang telah dilakukan oleh Ardyani, Anisa 2014 hasil penelitian menunjukan bahwa terdapat tujuh
kelompok faktor baru yang dapat mempengaruhi minat mahasiswa menjadi guru akuntansi nantinya yaitu a)
persepsi mahasiswa tentang profesi guru (24,66%), b) kesejahteraan guru (18,69%), c) prestasi belajar (15,26%), d)
pengalaman PPL (13,85%), e) teman bergaul (10,54%), f) lingkungan keluarga (4,32%), dan g) kepribadian
(2,62%). Berdasarkan persentase diatas persepsi mahasiswa tentang profesi guru merupakan persentase terbesar dan
tertinggi yang sangat berpengaruh terhadap minat mahasiswa untuk menjadi guru sebesar (24,66%) dan lingkungan
keluarga juga berpengaruh terhadap minat mahasiswa untuk menjadi guru sebesar (4,32%). Hal ini dikarenakan
positif atau negatifnya penilaian dan anggapan seseorang baik itu mahasiswa ataupun masyarakat terhadap profesi
guru turut mempengaruhi minat mahasiswa untuk menjadi guru. Penelitian terdahulu terkait minat menjadi guru yang dilakukan oleh Susanti, Yuli (2007) Hasil dari penelitian
tersebut menunjukan sebagian besar mahasiswa Pendidikan Ekonomi FE UNP dipengaruhi oleh faktor internal yaitu 221 Volume 2, Nomor 3, 5 September 2019 (3) Pengaruh persepsi mahasiswa tentang
profesi guru dan lingkungan keluarga terhadap minat menjadi guru PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU METODE PENELITIAN Penelitian ini tergolong pada penelitian deskriptif dan asosiatif. Penelitian dilakukan di Fakultas Ekonomi
Universitas Negeri Padang pada bulan April 2018. Populasi dalam penelitian ini adalah seluruh mahasiswa jurusan
pendidikan ekonomi yang terdaftar tahun 2014 sebanyak 89 orang. Pada saat melakukan penelitian di bulan April
2018 jumlah mahasiswa berkurang sebanyak 6 orang dikarenakan sudah menyelesaikan studi dan 1 orang pindah
jadi jumlah keseluruhan dalam penelitian ini berjumlah sebanyak 82 orang. Teknik penarikan sampel pada penelitian ini dilakukan secara Total Sampling, yaitu teknik pengambilan
sampel yang digunakan adalah total populasi. Dikarenakan jumlah mahasiswa terbatas maka penelitian ini dilakukan
secara keseluruhan dari populasi mahasiswa. Variabel dalam penelitian ini terdiri dari 2 variabel bebas, yaitu persepsi mahasiswa tentang profesi guru (X1)
dan lingkungan keluarga (X2) dan 1 variabel terikat, yaitu minat menjadi guru (Y). Instrumen yang digunakan
dalam penelitian ini adalah angket dengan pengukurannya menggunakan skala likert yang terdiri dari 5 (lima)
kategori pernyataan angket. Uji validitas dan reliabilitas instrumen dilakukan terlebih dahulu di fakultas ilmu
pendidikan universitas negeri padang terhadap 30 responden sebelum melakukan penelitian menggunakan program
SPSS versi 21.0, dengan hasil perolehan 3 item dalam variabel persepsi mahasiswa tentang profesi guru tidak valid,
sehingga item tersebut ada yang diperbaiki dan ada yang dibuang pernyataannya dan 9 item dalam variabel PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU 222 HASIL DAN PEMBAHASAN Rata-rata skor variabel persepsi mahasiswa tentang profesi guru adalah 4,40 dengan Tingkat Capaian
Responden (TCR) sebesar 88,00. Hal ini menunjukkan bahwa variabel lingkungan keluarga masuk dalam kategori
sangat baik. Artinya, persepsi mahasiswa tentang profesi guru sudah sangat baik, sehingga perlu dipertahankan dan
ditingkatkan. Rata-rata skor variabel lingkungan keluarga adalah 4,28 dengan Tingkat Capaian Responden (TCR) sebesar
85,70. Hal ini menunjukkan bahwa variabel lingkunga keluarga masuk dalam kategori sangat baik. Artinya, kondisi
lingkungan keluarga mahasiswa sudah baik, sehingga perlu dipertahankan dan ditingkatkan. rata-rata skor variabel minat menjadi guru adalah 3,93 dengan tingkat capai responden (TCR) sebesar 78,69. Hal ini menunjukkan bahwa variabel minat menjadi guru termasuk kedalam kategori baik. Artinya, minat menjadi
guru mahasiswa sudah baik, sehingga perlu dipertahankan dan ditingkatkan Analisis regresi berganda dilakukan untuk mengetahui pengaruh dua variabel bebas atau lebih terhadap
variabel terikat. Tabel 2. Analisis Regresi Linear Berganda Tabel 2. Analisis Regresi Linear Berganda Tabel 2. Analisis Regresi Linear Berganda
Coefficientsa
Model
Unstandardized
Coefficients
Standardized
Coefficients
t
Sig. B
Std. Error
Beta
1
(Constant)
-24,626
8,497
-2,898
,005
X1
,346
,148
,238
2,334
,022
X2
,546
,095
,584
5,728
,000
a. Dependent Variable: Y
Sumber: Pengolahan data primer 2018 Sumber: Pengolahan data primer 2018 erdasarkan hasil pengolahan regresi berganda dapat dirumuskan model regresi penelitian ini, yaitu: 𝑌= −24,626 + 0,346𝑋1 + 0,546𝑋2 + 𝑒 ,
,
1
,
2
Pada persamaan regresi diketahui nilai konstanta sebesar -24,626. Artinya tanpa ada variabel persepsi
mahasiswa tentang profesi guru (X1) dan lingkungan keluarga (X2) maka minat menjadi guru adalah -24,626. Sehingga diperoleh hasil dari tabel 2 di atas bahwa pengaruh persepsi mahasiswa tentang profesi guru (X1) terhadap
minat menjadi guru adalah sebesar 0,346 dengan nilai Sig 0,022 < α = 0,05 Hal ini menunjukkan bahwa persepsi
mahasiswa tentang profesesi guru berpengaruh signifikan terhadap minat menjadi guru. Jadi, semakin positif
persepsi mahasiswa tentang profesi guru, maka semakin positif pula minat menjadi guru pada diri mahasiswa. Pengaruh lingkungan keluarga (X2) terhadap minat menjadi guru adalah sebesar 0,546 dengan Sig 0,000< α = 0,05
Hal ini menunjukkan bahwa lingkungan keluarga berpengaruh signifikan terhadap minat menjadi guru. Jadi,
semakin baik lingkungan keluarga mahasiswa, maka semakin baik pula minat menjadi guru. g
g
g
p
j
g
Kontribusi dari variabel bebas terhadap variabel terikat dapat dilihat dari adjusted
menggunakan program SPSS versi 21.0. Tabel 3. Koefisien Determinasi Tabel 3. Koefisien Determinasi
Model Summary
Model
R
R Square
Adjusted R Square
Std. Volume 2, Nomor 3, 5 September 2019 lingkungan keluarga yang tidak valid 3 dibuang dan 6 diperbaiki pernyataannya dan 6 item variabel minat menjadi
guru tidak valit 4 dibuang 2 diperbaiki pernyataannya. Reliabilitas instrumen dilakukan dengan melihat nilai
cronbach’s alpha, dengan hasil klasifikasi indeks reliabilitas dalam kategori tinggi dan sangat tinggi. Data dianalisis
dengan menggunakan analisis deskriptif dan asosiatif. Adapun variabel yang akan dideskripsikan adalah persepsi
mahasiswa tentang profesi guru dan lingkungan keluarga, minat menjadi guru. HASIL DAN PEMBAHASAN Error of the Estimate
1
,772a
,596
,586
5,33712
a. Predictors: (Constant), X2, X1
Sumber: pengolahan data primer 2018 PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU 223 Volume 2, Nomor 3, 5 September 2019 Volume 2, Nomor 3, 5 September 2019 Berdasarkan tabel 2 di atas dapat dilihat bahwa hasil regresi menunjukkan bahwa hubungan persepsi
mahasiswa tentang profesi guru dan lingkungan keluarga terhadap minat menjadi guru adalah sebesar 0,772. Besarnya sumbangan bersama-sama yang diberikan oleh persepsi mahasiswa tentang profesi guru dan lingkungan
keluarga terhadap minat menjadi guru adalah sebesar 59,6% sedangkan sisanya sebesar 40,4% dipengaruhi oleh
faktor lain yang tidak diteliti dalam penelitian ini. Pengaruh variabel bebas (persepsi mahasiswa tentang profesi guru dan lingkungan keluarga) secara bersama-
sama terhadap variabel terikat (minat menjadi guru) dapat dilihat dengan uji F menggunakan program SPSS versi
21.0. Tabel 3. Uji F
ANOVAa
Model
Sum of Squares
Df
Mean Square
F
Sig. 1
Regression
3318,290
2
1659,145
58,246
,000b
Residual
2250,307
79
28,485
Total
5568,598
81
a. Dependent Variable: Y
b. Predictors: (Constant), X2, X1
Sumber: pengolahan data primer 2018 PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU Tabel 3. Uji F Tabel 3. Uji F Sumber: pengolahan data primer 2018 Dari tabel 3 dapat dilihat hasil pengolahan data nilai signifikansi hipotesis secara simultan sebesar 0,000 dan
nilai Fhitung sebesar 58,246. g
Pengaruh (secara parsial) setiap variabel bebas terhadap variabel terikat dapat dilihat dari uji t menggunakan
program SPSS versi 21.0. Pengaruh (secara parsial) setiap variabel bebas terhadap variabel terikat dapat dilihat dari uji t menggunakan
program SPSS versi 21.0. Tabel 4. Uji t Tabel 4. Uji t
Coefficientsa
Model
Unstandardized
Coefficients
Standardized
Coefficients
T
Sig. B
Std. Error
Beta
1
(Constant)
-24,626
8,497
-2,898
,005
X1
,346
,148
,238
2,334
,022
X2
,546
,095
,584
5,728
,000
a. Dependent Variable: Y
Sumber: Pengolahan data primer 2018 Sumber: Pengolahan data primer 2018 Dari tabel 4 dapat dilihat bahwa persepsi mhasiswa tentang profesi guru (X1) berpengaruh signifikan terhadap
minat menjadi guru (Y) dengan nilai signifikansi sebesar 0,022. Lingkungan keluarga (X2) berpengaruh signifikan
terhadap minat menjadi guru (Y) dengan nilai signifikansi sebesar 0,000. Dari tabel 4 dapat dilihat bahwa persepsi mhasiswa tentang profesi guru (X1) berpengaruh signifikan terhadap
minat menjadi guru (Y) dengan nilai signifikansi sebesar 0,022. Lingkungan keluarga (X2) berpengaruh signifikan
terhadap minat menjadi guru (Y) dengan nilai signifikansi sebesar 0,000. Berikut adalah kesimpulan pengujian hipotesis: Berikut adalah kesimpulan pengujian hipotesis: H1 : Pengujian hipotesis pertama dilakukan untuk mengetahui pengaruh persepsi mahasiswa tentang profesi guru
(X1) terhadap minat menjadi guru (Y). Hasil pengujian menghasilkan nilai Sig 0,22 <α = 0,05, maka Ho
ditolak. Hal ini berarti bahwa persepsi mahasiswa tentang profesi guru berpengaruh signifikan terhadap minat
menjadi guru pada mahasiswa Jurusan Pendidikan Ekonomi Fakultas Ekonomi UNP angkatan tahun 2014. H2 : Pengujian hipotesis kedua dilakukan untuk mengetahui pengaruh lingkungan keluarga (X2) terhadap minat
menjadi guru (Y). Hasil pengujian menghasilkan nilai Sig 0,000 < α = 0,05, maka Ho ditolak. Hal ini berarti
bahwa lingkungan keluarga berpengaruh signifikan terhadap minat menjadi guru pada mahasiswa Jurusan
Pendidikan Ekonomi Fakultas Ekonomi UNP angkatan tahun 2014. H3 : Pengujian hipotesis ketiga dilakukan untuk mengetahui pengaruh persepsi mahasiswa tentang profesi guru (X1)
dan lingkungan keluarga (X2) terhadap minat menjadi guru (Y). Hasil pengujian menghasilkan Nilai Sig 0,005
< α = 0,05, dan nilai Fhitung> Ftabel yaitu sebesar 3,11 maka H0 ditolak. Dengan demikian, persepsi mahasiswa
tentang profesi guru dan lingkungan keluarga secara bersama-sama berpengaruh terhadap minat menjadi guru
pada mahasiswa Jurusan Pendidikan Ekonomi Fakultas Ekonomi UNP angkatan tahun 2014. Volume 2, Nomor 3, 5 September 2019 sumbangan secara bersama-sama yang diberikan oleh persepsi mahasiswa tentang profesi guru dan lingkungan
keluarga terhadap minat menjadi guru adalah sebesar 59,6% sedangkan sisanya sebesar 40,4% dipengaruhi
oleh faktor lain yang tidak diteliti dalam penelitian ini. garuh Persepsi Mahasiswa Tentang Profesi Guru (X1) terhadap Minat Menjadi Guru (Y) Berdasarkan hasil penelitian, diketahui bahwa persepsi mahasiswa tentang profesi guru berpengaruh signifikan
terhadap minat menjadi guru. hal ini menunjukkan apabila persepsi mahasiswa tentang profesi guru semakin baik,
maka minat menjadi guru akan semakin meningkat. Sesuai dengan pernyataan Dalyono (2007:55) menjelaskan bahwa minat tidak terbentuk begitu saja dalam diri
seseorang. Ada beberapa faktor yang mempengaruhi minat salah satunya dipengaruhi oleh faktor internal yaitu
faktor yang berasal dari dalam diri mahasiswa yang mempengaruhi minat salah satunyaadalah persepsi. Sesuai
dengan pernyataan Alim, Arif Firman (2014:6) “Persepsi mempengaruhi minat melalui pandangan ataupun penilaian
mahasiswa tentang suatu objek, peristiwa, maupun kejadian yang dilihat, didengar dan dirasakan. Artinya persepsi
mempengaruhi minat menjadi guru melalui pandangan ataupun penilaian mahasiswa terhadap profesi guru. oleh
karena itu, mahasiswa diharapkan harus memiliki persepsi yang baik dan positif terhadap profesi guru supaya
semangat mahasiswa lebih terpacu untuk lebih giat lagi dalam belajar demi menambah ilmu pengetahuan keguruan
mahasiswa sehingga tercapainya tujuan dan cita-cita mahasiswauntuk menjadi guru yang profesional Pernyataan tersebut juga relevan dengan penelitian yang dilakukan oleh Destria, Yola (2015), yang mana
kesimpulan dari penelitian tersebut bahwa secara parsial hipotesis persepsi mahasiswa tentang program pendidikan
profesi guru berpengaruh positif dan siknifikan terhadap minat menjadi guru mahasiswa program studi pendidikan
ekonomi FE UNP. Baik buruknya persepsi mahasiswa tentang profesi guru akan mempengaruhi minatnya menjadi
guru. Dalam meningkatkan minat menjadi guru dibutuhkan hasrat yang tinggi, membutuhkan pengetahun informasi
yang baik tentang profesi guru, ketertarikan yang kuat dan keinginan yang besar terhadap profesi guru. Kenyataan
yang ada sekarang ini adalah mahasiswa banyak yang memandang sebelah mata mengenai profesi guru. hal ini yang
menyebabkan rendahnya minat mahasiswa terhadap profesi guru. Oleh karena itu diharapkan kepada mahasiswa
pendidikan harus merubah cara pandang menilai profesi guru bukan saja dari penghasilan yang didapatkan oleh guru
manun banyak hal positif yang didapatkan dibalik profesi guru tersebut. Berdasarkan hasil penelitian diatas, dapat diketahui bahwa persepsi mahasiswa tentang profesi guru
berpengaruh signifikan terhadap minat menjadi guru pada mahasiswa jurusan pendidikan ekonomi fakultas ekonomi
universitas negeri padang angkatan tahun 2014. Tabel 3. Uji F Dan besarnya PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP 224 EcoGen PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU Pengaruh Lingkungan Keluarga (X2) terhadap Minat Menjadi Guru (Y) Berdasarkan hasil penelitian, diketahui bahwa lingkungan keluarga berpengaruh signifikan terhadap minat
menjadi guru. Hal ini menunjukkan bahwa apabila lingkungan keluarga semakin baik, maka minat menjadi guru
pada Mahasiswa Pendidikan Ekonimi Jurusan Pendidikan Ekonomi Fakultas Ekonomi Universetas Negeri Padang
Angkatan Tahun 2014 akan meningkat juga. Hasil penelitian ini sesuai dengan teori yang diungkapkan oleh Hadinoto (1998:189) faktor yang
mempengaruhi minat seseorang salah satunya adalah faktor dari luar (ekstrinsik) yaitu suatu perbuatan yang
dilakukan atas dasar dorongan orang lain atau paksaan dari luar seperti lingkungan keluarga. Dapat kita ketahui
setiap lingkungan keluarga mahasiswa memiliki latar belakang kebudayan yang berbeda-beda seperti keluarga
mahasiswa yang kebanyakan berprofesi sebagai guru nah hal ini bisa mempengaruhi dan menumbuhkan minat
mahasiswa untuk lebih berminat menjadi guru karena adanya pengetahun mengenai profesi guru, adanya informasi
yang positif mengenai profesi guru dan adanya dorongan moral atau partisipasi dari keluarga seperti orang tua dan
saudara , menjadi inspirasi atau panutan bagi anak terhadap minat menjadi guru. hal ini secara tidak langsung dapat
menumbuhkan atau mempengaruhi minat mahasiswa untuk menjadiseorang guru nantinya Hasil penelitian ini sejalan dengan penelitian yang telah dilakukan oleh Sairoh (2016) hasil penelitiannya
menunjukkan bahwa lingkungan keluarga berpengaruh signifikan terhadap minat menjadi guru pada Mahasiswa PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU 225 Volume 2, Nomor 3, 5 September 2019 Pendidikan Ekonomi angkatan 2011 Fakultas Ekonomi Universitas Negeri yogyakarta. hal ini berarti, semakin baik
lingkungan keluarga maka minat menjadi guru pada mahasiswa juga semakin baik. Penelitian ini juga sejalan
dengan penelitian Ira (2005) disimpulkan bahwa lingkungan keluarga berpengaruh signifikan terhadap minat
menjadi guru pada siswa kelas XII di SMA N Nalumsari. Timbulnya minat karena adanya interaksi dengan orang lain terutama kepada lingkungan keluarga. Lingkungan
keluarga mempengaruhi individu, tetapi individu sebagai organisme yang hidup tidak begitu saja secara otomatis
menerima pengaruh tersebut tetapi didalam dirinya ada bermacam-macam kemampuan antara lain daya menyeleksi,
memperhitungkan, memutuskan, dan sebagainya. Oleh karena itu peran individu itu sendiri juga besar sekali dalam
menerima pengaruh tersebut. Jadi salah satu faktor yang dapat mempengaruhi dan mendukung minat minat menjadi
guru pada mahasiswa adalah lingkungan keluarga. Pengaruh Persepsi Mahasiswa Tentang Profesi Guru (X1) dan Lingkungan Keluarga (X2) terhadap Minat
Menjadi Guru (Y) Berdasarkan hasil analisis yang telah dilakukan dapat diketahui bahwa persepsi mahasiswa tentang profesi
guru dan lingkungan keluarga berpengaruh signifikan terhadap minat menjadi guru. hal ini apabilapersepsi
mahasiswa tentang profesi guru dan lingkungan keluarga baik, maka minat menjadi guru pada mahasiswa akan
meningkat juga. Kontribusi variabel persepsi mahasiswa tentang profesi guru dan lingkungan keluarga terhadap minat menjadi
guru sebesar 0,595 atau 59,6% dan sisanya 40,4%merupakan kontribusi variabel lain diluar variabel
persepsimahasiswa tentang profesi guru dan lingkungan keluarga.dari kedua variabel bebas yaitu persepsi
mahasiswa tentang profesi guru lebih besar dibandingkan dengan lingkungan keluarga dalam mempengaruhi
variabel terikat yaitu minat menjadi guru. hal ini menunjukkan bahwa persepsi mahasiswa tentang profesi guru
mempunyai pengaruh yang cukup besar dalam mempengaruhi minat menjadi guru. Berdasarkan hasil analisis diatas dapat diketahui bahwa minat seseorang dipengaruhi oleh beberapa faktor,
diantaranya yaitu persepsi mahasiswa tentang profesi guru dan lingkungan keluarga. Seperti pendapat yang
dikemukakan oleh Dalyono (2007:55) selain persepsi yang mempengaruhi minat menjadi guru, lingkungan keluarga
dan masyarakat juga termasuk mempengaruhi minat seseorang. Teori ini sejalan dengan Hadinoto (1998:189) faktor
yang mempengaruhi minat seseorang salah satunya adalah faktor dari luar (ekstrinsik) yaitu suatu perbuatan yang
dilakukan atas dasar dorongan orang lain atau paksaan dari luar seperti lingkungan keluarga. Dapat kita ketahui
setiap lingkungan keluarga mahasiswa memiliki latar belakang kebudayan yang berbeda-beda seperti keluarga
mahasiswa yang kebanyakan berprofesi sebagai guru nah hal ini bisa mempengaruhi dan menumbuhkan minat
mahasiswa untuk lebih berminat menjadi guru karena adanya pengetahun mengenai profesi guru, adanya informasi
yang positif mengenai profesi guru dan adanya dorongan moral atau partisipasi dari keluarga seperti orang tua dan
saudara , menjadi inspirasi atau panutan bagi anak terhadap minat menjadi guru. hal ini secara tidak langsung dapat
menumbuhkan atau mempengaruhi minat mahasiswa untuk menjadi seorang guru nantinya. Dapat disimpulkan jika
seseorang merasa profesi guru sesuai dengan bakat yang ia miliki dan hati nuraninya terpanggil maka dengan
sendirinyaakan muncul minat sebagai guru. SIMPULAN Berdasarkan hasil penelitian dan pembahasan, maka kesimpulan yang dapat diambil dari pengaruh lingkungan
keluarga dan minat belajar adalah : 1. Persepsi mahasiswa tentang profesi guru berpengaruh signifikan terhadap minat menjadi guru. Artinya persepsi
mahasiswa tentang profesi guru secara langsung sangat besar pengaruhnya terhadap pembentukan minat
menjadi guru. 1. Persepsi mahasiswa tentang profesi guru berpengaruh signifikan terhadap minat menjadi guru. Artinya persepsi
mahasiswa tentang profesi guru secara langsung sangat besar pengaruhnya terhadap pembentukan minat
menjadi guru. 2. Lingkungan keluarga berpengaruh signifikan terhadap minat menjadi guru. Artinya lingkungan keluarga secara
tidak langsung mampu memberikan pengaruh yang besar terhadap minat mahasiswa untuk menjadi guru. 2. Lingkungan keluarga berpengaruh signifikan terhadap minat menjadi guru. Artinya lingkungan keluarga secara
tidak langsung mampu memberikan pengaruh yang besar terhadap minat mahasiswa untuk menjadi guru. PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU Saran Berdasarkan hasil penelitian dan kesimpulan diatas, maka untuk meningkatkan minat menjadi guru pada
mahasiswa Jurusan Pendidikan Ekonomi Fakultas Ekonomi Universitas Negeri Padang penulis memberikan saran-
saran sebagai berikut: 1. Sesuai dengan hasil penelitian, bahwa profesi guru merupakan pekerjaan yang membutuhkan keahlian khusus
dalam bidang pendidikan, pengajaran, dan pelatihan. Oleh karena itu hendaknya sebagai calon guru mahasiswa
pendidikan harus memahami betul tugas penting dari seorang guru tersebut. Dimana guru merupakan
pekerjaan yang mulia dan dapat mencerdaskan generasi bangsa serta membangun sumber daya manusia yang
berkualitas. Sehingga diharapkan kepada mahasiswa janganlah melihat profesi guru dari hasil yang di dapat
tetapi lihatlah pengabdian yang telah beliau berikan kepada masyarakat. Oleh karena itu dibutuhkan persepsi
yang baik terhadap profesi guru sehingga dapat meningkatkan minat menjadi guru menjadi salah satu cita-cita
yang diinginkan. y
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2. Bagi orang tua mahasiswa hendaknya dengan mereka berprofesi sebagai guru hal ini menjadi salah satu faktor
pendukung dan langkah awal buat mahasiswa untuk lebih tertarik menjadi guru nantinya dengan memberikan
pandangan positif mengenai profesi guru kepada anaknya. y
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2. Bagi orang tua mahasiswa hendaknya dengan mereka berprofesi sebagai guru hal ini menjadi salah satu faktor
pendukung dan langkah awal buat mahasiswa untuk lebih tertarik menjadi guru nantinya dengan memberikan
pandangan positif mengenai profesi guru kepada anaknya. p
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p
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p
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y
3. Bagi peneliti berikutnya yang tertarik melakukan penelitian yang sejenis diharapkan dapat me
pedoman sehingga dapat mengembangkan penelitian ini lebih luas lagi terkait minat menjadi g 4. Penelitian ini masih terbatas pada ruang lingkup yang kecil, sehingga diharapkan pada peneliti selanjutnya
untuk meneliti faktor-faktor lain yang mempengaruhi minat menjadi guru, dan demi kesempurnaan penelitian
ini, penulis berharap ada yang mengadakan penelitian lebih lanjut dengan alat uji yang lebih baik. Volume 2, Nomor 3, 5 September 2019 Volume 2, Nomor 3, 5 September 2019 3. Persepsi mahasiswa tentang profesi guru dan lingkungan keluarga secara bersama-sama berpengaruh signifikan
terhadap minat menjadi guru. Artinya persepsi mahasiswa tentang profesi guru dan lingkungan keluarga
mempengaruhi minat menjadi guru. Besarnya sumbangan secara bersama-sama yang diberikan oleh persepsi
mahasiswa tentang profesi guru dan lingkungan keluarga terhadap minat menjadi guru adalah sebesar 0,596
atau 59,6% dan sisanya sebesar 40,4% dipengaruhi oleh faktor lain yang tidak diteliti dalam penelitian ini. 3. Persepsi mahasiswa tentang profesi guru dan lingkungan keluarga secara bersama-sama berpengaruh signifikan
terhadap minat menjadi guru. Artinya persepsi mahasiswa tentang profesi guru dan lingkungan keluarga
mempengaruhi minat menjadi guru. Besarnya sumbangan secara bersama-sama yang diberikan oleh persepsi
mahasiswa tentang profesi guru dan lingkungan keluarga terhadap minat menjadi guru adalah sebesar 0,596
atau 59,6% dan sisanya sebesar 40,4% dipengaruhi oleh faktor lain yang tidak diteliti dalam penelitian ini. PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU 226 DAFTAR PUSTAKA Alim, Arif Firman. 2014. Faktor-Faktor Yang Mempengaruhi Minat Profesi Guru Mahasiswa Program Studi
Pendidikan Teknik Bangunan (S1) Jurusan Teknik Sipil FT UNP. Artikel Penelitian. Uuniversitas Negeri
Padang. Ardyani, Anisa dan Latifah, Iyna. 2014. Analisis Faktor-Faktor Yang Mempengaruhi Minat Mahasiswa Menjadi
Guru Akuntansi Pada Mahasiswa Program Studi Pendidikan Akuntansi Angkatan 2010 Universitas Negeri
Semarang. Artikel Penelitian. Universitas Negeri Semarang. Dalyono, M. 2007. Psikologi Pendidikan. Jakarta: Rineka Cipta. Destria, Yola. 2015. Pengaruh lingkungan keluarga dan persepsi mahasiswa tentang program pendidikan profesi
guru (PPG) terhadap minat menjadi guru mahasiswa program studi pendidikan ekonomi FE UNP. Skripsi. Uuniversitas Negeri Padang. Ira. 2015. Persepsi Siswa Tentang Profesi Guru Dan Lingkungan Keluarga Pengaruhnya Terhadap Minat Menjadi
Guru Pada Siswa Kelas XII Sekolah Menenngah Atas Negeri 1 Nalumsari. Jurnal Ilmu Pendidikan. FKIP
Universetas Muhammadiyah Surakarta. Sairoh. 2016. Pengaruh Pengalaman PPL, IPK, Lingkungan keluarga dan Informasi Dunia Kerja Terhadap Minat
Menjadi Guru Pada Mahasiswa Pendidikan Ekonomi Angkatan 2011 FE UNY. Artikel Penelitian. Universitas Negeri Yogyakarta. Sardiman, A.M. 2012. Interaksi Dan Motivasi Belajar Mengajar. Jakarta: PT. Raja Grafindo Pe Sardiman, A.M. 2012. Interaksi Dan Motivasi Belajar Mengajar. Jakarta: PT. Raja Grafindo Persada. Slameto. 2010. Belajar dan Faktor-Faktor Yang Mempengaruhinnya. Jakarta: Rineka Cipta ,
j
g j
j
Slameto. 2010. Belajar dan Faktor-Faktor Yang Mempengaruhinnya. Jakarta: Rineka Cipta Slameto. 2010. Belajar dan Faktor-Faktor Yang Mempengaruhinnya. Jakarta: Rineka Cipta Susanti ,Yuli. 2013. Faktor Internal Dan Faktor Eksternal Yang Mempengaruhi Minat Mahasiswa Program Studi
Pendidikan Ekonomi FE UNP Untuk Menjadi Guru. Skripsi. Universitas Negeri Padang. Undang-undang Republik Indonesia. Nomor 14 Tahun 2005. Tentang Guru dan Dosen. Jakarta. Undang-undang Republik Indonesia. Nomor 14 Tahun 2005. Tentang Guru dan Dosen. Jakarta. PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU PENGARUH PERSEPSI MAHASISWA TENTANG PROFESI GURU DAN LINGKUNGAN KELUARGA TERHADAP
MIINAT MENJADI GURU
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HOME HYGIENE AND TUBERCULOSIS.
|
Lancet
| 1,906
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public-domain
| 1,817
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To the Editors of THE LANCET. To the Editors of THE LANCET. SIRS,-As one now residing in the eastern counties I can
fully endorse the statements in the letter quoted by you in
the course of your article on this subject which appeared in
THE LANCET of Jan. 27th, p. 248. There are, however, two
points to which I should like to draw your attention : one is the
prevalence of pulmonary tuberculosis as a result of families
occupying badly ventilated cottages previously occupied by
those in all stages of the disease, the other is the obvious
connexion between the state of the labourer’s cottage and
the high rate of illegitimacy in those districts in which the
cottage accommodation leaves much to be desired. For
every 1000 births registered in the county of Norfolk 74 are
illegitimate, in Herefordshire 85, and in Shropsbire 80, all
of which counties are great agricultural centres. In com-
parison Middlesex has 34, whilst such great industrial
counties as Durham, Derby, and Lancashire average but 41. It is a well-known fact that the farm labourer in Scotland is
very poorly housed and in one rural district in that country
the percentage of illegitimate children is 18’4. From these
figures it can be seen that the proportion is very much higher
in agricultural districts. That the sleeping accommodation
is bad will not alone give us the explanation ; most of the
mothers are quite young girls and there can be no doubt
that it is the want of proper dwelling rooms that is the
chief factor in causing such girls to prefer being out and
about rather than herd together with their little brothers and
sisters at home. As a result of deficient sleeping room
children are brought up to disregard those rules which
decorum demands and are invariablv the ones from whom
the next generation of illegitimate children are born. I need
not dwell upon the results of this deplorable amount of co
e c
g
p ope
p e e t
e
The number of old-standing and recent cases thus dealt
with in the three years the system has been in operation
exceeds 200. In over 160 of these voluntary notification was
made to the medical officer of health of St. Marylebone,
and a secondary notification to that official when subse-
quently any of these patients either changed their residence,
died, or removed to hospital or infirmary. HOME HYGIENE AND TUBERCULOSIS. As a member of the weekly board of
management of the Exeter Dispensary for 30 years I crave
and shall be grateful for information upon this point. upon
point. I am
Sirs, yours faithfully, upon
point. I am, Sirs, yours faithfully, Exeter, Jan. 25th, 1906. ithfully,
HARRY HEMS. HARRY HEMS. * * We understand that most of the dispensaries com-
parable to the Exeter Dispensary in Liverpool, Manchester,
Birmingham, and Leeds are closed in the evenings, save,
of course, for the treatment of casualties. Certain provident
dispensaries are, however, open. Our readers can perhaps
give Mr. Hems fuller information -ED. L. My board of directors, however, feel strongly that should
any scheme as that now to be taken in hand by the Brompton
Hospital be entered upon it should be applied to London as a
whole, when it would without doubt prove of great and im-
mediate value to the public health. In this connexion they
would point out that what could be undertaken in this
dispensary with so much success and encouragement could
doubtless be also undertaken by the numerous local public
or " general dispensaries," many of them, as in our case,
holding an honourable record of over a century’s work. These dispensaries, equipped for the treatment of patients
either at the institution or at their own homes, possess a
knowledge of the environment of the sick-an advantage
which the general hospital and other forms of medical
charities cannot possibly acquire. HYGIENE OF COTTAGES IN AGRI-
CULTURAL DISTRICTS. SIRS,—The directors of St. Marylebone General Dis-
pensary are desirous of expressing tle:r appreciation of the
importance and utility of the proposed innovation in
relation to the spread of tuberculosis (a notice of which
appeared in the press of Jan. 30th and THE LANCET,
Feb. 3rd, p. 316) and which is about to be adopted by
the committee of the Brompton Hospital in conjunction
with Dr. A. Newsholme of Brighton. The "proposed
innovation," however, forms a part of the generally
accepted requirements for any complete scheme for the
prevention of consumption and its value has long been
recognised by the medical officers of the above dispensary. For a period of over three years our late senior physician,
Dr. J. Edward Squire, assisted by our resident medical officer,
Dr. E. B. Hulbert, with the entire concurrence and coöpera-
tion of our honorary physicians, has been actively ergaged
in carrying out a fairly complete scheme of visitation
at the homes of consumptive patients. The object has
been to see that proper precautions are taken to prevent the
spread of the disease, to recommend sanitary and hygienic
requirements for diminishing predisposition in those closely
associated with the consumptive, and to advise other
members of the family or inmates of the house to attend at
the dispensary for examination for early detection of any
commencing disease and for proper preventive treatment. To the Editors of THE LANCET. R. C. S. Eng., L R.C.P. Lond. Bucklersbury, E.C., Feb. 4th, 1906. J. A. BARNES, M.R.C.S.Eng., L R.C.P. Lond. , you s
a t
u
y,
J. A. BARNES, M. R. C. S. Eng., L R.C.P. Lond. Bucklersbury, E.C., Feb. 4th, 1906. J. A. BARNES, M.R.C.S.Eng., L R.C.P. Lond. ,
, y
y,
FRANK STOKES, Secretary. St. Marylebone General Dispensary, Feb. 6th, 1906. FRANK STOKES, Secretary. FRANK STOKES, Secretary. St. Marylebone General Dispensary, Feb. 6th, 1906. FRANK STOKES, Secretary. STOKES,
St. Marylebone General Dispensary, Feb. 6th, 1906. STOKES, HOME HYGIENE AND TUBERCULOSIS. HOME HYGIENE AND TUBERCULOSIS. 404 domicile (including other residents in tenement houses),
intimate friends, or fellow workers. Disinfection of premises
on death or removal is advised and arranged for, and all
sufferers and suspects are urged to (and do) attend at the
dispensary at frequent intervals for examination and treat-
ment ; care is taken to keep in touch with them, even to
following up many who have drifted into our Marylebone
Infirmary or Mount Vernon Hospital. The results obtained
have more than compensated for the labour incurred, and
did space permit many instances of direct value in apprecia-
tion of such a scheme could be quoted, but as the results and
details of this investigation will, no doubt, shortly be made
public, it is hardly necessary to press the point further in
this letter. domicile (including other residents in tenement houses),
intimate friends, or fellow workers. Disinfection of premises
on death or removal is advised and arranged for, and all
sufferers and suspects are urged to (and do) attend at the
dispensary at frequent intervals for examination and treat-
ment ; care is taken to keep in touch with them, even to
following up many who have drifted into our Marylebone
Infirmary or Mount Vernon Hospital. The results obtained
have more than compensated for the labour incurred, and
did space permit many instances of direct value in apprecia-
tion of such a scheme could be quoted, but as the results and
details of this investigation will, no doubt, shortly be made
public, it is hardly necessary to press the point further in
this letter. asserts that no dispensary exists in this country where
evening consultations are available. Is it not possible that
Mr. Bell is in error in this impression ? If so, may I ask the
name of a dispensary or dispensaries where patients do
receive the benefits of such charities after their ordinary
working hours ? As a member of the weekly board of
management of the Exeter Dispensary for 30 years I crave
and shall be grateful for information upon this point. asserts that no dispensary exists in this country where
evening consultations are available. Is it not possible that
Mr. Bell is in error in this impression ? If so, may I ask the
name of a dispensary or dispensaries where patients do
receive the benefits of such charities after their ordinary
working hours ? To the Editors of THE LANCET. To the Editors of THE LANCET. SIRS,-With great interest I read Dr. M. Eden Paul’s note
in THE LANCET of Feb. 3rd of a case of aberrant vaccinia,
more especially as I happened to see a case myself about
three years ago. I was then in India in charge of a native
regiment. Owing to a small outbreak of small-pox it was
found necessary to vaccinate the whole of the native garrison
who had not had small-pox or been recently vaccinated. Five days after this had been done a sepoy came to see me
with a "sore " over the right malar bone. He was one of the
recently vaccinated and the sore ran the typical course in
every way of a vaccinia vesicle. With some difficulty (having
warned all the sepoys against doing it) I elicited the fact
that after the operation the man had attempted to allay the
irritation by scratching his arm and also that he had a small
abrasion on his cheek at that time. Judging from its appear-
ance and course I have no doubt at all that the " sore was
a vaccinia vesicle. It was particularly interesting, in my
opinion, from the fact that it was undoubtedly a case of
auto-inoculation. This is the only occasion on which I have
had an opportunity of seeing vaccination on a large scale
and I should feel very interested to hear if such cases are at
all common.-I am. Sirs, yours faithfully, possibly acquire. My directors cannot but regret that notwithstanding the
undoubted value of the work of "general dispensaries and
the facilities they have for active cooperation in the pre-
vention and cure of disease their importance in that field of
labour should be so underrated (even to the exclusion from
the benefits of the King Edward’s Hospital Fund). As a
consequence, after the provision of necessary funds for every-
day maintenance there is little left for clinical research or
general aid in the vital work of preserving the public health,
but there is little doubt that the staff of these general
dispensaries are as actively alive to matters affecting the
public interest and welfare as those attached to the better-
known and larger institutions, and where funds permit the
general dispensaries are not found behindhand in up to-date
and progressive treatment. e
I am, Sirs, yours faithfully, , yours faithfully,
J. A. BARNES, M. To the Editors of THE LANCET. Cards of " advice
to consumptives " are freely distributed ; clinical histories
are taken in all cases with special reference to relatives,
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Diminished sensitivity of audiovisual temporal order in autism spectrum disorder
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Frontiers in integrative neuroscience
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cc-by
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Diminished sensitivity of audiovisual temporal order in autism spectrum disorder
de Boer-Schellekens, L.; Eussen, M.; Vroomen, J.
Published in:
Frontiers in Integrative Neuroscience
DOI:
10.3389/fnint.2013.00008
Publication date:
2013
Document Version
Publisher's PDF, also known as Version of record
Link to publication in Tilburg University Research Portal
Citation for published version (APA):
de Boer-Schellekens, L., Eussen, M., & Vroomen, J. (2013). Diminished sensitivity of audiovisual temporal order
in autism spectrum disorder. Frontiers in Integrative Neuroscience, 7, Article 8.
https://doi.org/10.3389/fnint.2013.00008 Diminished sensitivity of audiovisual temporal order in autism spectrum disorder
de Boer-Schellekens, L.; Eussen, M.; Vroomen, J. Diminished sensitivity of audiovisual temporal order in autism spectrum disorder
de Boer-Schellekens, L.; Eussen, M.; Vroomen, J. Citation for published version (APA):
de Boer-Schellekens, L., Eussen, M., & Vroomen, J. (2013). Diminished sensitivity of audiovisual temporal order
in autism spectrum disorder. Frontiers in Integrative Neuroscience, 7, Article 8. https://doi.org/10.3389/fnint.2013.00008 Citation for published version (APA):
de Boer-Schellekens, L., Eussen, M., & Vroomen, J. (2013). Diminished sensitivity of audiovisual temporal order
in autism spectrum disorder. Frontiers in Integrative Neuroscience, 7, Article 8.
https://doi.org/10.3389/fnint.2013.00008 Tilburg University Tilburg University INTRODUCTION incoming information in its context, pulling information together
for higher-level meaning, often at the expense of memory for
detail. Frith (1989) proposed that people with autism show
detail-focused local processing in which features are perceived
and retained at the expense of global configuration and contex-
tualized meaning. Some have linked this to functional and/or
neuroanatomical under-connectivity between brain areas (Brock
et al., 2002; Courchesne and Pierce, 2005). The distribution of
attention to global/local features may be different in autism, lead-
ing one to predict relatively good performance where attention
to local information is advantageous, but poor performance on
tasks requiring the recognition of global meaning or integration
of stimuli in context (Happé, 1999). Iarocci and McDonald (2006)
suggest that many of the leading theories of autism allude to
dynamic constructs and conceptualizations such as central coher-
ence, temporal binding, shifting attention, enhanced perception,
and neural modulation and connectivity that may all eventually
involve multisensory processing and integration. In the multisensory world we live in, we are constantly bom-
barded with information that reaches us through our different
senses. The brain has to synthesize this mix of sensory informa-
tion into one coherent multisensory percept. An example of this
intersensory process is a speaker that can be seen and heard at
the same time. This results in an ensemble of multiple features
across the different senses that our brain has access to (i.e., lip
movement, facial expression, speed, and temporal structure of the
speech sound) that ultimately leads to an increase in perceptual
reliability. This sensory synthesis is a constantly occurring phe-
nomenon that shapes our view of the world and it is therefore
crucial for our everyday, social and adaptive behavior (Wallace,
2004). It also raises the question how our brain integrates this
wealth of sensory information and how a coherent representation
of the world is obtained (Keetels and Vroomen, 2012). Another
question is what happens if the brain is impaired in integrating
this mix of sensory input. This is one of the issues raised in contemporary research
in autism spectrum disorder (ASD). In addition to traditional
impairments in communication, social behavior, and stereotyped
repetitive movements, abnormalities in sensory processing are
often reported in ASD. Indeed, several contemporary theories
on ASD reflect the idea that sensory deficits are core symptoms
of autism as well (Kern et al., 2007; Crane et al., 2009). ORIGINAL RESEARCH ARTICLE published: 27 February 2013
doi: 10.3389/fnint.2013.00008 Liselotte de Boer-Schellekens1, Mart Eussen 2 and Jean Vroomen1* 1 Department of Cognitive Neuropsychology, Tilburg University, Tilburg, Netherlands
2 Yulius Mental Health Organization, Dordrecht, Netherlands We examined sensitivity of audiovisual temporal order in adolescents with autism
spectrum disorder (ASD) using an audiovisual temporal order judgment (TOJ) task. In order
to assess domain-specific impairments, the stimuli varied in social complexity from simple
flash/beeps to videos of a handclap or a speaking face. Compared to typically-developing
controls, individuals with ASD were generally less sensitive in judgments of audiovisual
temporal order (larger just noticeable differences, JNDs), but there was no specific
impairment with social stimuli. This suggests that people with ASD suffer from a more
general impairment in audiovisual temporal processing. Frontiers in Integrative Neuroscience General rights
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and investigate your claim Download date: 24. Oct. 2024 Edited by: Edited by:
Mark T. Wallace, Vanderbilt
University, USA
Reviewed by:
Sophie Molholm, Albert Einstein
College of Medicine, USA
David I. Shore, McMaster
University, Canada
*Correspondence:
Jean Vroomen, Department of
Cognitive Neuropsychology, Tilburg
University, Warandelaan 2,
PO Box 90153, 5000 LE Tilburg,
Netherlands. e-mail: j.vroomen@uvt.nl Edited by:
Mark T. Wallace, Vanderbilt
University, USA
Reviewed by:
Sophie Molholm, Albert Einstein
College of Medicine, USA
David I. Shore, McMaster
University, Canada
*Correspondence:
Jean Vroomen, Department of
Cognitive Neuropsychology, Tilburg
University, Warandelaan 2,
PO Box 90153, 5000 LE Tilburg,
Netherlands. e-mail: j.vroomen@uvt.nl y
Mark T. Wallace, Vanderbilt
University, USA Mark T. Wallace, Vanderbilt
University, USA Reviewed by:
Sophie Molholm, Albert Einstein
College of Medicine, USA
David I. Shore, McMaster
University, Canada Keywords: autism spectrum disorder, multisensory integration, audiovisual processing, temporal intersensory
sensitivity, social (un)impairment INTEGRATIVE NEUROSCIENCE INTEGRATIVE NEUROSCIENCE ORIGINAL RESEARCH ARTICLE
bli h d 27 F b
2013 INTRODUCTION In this
view, sensory deficits might have downstream effects on the devel-
opment of the perceptual system that may eventually lead to
adverse consequences for communication and social interaction
(Mottron and Burack, 2001; Bertone et al., 2005). Empirical evidence of multisensory processing deficits in ASD
is growing and there are now several clinical and anecdotal reports
that the sensory abnormalities that are observed among individ-
uals with ASD involve more than one sensory modality (e.g.,
Frith, 1989; O’Neill and Jones, 1997; Brock et al., 2002; Happé,
2005). One piece of evidence in support of this notion comes
from studies on the multisensory integration (MSI) of speech
and emotions as perceived from the face and the voice. Several
studies reported that persons with ASD may have less MSI (de
Gelder et al., 1991; Bebko et al., 2006; Smith and Bennetto, 2007;
Magnée et al., 2008; Mongillo et al., 2008; Russo et al., 2010). As an example, de Gelder et al. (1991) reported that individuals
with ASD were normal in auditory speech perception and were
unimpaired in silent lipreading, but when the auditory and visual
information streams were combined, there was very little effect of
the lipread information on auditory speech perception. Magnée Early clinical observations dating back to Kanner (1943)
already emphasized sensory avoidance and a tendency to over-
focus on local attributes. More recent accounts have proposed
that autism is characterized by weak “central coherence” (Frith,
1989). Central coherence is the everyday tendency to process February 2013 | Volume 7 | Article 8 | 1 Frontiers in Integrative Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Diminished temporal sensitivity in ASD de Boer-Schellekens et al. and found important deficits in duration judgments compared
to TD children. In a study by Bebko et al. (2006), intermodal
perception of audiovisual temporal synchrony in young children
with autism was compared with children without impairments
and a group of children with other developmental disabilities. The ASD group displayed reduced or no preference at all for syn-
chronous over asynchronous audiovisual speech and non-speech
stimuli, possibly because children with ASD are not (yet) sensi-
tive to audiovisual temporal synchrony. Two recent studies, by
Kwakye et al. (2011) and Foss-Feig et al. (2010), proposed that
individuals with ASD may have an extended window of multi-
sensory temporal binding. The study by Foss-Feig et al. (2010)
used the sound-induced double-flash illusion in children with
ASD. INTRODUCTION Research in ASD’s (multi)sensory processing has mainly
focused on the integration of higher-level information like speech
or faces, but there has been far less interest in the underlying
mechanism and the constraints under which information from
different modalities is combined. In research on MSI, it is gener-
ally agreed that (near) temporal synchrony is the most important
factor for MSI to occur (e.g., Welch and Warren, 1980; Stein
and Meredith, 1993; Radeau, 1994). Intersensory integration thus
will only occur if information from the different sensory modali-
ties arrives at approximately the same time in the brain because
otherwise two separate events are perceived. However, tempo-
ral synchrony between the senses is not straightforward, because
there is no evidence of a dedicated sense organ that registers time
in an absolute scale. It is well-known that the neural transduction
times of the various sensory modalities differ significantly, and the
brain thus has to overcome differences in transduction and neural
transmission time (Pöppel, 1997; Vroomen and de Gelder, 2004). It is conceivable that if there are fundamental disturbances in the
temporal orchestration of multisensory events, this will lead to
deficits in multisensory processing as well. Brock et al. (2002)
indeed theorized that the critical deficit of MSI in people with
ASD may lie in the temporal synchronization among both local
and distributed neural networks. These networks can show strong
patterns of entrainment in response to a given sensory stimulus
(i.e., a focus of activation in one area is soon followed in a strongly
time-locked fashion by a focus in a second connected brain area),
and this temporal synchronization among brain regions is likely
to be critically important in the binding of multisensory stim-
uli into unified perceptual constructs (Senkowski et al., 2008). A
critical question is thus whether people with ASD do indeed suf-
fer from intersensory temporal deficits that may underlie other
impairments in MSI. In this current study we presented adolescents with ASD and
TD controls an audiovisual TOJ task to examine their sensitiv-
ity of intersensory temporal order in a direct way. Three kinds of
stimuli were used that are known to differ on a number of poten-
tially relevant dimensions: a flash/beep, a video of a handclap
with the corresponding sound, and a video of a face articulating
a syllable with the corresponding speech sound. INTRODUCTION In this illusion, pairing of a single visual stimulus (i.e., flash)
with several auditory stimuli (i.e., beeps) often results in the per-
ception of two or more flashes (Shams et al., 2000). Foss-Feig
et al. (2010) reported that children with ASD had this flash-beep
illusion over an extended range of stimulus onset asynchronies
(SOAs) relative to TD children. Kwakye et al. (2011) also reported
an extended window of temporal integration in children with
ASD using temporal order judgment (TOJ) tasks with visual,
auditory, and audiovisual stimuli. The authors reported no dif-
ferences in sensitivity for visual temporal order, but thresholds
were higher in ASD on the auditory TOJ task. In the multisensory
TOJ task, the authors relied on the phenomenon known as tem-
poral ventriloquism (Scheier et al., 1999), where click sounds can
improve sensitivity for visual temporal order if the clicks are pre-
sented within a certain time window. Children with ASD showed
performance improvements over a wider range of temporal inter-
vals than TD children, thus reinforcing the idea that children with
ASD have a wider temporal window of MSI. et al. (2008) also observed that high-functioning adults with ASD
had difficulties integrating heard and lipread speech that could
not be attributed to problems in abnormal low-level integration. Possibly, then, people with ASD may have a generalized deficit
integrating information from different modalities (Kern et al.,
2007; Mongillo et al., 2008; Oberman and Ramachandran, 2008;
Foxe and Molholm, 2009; Brandwein et al., 2012). Others, however, found MSI in people with ASD to be nor-
mal (Van der Smagt et al., 2007; Grossman et al., 2009; Foss-Feig
et al., 2010). Williams et al. (2004) reported that children with
autism normally utilized visual information in identifying audi-
tory speech. Children with autism could also determine—as typi-
cally developing (TD) controls did—whether the specific sound
of a bouncing ball matched its physical appearance (Mongillo
et al., 2008). Equivalent amounts of MSI between an ASD- and
TD-group were also reported when participants were asked to
detect the direction of two laterally separated beeps in the pres-
ence of concurrent visual apparent motion, or the number of
flashes when concurrent beeps were present (Keane et al., 2010). When these results are taken together, there is thus no consensus
as to what extent MSI is impaired in autism. Frontiers in Integrative Neuroscience INTRODUCTION The asynchrony
between the audio and video was varied, and participants judged
whether the auditory stimulus came “early” or “late” with respect
to the video. By using different stimuli (flash/beep, handclap,
speech), we varied the complexity of the stimuli that allowed us
to examine whether people with ASD suffer from a general or
a more specific impairment in audiovisual temporal processing. Previous studies with TD participants have shown that people
are more sensitive to audiovisual timing differences of artificial
stimuli than audiovisual speech (Dixon and Spitz, 1980) and that
judging temporal order in audiovisual speech is particularly dif-
ficult, possibly because it lacks fast visual and auditory transients
that can serve as temporal markers (Stekelenburg and Vroomen,
2007). The handclap condition was expected to be relatively easy
for TD participants because it not only contains a fast audiovisual
transient, but also visual information that predicts sound onset
that may serve as a temporal anchor (Vroomen and Keetels, 2010). If individuals with ASD have impairments in action understand-
ing (Smith and Bryson, 1994; Iacoboni and Dapretto, 2006; Zalla
et al., 2010) that disrupt their ability to predict others’ actions, At present, autism studies on temporal processing are very
limited, but some indeed report differences in various aspects of
temporal functioning. Szelag et al. (2004) studied temporal pro-
cessing in the time domain of a few seconds in children with ASD February 2013 | Volume 7 | Article 8 | 2 www.frontiersin.org www.frontiersin.org www.frontiersin.org Diminished temporal sensitivity in ASD de Boer-Schellekens et al. (mean age = 19.2, SD = 2.4). The clinical participants were all in
residential care and recruited from “De Steiger” (part from men-
tal health institution “Yulius”), a residential unit in Dordrecht,
The Netherlands, serving patients with ASD exclusively. Ten ado-
lescents were administered the Wechsler Adult Intelligence Scale
(WAIS-III) and six the Wechsler Intelligence Scale for Children
(WISC-III) (see Table 1 for individual demographics per group). The severity of autistic symptoms was quantified with a check-
list of the 12 DSM-IV (APA, 1994) diagnostic criteria (sub A)
for 299.0 autistic disorder (see also Teunisse et al., 2001; Berger
et al., 2003). Based on this checklist and on the expertise of a
professional clinical team, two of the participants in the ASD
group met DSM-IV criteria for autistic disorder, 10 for Pervasive-
Developmental-Disorder Not-Otherwise-Specified (PDD-NOS)
and four for Asperger’s disorder (see also Table 2 for over-
all group demographics). INTRODUCTION Participants in the TD-control group
were recruited from Tilburg University and were non-psychiatric,
eleven males (mean age = 19.6 years) and five females (mean one may expect individuals with ASD to profit less from this
predictive information in the handclap condition. Finally, peo-
ple with ASD may have specific problems judging audiovisual
synchrony in faces because they are especially handicapped in
processing socially relevant stimuli (Kanner, 1943; Swettenham
et al., 1998; Klin et al., 2002; Bebko et al., 2006; Riby and Hancock,
2008). Numerous studies have indeed demonstrated that individ-
uals with ASD exhibit abnormalities in perceiving and attending
facial and social stimuli (Osterling and Fawson, 1994; Dawson
et al., 1998; Schultz et al., 2000; Golarai et al., 2006). We therefore
expected that people with ASD may have specific deficits judging
temporal order of audiovisual speech because it is both a complex
and social stimulus. www.frontiersin.org February 2013 | Volume 7 | Article 8 | 3 www.frontiersin.org
February 2013 | Volume 7 | Article 8 | 3 Frontiers in Integrative Neuroscience
www.frontiersin.org Frontiers in Integrative Neuroscience PARTICIPANTS Sixteen high-functioning adolescents with ASDs were included,
11 males and 5 females, ranging in age between 16 and 22 years Table 1 | Individual demographics and Just Noticeable Difference (JND) in ms per group. Sub
Age
Gender
Diagnosis
VIQ
PIQ
TIQ
JND Speech
JND Handclap
JND Flash
ASD GROUP
X1
19
M
AUTISM
110
85
98
182.14
66.78
85.40
X2
20
F
AUTISM
102
99
100
202.69
70.22
125.38
X3
19
M
PDD-NOS
81
87
83
150.69
173.64
215.35
X4
18
M
PDD-NOS
89
79
83
229.21
74.27
145.96
X5
16
M
ASPERGER
114
112
115
320.7
130.64
106.77
X6
16
M
ASPERGER
107
117
114
102.09
86.54
100.18
X7
18
F
PDD-NOS
121
140
134
90.37
183.55
206.65
X8
20
M
PDD-NOS
109
123
116
65.64
66.94
72.07
X9
20
M
ASPERGER
108
104
107
61.86
51.72
102.54
X10
21
M
ASPERGER
94
104
98
113.59
61.09
63.86
X11
16
F
PDD-NOS
129
109
115
98.01
60.49
85.29
X12
16
F
PDD-NOS
107
102
106
110.87
81.21
127.14
X13
22
M
PDD-NOS
115
96
107
132.28
66.19
89.61
X14
22
M
PDD-NOS
92
99
98
140.17
53.91
71.06
X15
20
M
PDD-NOS
98
99
97
98.87
66.81
76.63
X16
24
F
ASPERGER
132
119
128
79.30
70.61
129.87
TD GROUP
X20
18
M
–
122
113
120
180.62
100.07
103.37
X21
18
F
–
110
108
109
92.49
75.61
65.12
X22
18
F
–
114
109
112
99.53
66.20
55.72
X23
18
M
–
110
104
107
66.10
56.71
56.26
X24
21
M
–
127
101
117
71.51
54.20
63.53
X25
22
M
–
90
107
96
67.50
60.91
115.98
X26
18
F
–
102
100
101
91.17
55.93
85.10
X27
20
M
–
109
113
111
93.32
57.05
104.27
X28
18
F
–
113
101
108
135.55
63.42
89.48
X29
18
M
–
122
111
119
96.56
65.39
65.80
X30
21
M
–
99
94
96
137.97
98.10
90.67
X31
20
M
–
106
108
107
176.26
107.12
77.84
X32
20
F
–
97
93
94
129.75
56.33
147.56
X33
19
M
–
103
90
97
97.65
79.89
72.56
X34
19
M
–
114
85
101
123.63
91.13
122.87
X35
20
M
–
111
110
111
52.36
53.13
60.71 Table 1 | Individual demographics and Just Noticeable Difference (JND) in ms per group. Diminished temporal sensitivity in ASD de Boer-Schellekens et al. PARTICIPANTS The duration of the auditory sample was 325 ms for/bi/and
120 ms for the handclap (for more details on these stimuli see
Stekelenburg and Vroomen, 2007, who originally recorded and
used these stimuli). The sound pressure level of /bi/ was 63 dB(A)
and 67 dB(A) for handclap (see Figure 1B). The SOA between the
auditory and visual part of each stimulus stimuli varied in 10 steps
(±320, ±240, ±160, ±80, ±40ms, with negative values indicating
sound first). This resulted in 10 unique trials each randomly pre-
sented 16 times in two blocks of 80 trials each for each of the three
stimulus conditions. The three stimulus conditions were blocked age = 18.4 years), age range 18–22 years. Both groups were
matched on age, gender and IQ. None of the participants had
a history of serious medical, neurological or psychiatric illness
(apart from ASD), seizure disorder, trauma, or use of medication
affecting the nervous system. All reported normal or corrected-
to-normal vision and hearing and were tested individually. The
ASD group received gift vouchers for their participation, the TD
group received course credits in return. All participants were
naïve to both the experimental procedure and the purpose of the
study and gave written consent prior to participating (in case of
immature participants, written consent was also given by par-
ents). The study was carried out in accordance with the ethical
standards of the Declaration of Helsinki and was approved by the
Medical Review Ethics Committee of the St. Elisabeth Hospital,
Tilburg, The Netherlands. PARTICIPANTS The flash/beep condition
started with the presentation of a gray placeholder (diameter of
3.5 cm) against a dark background in the middle of the screen. After 1000–1500 ms either a 7 ms sound burst of 69 dB(A) or a
white square (diameter of 1.5 cm) in the placeholders position
were presented with variable SOAs (sound first or flash first). The speech stimulus consisted of the pronunciation of the syl-
lable/bi/by a Dutch female speaker whose face was entirely visible
on the monitor. In the handclap condition, a single clap of two
hands was presented. The videos were presented at a rate of 25
frames/s. The duration of the videos was 3 s, including a 200 ms
fade-in and fade-out, and a still image (400–1100 ms) at the
start. The duration of the auditory sample was 325 ms for/bi/and
120 ms for the handclap (for more details on these stimuli see
Stekelenburg and Vroomen, 2007, who originally recorded and
used these stimuli). The sound pressure level of /bi/ was 63 dB(A)
and 67 dB(A) for handclap (see Figure 1B). The SOA between the
auditory and visual part of each stimulus stimuli varied in 10 steps
(±320, ±240, ±160, ±80, ±40ms, with negative values indicating
sound first). This resulted in 10 unique trials each randomly pre-
sented 16 times in two blocks of 80 trials each for each of the three
stimulus conditions. The three stimulus conditions were blocked handclap, and speech (see Figure 1A). The flash/beep condition
started with the presentation of a gray placeholder (diameter of
3.5 cm) against a dark background in the middle of the screen. After 1000–1500 ms either a 7 ms sound burst of 69 dB(A) or a
white square (diameter of 1.5 cm) in the placeholders position
were presented with variable SOAs (sound first or flash first). The speech stimulus consisted of the pronunciation of the syl-
lable/bi/by a Dutch female speaker whose face was entirely visible
on the monitor. In the handclap condition, a single clap of two
hands was presented. The videos were presented at a rate of 25
frames/s. The duration of the videos was 3 s, including a 200 ms
fade-in and fade-out, and a still image (400–1100 ms) at the
start. PARTICIPANTS Table 2 | Overall demographics and comparison per group and mean Just Noticeable Difference (JND) and Point of Subjective Simultaneity
(PSS) in ms and Standard deviations per condition. ASD group
TD group
Comparison
Mean
SD
Mean
SD
t(30)
p
Age (in years)
19.2
(2.4)
19.3
(1.3)
0.09
0.93
Verbal IQ
106.8
(13.9)
109.0
(9.7)
0.53
0.60
Performal IQ
104.6
(15.4)
103.3
(8.9)
−0.31
0.76
Total IQ
106.2
(14.1)
106.6
(8.4)
0.11
0.92
Medication
N
Mean daily dosage
Clonidine
1
0.125 mg
Paroxetine
1
20 mg
Sertraline
2
75 mg
Risperdal
1
1.5 mg
Alprazolam
1
0.25 mg
Diagnoses
N
Autism
2
PDD-NOS
9
Asperger
5
JNDs PER CONDITION IN ms
Speech
136.2
(68.5)
107.0
(37.6)
Handclap
86.0
(41.5)
71.3
(18.3)
Flash/beep
112.7
(45.1)
86.0
(26.8)
PSSs PER CONDITION IN ms
Speech
11.7
(92.4)
42.2
(77.3)
Handclap
24.0
(64.2)
15.9
(47.5)
Flash/beep
27.0
(90.0)
65.1
(90.7) Table 2 | Overall demographics and comparison per group and mean Just Noticeable Difference (JND) and Point of Subjective Simultaneity
(PSS) in ms and Standard deviations per condition. nd comparison per group and mean Just Noticeable Difference (JND) and Point of Subjective Simultaneity
ions per condition. Table 2 | Overall demographics and comparison per group and mean Just Noticeable Difference (JND) and Point of Subjective Simultaneity
(PSS) in ms and Standard deviations per condition. ASD group
TD group
Comparison
Mean
SD
Mean
SD
t(30)
p
Age (in years)
19.2
(2.4)
19.3
(1.3)
0.09
0.93
Verbal IQ
106.8
(13.9)
109.0
(9.7)
0.53
0.60
Performal IQ
104.6
(15.4)
103.3
(8.9)
−0.31
0.76
Total IQ
106.2
(14.1)
106.6
(8.4)
0.11
0.92
Medication
N
Mean daily dosage
Clonidine
1
0.125 mg
Paroxetine
1
20 mg
Sertraline
2
75 mg
Risperdal
1
1.5 mg
Alprazolam
1
0.25 mg
Diagnoses
N
Autism
2
PDD-NOS
9
Asperger
5
JNDs PER CONDITION IN ms
Speech
136.2
(68.5)
107.0
(37.6)
Handclap
86.0
(41.5)
71.3
(18.3)
Flash/beep
112.7
(45.1)
86.0
(26.8)
PSSs PER CONDITION IN ms
Speech
11.7
(92.4)
42.2
(77.3)
Handclap
24.0
(64.2)
15.9
(47.5)
Flash/beep
27.0
(90.0)
65.1
(90.7)
age = 18.4 years), age range 18–22 years. Both groups were
matched on age, gender and IQ. None of the participants had
a history of serious medical, neurological or psychiatric illness
(apart from ASD), seizure disorder, trauma, or use of medication
affecting the nervous system. All reported normal or corrected-
to-normal vision and hearing and were tested individually. Frontiers in Integrative Neuroscience PARTICIPANTS The
ASD group received gift vouchers for their participation, the TD
group received course credits in return. All participants were
naïve to both the experimental procedure and the purpose of the
study and gave written consent prior to participating (in case of
immature participants, written consent was also given by par-
ents). The study was carried out in accordance with the ethical
standards of the Declaration of Helsinki and was approved by the
Medical Review Ethics Committee of the St. Elisabeth Hospital,
Tilburg, The Netherlands. STIMULI
Visual stimuli were presented on a 15′ laptop monitor [Dell
Inspiron 6000, controlled by E-Prime (Psychology Software Tools,
Inc.; www.pstnet.com/eprime)] positioned on eye-level, approx-
imately 60 cm in front of the participants. The sounds came from
the laptop speakers. There were three types of stimuli: flash/beep,
handclap, and speech (see Figure 1A). The flash/beep condition
started with the presentation of a gray placeholder (diameter of
3.5 cm) against a dark background in the middle of the screen. After 1000–1500 ms either a 7 ms sound burst of 69 dB(A) or a
white square (diameter of 1.5 cm) in the placeholders position
were presented with variable SOAs (sound first or flash first). The speech stimulus consisted of the pronunciation of the syl-
lable/bi/by a Dutch female speaker whose face was entirely visible
on the monitor. In the handclap condition, a single clap of two
hands was presented. The videos were presented at a rate of 25
frames/s. The duration of the videos was 3 s, including a 200 ms
fade-in and fade-out, and a still image (400–1100 ms) at the
start. The duration of the auditory sample was 325 ms for/bi/and
120 ms for the handclap (for more details on these stimuli see
Stekelenburg and Vroomen, 2007, who originally recorded and
used these stimuli). The sound pressure level of /bi/ was 63 dB(A)
and 67 dB(A) for handclap (see Figure 1B). The SOA between the
auditory and visual part of each stimulus stimuli varied in 10 steps
(±320, ±240, ±160, ±80, ±40ms, with negative values indicating
sound first). This resulted in 10 unique trials each randomly pre-
sented 16 times in two blocks of 80 trials each for each of the three
stimulus conditions. The three stimulus conditions were blocked handclap, and speech (see Figure 1A). RESULTS Trials of the practice session were excluded from analyses. The
individual proportion of sound-early responses was calculated for
each combination of stimulus condition and SOA, and then con-
verted into equivalent Z-scores (for averaged raw data of both
groups for each condition, see Figure 2). For both groups JNDs
were initially computed by fitting both logistic and linear func-
tions. The results of the logistic and linear slopes and JNDs were
equivalent. The linear function provided a significantly better fit
to the data of the three conditions than the logistic function for
both groups, and was therefore used as the principle function. The
mean and standard deviations of R² for the control group were
0.79 (±0.08), 0.81 (±0.08) and 0.85 (±0.11) for the three con-
ditions (speech, handclap and flash/beep) and 0.76 (±0.14), 0.82
(±0.10) and 0.81 (±0.15) for the ASD group. For each condi-
tion, the best fitting straight line was then calculated over the ten
SOAs. Two subjects (one from each group) were excluded from
further analyses, because their results did not conform to a typi-
cal s-shaped function. The lines’ slopes and intercepts were used
to determine the just noticeable difference (JND = 0.675/slope)
and the point of subjective simultaneity (PSS). The JND repre-
sents the smallest interval between two stimuli needed by par-
ticipants to correctly judge which stimulus came first. A smaller
JND thus represents good sensitivity as smaller stimulus differ-
ences are required for correctly judging temporal order. The PSS
represents the average interval by which one stimulus had to
lead the other for being perceived as simultaneous. The group-
averaged JNDs for each condition are presented in Figure 3. As
is clearly visible, the ASD group had overall larger JNDs than
the TD group. This indicates that individuals with ASD were
less sensitive to judge audiovisual temporal synchronies. This
was confirmed in a 2 (group) × 3 (condition) ANOVA on the FIGURE 1 | (A) Examples of the stimuli used in the three conditions;
speech, handclap, and flash/beep. (B) Depiction a “sound first” [the sound
(handclap) is presented at −240ms] trial in the handclap condition. FIGURE 1 | (A) Examples of the stimuli used in the three conditions;
speech, handclap, and flash/beep. (B) Depiction a “sound first” [the sound
(handclap) is presented at −240ms] trial in the handclap condition. STIMULI Visual stimuli were presented on a 15′ laptop monitor [Dell
Inspiron 6000, controlled by E-Prime (Psychology Software Tools,
Inc.; www.pstnet.com/eprime)] positioned on eye-level, approx-
imately 60 cm in front of the participants. The sounds came from
the laptop speakers. There were three types of stimuli: flash/beep, February 2013 | Volume 7 | Article 8 | 4 Frontiers in Integrative Neuroscience www.frontiersin.org Diminished temporal sensitivity in ASD de Boer-Schellekens et al. and presented in an ABCCBA design, with stimulus order coun-
terbalanced across participants. The SOA varied randomly within
each block. and presented in an ABCCBA design, with stimulus order coun-
terbalanced across participants. The SOA varied randomly within
each block. relative to the visual stimulus. Responses were given by pressing
one of two keys (“sound early,” “sound late”) on a response box. Responses were unspeeded with emphasis on accuracy. A practice
session preceding the test was given in which trials were pre-
sented with the two longest SOAs for each condition. During
practice, participants received feedback (“wrong” or “correct”)
after each trial. Practice continued until six consecutive correct
answers were given. Then testing started without feedback. PROCEDURE Participants were individually tested in a quiet test room (either
at Tilburg University or in “De Steiger” clinic, Yulius). The partic-
ipants’ task was to judge whether the sound came “early” or “late” FIGURE 1 | (A) Examples of the stimuli used in the three conditions;
speech, handclap, and flash/beep. (B) Depiction a “sound first” [the sound
(handclap) is presented at −240ms] trial in the handclap condition. RESULTS FIGURE 2 | Averaged raw data of both the TD and ASD group for each condition (“speech (A),” “handclap (B),” and “flash/beep (C)”). ged raw data of both the TD and ASD group for each condition (“speech (A),” “handclap (B),” and “flash/beep (C)”). February 2013 | Volume 7 | Article 8 | 5 Frontiers in Integrative Neuroscience www.frontiersin.org Diminished temporal sensitivity in ASD de Boer-Schellekens et al. FIGURE 3 | Group-averaged JNDs as a function of the interval between
the sound and video and (bars represent 1 standard error of the mean). handclap and flash/beep). However, there was no trace of a spe-
cific impairment, as the JNDs of both groups were equally affected
by the different stimuli. This thus suggests that people with
ASD may suffer from a more general impairment in audiovisual
temporal processing. Our findings fit a study by Bebko et al. (2006) who showed that
children with ASD had impairments in the detection of violations
of temporal synchrony of audiovisual linguistic stimuli if com-
pared to TD children and children with non-autistic developmen-
tal delays. They used a preferential looking paradigm in which the
children viewed two screens displaying identical video tracks, but
one offset from the other by 3 s, and with the single audio track
that matched to only one of the displays. Even though their study
showed (very small) contrasting results for non-linguistic stimuli
(which can be explained by choice of paradigm and the essential
difference in timing durations of the asynchrony in audiovisual
stimuli, 3 s compared to our range of 320–340 ms), there are
indications of impaired temporal sensitivity for synchrony. FIGURE 3 | Group-averaged JNDs as a function of the interval between
the sound and video and (bars represent 1 standard error of the mean). Grossman et al. (2009) wanted to test whether high-
functioning ASD adolescents were able to integrate AV informa-
tion of meaningful, phrase-length language in a task of onset
asynchrony detection. They found no significant differences
between adolescents with ASD and their TD peers in accuracy
of onset asynchrony detection. The authors used video clips of
complete phrases, using simple, commonly occurring words. The
clips were manipulated to have the video precede the correspond-
ing audio by audio delays from 120–500 ms. Like the Bebko et al. (2006) study, the delays in this study were substantially larger
compared to our study (40–320 ms). RESULTS This temporal component
could be an explanation for the contrasting results between the
studies. As Figure 2 of our data shows, the ASD group scores
near 85-95% correct on the large SOAs (at least ±320 and ±240). This percentage drops at the smaller SOAs (just like in the TD
group). These results show that the ASD group is capable of
judging temporal asynchrony, but their larger JNDs reveal that
they need more time between the audiovisual stimuli to do so
(but in a much smaller timeframe than the studies described
before). A possible interpretation of these results could be that
people with ASD continue to bind two stimuli as part of one
event. JNDs. There was a main effect of group F(1, 31) = 4.399, p < 0.05,
ηp2 = 0.13, indicating that, on average, the ASD group had larger
JNDs than the TD group (group averages of 116.6 ms and 88.1 ms
for ASD and controls, respectively). There was also a main effect
of condition F(2, 30) = 12.058, p < 0.001, ηp2 = 0.29, because
sensitivity differed among the three different stimuli, while the
theoretically important Group × Condition interaction was not
significant, F < 1. As also visible in Figure 3, both groups showed
the smallest JNDs (best sensitivity) in the handclap condition. Independent t-tests across groups comparing the three condi-
tions confirmed that the difference in JND (43 ms) between the
speech and handclap condition, t(31) = 4.619, p < 0.001, and the
20.4 ms difference between the flash/beep and handclap condi-
tion, t(31) = −4.067, p < 0.001, were significant. The difference
in JND (22.3 ms) between the speech and flash/beep condition
was also significant, t(31) = 2.093, p < 0.05. To summarize, we
succeeded in creating audiovisual stimuli that varied in their
difficulty of judging the temporal order of their components. Individuals with ASD were, in general, less sensitive perceiving
small audiovisual timing differences than controls, but they were
not specifically impaired with audiovisual speech. For completeness, similar analyses were also run on the PSSs
(see Table 2). A 2 (group) × 3 (conditions) overall ANOVA on
the PSSs showed that there was no effect of Group (F = 1.137,
p > 0.05), Condition (F = 1.17, p > 0.05), and no interaction
between the two factors (F < 1). RESULTS The point at which the audio-
visual stimuli were perceived to be maximally synchronous thus
did not differ between group and stimuli. An extended window of temporal binding of two intermodal
stimuli in people with ASD has also been proposed by Kwakye
et al. (2011) and Foss-Feig et al. (2010). Results of both their
flash-beep illusion and TOJ studies revealed that children with
ASD have altered multisensory temporal function as they showed
extended illusion ranges in performance in the multisensory
tasks. For example, in the TOJ task performance gains of the
children with ASD manifested themselves as improvements in
accuracy and as faster responses relative to the unisensory (i.e.,
visual-only) baseline condition across an increased range of mul-
tisensory delays [important to mention here is that the SOAs in
these studies are comparable with those in our study (a range of
0–500 ms)]. It seems conceivable that the diminished sensitivity
to temporal asynchrony we found here could result in an enlarged
multisensory temporal binding window. Alternatively, though, it
might also be a result of some temporal binding deficit, as pro-
posed by Brock et al. (2002). They suggest that activities within Frontiers in Integrative Neuroscience DISCUSSION Here we examined sensitivity of audiovisual temporal asyn-
chronies in adolescents with ASD and TD controls in an audio-
visual TOJ task. The results showed that the ASD group had
larger JNDs (= lower sensitivity) than the TD group, indicat-
ing that the ASD group had more difficulty judging audiovisual
synchrony. Furthermore, we hypothesized that people with ASD
might be specifically impaired when social stimuli were involved,
and therefore used different stimulus classes (audiovisual speech, February 2013 | Volume 7 | Article 8 | 6 Frontiers in Integrative Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Diminished temporal sensitivity in ASD de Boer-Schellekens et al. were presented a short non-word “bi” as pronounced by a female
face and they only had to judge whether the sound came before
or after the lips moved. The spoken stimuli thus had no further
meaningful content, and this focus on low-level aspects of the
stimulus might overshadow its social relevance. Another expla-
nation might be more temporal, as the duration of our videos
was relatively short (3 s) compared to other studies (e.g., Dawson
et al., 1998; Bebko et al., 2006). Participants were thus exposed to
much shorter fragments of faces which may ease processing load. networks of interconnected sensory areas are not as strongly
correlated in ASD, resulting in disruptions in the binding of per-
ceptual information. Kwakye et al. (2011) further speculate that
it may be the case that these neural signals are not so drastically
uncorrelated as to cause decoupling across regions (as initially
hypothesized by Brock et al., 2002), but instead occur in such
a way as to necessitate an extended temporal binding window
within which two stimuli can continue to be bound as part of one
event. Clearly, further research is needed for additional informa-
tion on these mechanisms and theories on networks connections. There are some obvious limitations in our study. Firstly, we
only investigated a very specific group of high-functioning ado-
lescents with ASD and it remains to be examined whether this
can be generalized to other subtypes of ASD. Interpreting the
results is also complicated by the heterogeneity of the disorder,
even within each subtype. Therefore, our results may not apply
to other subpopulations of ASD such as children, adults or lower-
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those with typical development who are typically developed or to
those who are both developmentally impaired and non-autistic. Interestingly, our results show no differences in the judgment
of the audiovisual temporal order of specific stimuli between the
two groups. Both groups performed best with clapping hands,
followed by the artificial beep-flash, and worse with audiovisual
speech. These findings concur with the results of Stekelenburg
and Vroomen (2007). They used the same TOJ task (except for the
artificial condition) and found that JNDs for non-speech events
(clapping hands) were smaller than for speech (facial condition). This indicates that the temporal relation between audition and
vision of the handclap was more precisely defined. The authors
also pointed out that the clapping hands contained more anticipa-
tory visual motion (280 ms) than the speech stimuli (160 ms), and
faster transient onsets in audition and vision. They proposed that
judging temporal order in audiovisual speech is particularly diffi-
cult because it contains less visual anticipatory motion and lacks
fast auditory and visual transients. Apparently, predictive infor-
mation can be used by individuals with ASD in this task, despite
their putative impairments in action understanding. Although the exact causes of our findings are speculative, they
are in line with the majority of ASD studies on MSI which show
that individuals with ASD have altered MSI (de Gelder et al.,
1991; Bebko et al., 2006; Kern et al., 2007; Smith and Bennetto,
2007; Magnée et al., 2008; Mongillo et al., 2008; Oberman and
Ramachandran, 2008; Foxe and Molholm, 2009; Russo et al.,
2010), or altered multisensory temporal function (Foss-Feig et al.,
2010; Kwakye et al., 2011). Further research is clearly needed
to examine and characterize multisensory processes in ASD in
more detail and this may ultimately lead to a broader and better
understanding and diagnosis of this disorder. We also hypothesized that people with ASD might have spe-
cific problems judging audiovisual temporal order in audiovi-
sual speech because of the social component. Numerous studies
reported that individuals with ASD exhibit abnormalities in facial
and social stimuli (e.g., Osterling and Fawson, 1994; Dawson
et al., 1998; Schultz et al., 2000; Golarai et al., 2006). However,
we found no such impairments here with faces and, arguably, the
hand clap. ACKNOWLEDGMENTS We wish to thank the participating adolescents with ASD for their
time. We also thank Yulius Mental Health Organization, especially
the staff of De Steiger location Amazone, in particular Ad van der
Sijde, MD., and Sandra Kint. DISCUSSION An explanation might be that the participants’ task
in our experiment did not involve speech comprehension, face
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access article distributed under the terms
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https://openalex.org/W4280631734
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https://econjournals.com/index.php/ijeep/article/download/12961/6778
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English
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Energy Poverty Clustering by Using Power-cut Job Order Data of the Electricity Distribution Companies
|
International journal of energy economics and policy
| 2,022
|
cc-by
| 8,688
|
International Journal of Energy Economics and
Policy International Journal of Energy Economics and Policy, 2022, 12(3), 401-409. Tamer Emre*, Adnan Sozen Energy Systems Engineering Department, Gazi University, Ankara, Turkey. *Email: evretameremre@yahoo.com DOI: https://doi.org/10.32479/ijeep.12961 DOI: https://doi.org/10.32479/ijeep.12961 Received: 01 February 2022 DOI: https://doi.org/10.32479/ijeep.12961 Accepted: 17 April 2022 ABSTRACT Keywords: Energy Poverty, Fuel Poverty, Arrears on Utility Bills, Power-cut Job Order
JEL Classifications: Q4, H43, I3 This Journal is licensed under a Creative Commons Attribution 4.0 International License Keywords: Energy Poverty, Fuel Poverty, Arrears on Utility Bills, Power-cut Job Order
JEL Classifications: Q4, H43, I3 ABSTRACT The identification of the population suffering from energy poverty, which is more visible after Covid-19 pandemic following by the 2021 energy
crisis, is an essential requirement for producing systematic and sustainable solutions. Although European Union approaches to the problem with a
multi-indicator sets; this indicator sets have a large amount of secure and almost unreachable data, such as identity information, wage information,
health information, asset information (title deed, rental income), expenditure information, debt information, credit information, bank records, etc. Experienced two long term projects between 2014 and 2016 (problem definition for energy theft and the best practices searching 13 different country
examples including Brazil, Hungary, India, etc.) and 2016–2018 (energy poverty set and consumption characteristics in Turkey) over 6 million
end-user consumption and payment data brings us to confirm that. The primary indicator of energy poverty is the arrears on utility bills. The arrears
resulting from the affordability problem of the energy consumed trigger a power cut-off job order in the utility company. This research examines the
literature and country social assistance implementation data to see how an energy poverty level can be identified using details on arrears and power-
cut job orders. On this subject, power-cut job orders were constituted, because of arrears on utility bills, were subjected to statistical analysis, and the
compatibility of the trend data with the socio-economic development index was investigated. Cities with a less indexes have more utility bill arrears
in terms of both number and volume, according to correlation-test data. Urban cities are more visible in data since the non-urbanized cities have some
energy theft activities which show us no efficiency target for the consumption! Hence one of the strategical step for decreasing the non-technical losses
is having more registered customer, the relationship between the growth index and the number of customers is another intriguing finding. Separating
the consumption levels of arrears, it is found that 63% of total non-payment is depending on 18% of consumers. Trend analysis confirmed that every
energy consumption level has the absolute and fluctuated component inside. The number of people inside the absolute poverty cluster is coherent with
national and international approaches almost in the same number. The findings revealed that arrears on utility bills can be used specifically to assess
the population identified with energy dependency rather than relying on evidence from a variety of sources. 1. INTRODUCTION Each subscription may be on behalf of a different person in
the household, or records may not belong to individuals in
that household because of the flawed/missing data update l
g
p
c. Failure in the reliability of the surveys or questionnaires. In
case the data cannot be obtained, some methods like surveys or
questionnaires come into play. When the subject is a sensitive
and psychological word such as “poverty,” the real needy hide
the situation, and some parts of the non-needy part worsen it. The reliability of a questionnaire becomes unclear because it
is declaration-based. It is not possible to observe the problem
or produce a policy dependent on the declaration-based data. When such information is collected, the mass that is having
constantly affordability problems can be determined (according
to Moore’s absolute poverty approach [Moore, 2012]). However, the fluctuating poverty section suffering from this
periodically may cause an erroneous assessment. Having these
three main problems, we need to develop a certain systematic
methodology to set the people to the EP cluster. l
g
p
c. Failure in the reliability of the surveys or questionnaires. In
case the data cannot be obtained, some methods like surveys or
questionnaires come into play. When the subject is a sensitive
and psychological word such as “poverty,” the real needy hide
the situation, and some parts of the non-needy part worsen it. The reliability of a questionnaire becomes unclear because it
is declaration-based. It is not possible to observe the problem
or produce a policy dependent on the declaration-based data. When such information is collected, the mass that is having
constantly affordability problems can be determined (according
to Moore’s absolute poverty approach [Moore, 2012]). However, the fluctuating poverty section suffering from this
periodically may cause an erroneous assessment. Having these
three main problems, we need to develop a certain systematic
methodology to set the people to the EP cluster. While there is a description of spending over 10% of household
income on energy expenses in the literature, according to
Bouzarowski, one of the leading names in the field, it will be more
fitting to define indicators that would represent the real situation
in Eastern Block countries rather than this difficult to identify
situation (Bouzarovski, 2015). According to Bouzarovski, the
energy-poor people spend more than usual on energy because of
poor equipment (Bouzarovski, 2012). This situation is like the
older model vehicles consuming more fuel. 1. INTRODUCTION certainness to be gained in identifying the Energy Poor will
encourage preventative steps to be taken. Support structures can
be established, for this reason, energy efficiency projects can be
carried out, and public and private sector social projects in this
field can be created. Failure to fully identify those who are in
need leads to an attempt to address the problem through general
social arrangements and assistance. The solution process is limited
because the problematic cluster cannot be established. According
to Boardman, who is associated with the EP concept, this section Nowadays, there is a lot of research to ensure equitable access
to energy, which is essential for life. It would be appropriate
to consider the concept of Energy Poverty (EP) as an activity
within these studies. “Energy poverty exists when a household
lacks sufficient energy level and quality (Thomson, 2017),
(Erdoğdu, 2020) services in the home,” according to the admitted
description of EP (EU Energy Poverty Observatory, 2020). The International Journal of Energy Economics and Policy | Vol 12 • Issue 3 • 2022
401
This Journal is licensed under a Creative Commons Attribution 4.0 International License Emre and Sozen: Energy Poverty Clustering by Using Power-cut Job Order Data of the Electricity Distribution Companie in years of work, is in Boardman’s book “Fuel Poverty, From Cold
Homes to Affordable Warmth” published in 1991 (Emre, 2020),
(Boardman, Fuel Poverty: From Cold Homes to Affordable Warmth,
1991), (Liddell et al., 2012). Bradshaw said in 2008 that 71% of EP
was already poor (Emre, 2020), (Mahoney, 2020), (Bradshaw, 2008). This approach puts forward the idea that EP can be reached when the
poor are detected. UK Department of Energy and Climate Change
issued another document finding and using “the 10%” in 2010 (UK
Department of Energy and Climate Change, 2010), (Emre, 2020). Although later, some specialists like Boardman and Dubois said that
this study was a work that produced numbers to be nice, Hills said
10% criteria puts more population into the EP basket than reality
and suggested additional criteria (Emre, 2020), (Boardman, Fuel
poverty synthesis: Lessons learnt, actions needed, 2012), (Dubois,
2012), (Hills, 2012). Fahmy drew attention to his research about
how many EP is in the category that meets the 10% criterion when
pointing to Broadshaw’s report, stating that 7% of consumers met
the requirement (Fahmy, 2011), (Emre, 2020). 1. INTRODUCTION spends more than 10% of the household’s total income on energy
(Boardman, Fuel Poverty: From Cold Homes to Affordable
Warmth, 1991). The household’s income cannot be accurately
determined. The difficulties here stem from conditions as well
as the need to register all members of the household, assessing
the household’s properties and income, and the presence of an
unregistered, tax-free portion among them (Tax reports are usually
the primary source). The accuracy of the collected data is often a
real issue at this point. The current challenges can be tackled in
the following ways: a. Failure in data collection regarding the protection of
personal data. In some country practices like Italy, as
both pilot and the local studies, municipalities set up their
informatics infrastructures by signing protocols for sharing
this information with a common secure database for social
assistance evaluation together this information and evaluate
them (Emre, 2018). Although municipalities are the executives
of almost all infrastructure issues, they keep such data as
customers, addresses, contact details in separate databases,
and combine them only with protocols; they have also
difficulties with this operation In today’s circumstances, according to Boardman, real energy
expenditure has risen uncontrollably, and even this threshold
is difficult to meet (Boardman, Fuel poverty synthesis: Lessons
learnt, actions needed, 2012). According to Boardman, the
lowest-income 30% of households in England invested 70% of
the “required” normal energy expenditure in 2009 (Emre, 2020),
(Boardman, Fuel poverty synthesis: Lessons learnt, actions needed,
2012). In the following studies, we can see that the guidelines
were looked at in order to assess the EP cluster and that similar
programs and pilot applications were completed. Costa-Campi
et al. confirmed energy poverty can be labelled through income
level of the households (Costa-Campi, 2019). According to Li,
Energy Development Index (EDI) and Multidimensional Energy
Poverty Index (MEPI) were defined between 2002 and 2010 (Li,
2014). As a different approach, Dagoumas emphasized unpaid
invoices in its 2014 article (Dagoumas, 2014). It is an important
reminder that the main criterion should be the arrears on utility
bills. Moore, for the 1st time, expressed the definition of absolute
and relative poverty, which made him one of the most important
names in the subject (Moore, 2012). b. Failure in synchronized data and matching, even if all of the
data is combined into a single channel, matching it is a huge
challenge since the consumer in the household is not set. 1. INTRODUCTION Such finding was
confirmed by the afford of Emeç et al. of activities for the Turkish
case (Emec, 2015). This study aims to contribute to the current literature by exploring
the relationship between energy poverty and arrears on utility
bills. Since the data availability problems limit all mathematical
analysis to investigate and define the core problem, our study
depending on the real commercial outputs of the utilities will
reflect a clear approach. 2. RELATED LITERATURE stated accordingly the 2017
household budget survey data sets and their own survey results
EP clusters cover ¼ of the total households, which means “Half
of the lowest-income households face energy poverty.” It has been
said that this rate was 36% of households in 2003 and decreased
to 23% in 2017 (Selçuk, 2019). PwC Report on EP stated each
level of consumption has a unique EP threshold inside according
to analysis through consumption data through 3 million consumers
(Emre, 2018). •
Expenditure – where examinations of the energy costs faced
by households against absolute or relative thresholds provide a
proxy for estimating the extent of domestic energy deprivation •
Expenditure – where examinations of the energy costs faced
by households against absolute or relative thresholds provide a
proxy for estimating the extent of domestic energy deprivation •
Consensual approach – based on self-reported assessments
of indoor housing conditions, and the ability to attain certain
necessities relative to the society in which a household resides y
•
Direct measurement – where the level of energy services (such
as heating) achieved in the home is compared to a cluster
standard (Thema, 2020). According to them, researchers are mostly dependent on consensus
evidence about the effects of energy poverty, such as power bill
arrears and dampness in the household, and data accuracy must
increase in order to progress the calculation of energy poverty
on a European scale. This could involve amending existing
variables so that they are more useful indicators, for instance
changing from a binary response format to a Likert type scale
to detect the frequency of the problems (Thomson, 2017). In a
2017 article, Lenz and Grgurev claimed that people in certain EU
countries, especially Bulgaria, Croatia, and Romania, were unable
to adequately heat their homes. There are households in the EP
cluster with a rate of 45% in Bulgaria, 14% in Romania, and 10% in
Croatia (Lenz, 2017: 7(2)). Bouzarovski, in his book published in
2018 (the book was written by Bouzarovski, Thomson, and Petrova
and edited by Simcock), the subject was associated with energy
prices, low household productivity conditions, and low income
(Bouzarovski, 2018). With a more comprehensive approach, it is
impossible to deny that the problem has socioeconomic, regional,
and political dimensions (Emre, 2020). 2. RELATED LITERATURE “Those who have an
income below the threshold and consume above-average energy”
(NEPIM, Wafzig, M., Zimper, J., 2018) are described as “energy
poor” in Austria. Seebauer et al. studied on social housing policy
to alleviate energy policy in Austria (Sebastian Seebauer, 2019). Lakatos and Arsenopoulas studied a swot analysis for potential
financial supporting programmes for the energy poverty population
(Lakatos, 2019). According to this study, 10%, etc. Such static
methods can cause problems to be overlooked on a regular basis
(Emre, 2020). In 2017, the Turkish government launched a new version of an
existing social support system based on previous examples and
best practices (Republic of Turkey Ministry of Family and Social
Policy, 2017). In 2019, Presidential Declaration derived support
for about 2 million household electricity expenditure (Republic
of Turkey Ministry of Family, Labour and Social Services, 2019). Erdoğdu concluded that this figure is below the calculated number
of poor households in his calculation based on 2018 TUIK data
(Erdoğdu, 2020). Parliamentary Records state that electricity
utilities issued 330 million electricity bills to households in 2019. The 1.26% of this number was subject to power cut because of
arrears. Utility bill arrears about 2 billion and 94 million TL (Grand
National Assembly of Turkey, 2020). According to the Turkish
Minister of Energy and Natural Resources, 3.7 million consumers
(out of 45 million) have had their power turned off due to utility
bill arrears. The results for 2020 can represent a fluctuating EP
population as well as an absolute EP population with over-use1
(Turkish Newspaper Sözcü, 2021). Utility bill arrears are once again being highlighted by current
sources. EPOV Member State Reports on Energy Poverty
underlined the correlation between arrears on utility bills
and affordability (EU Energy Poverty Observatory, 2020). Regarding this report, it can be underlined that inability to keep
the home adequately warm and arrears in utility bills trends are
highly correlated. In this way, a simplification suggestion can
be developed to focus on arrears on utility bills to catch the
affordability cluster. EPOV uses 28 different standards to determine EP (Thomson,
2017). The number of criteria can be increased with today’s
technology, but their meaning should be questioned. The
synchronicity of these data is a separate issue for indices like
the European Union Statistics on Income and Living Conditions
(SILC) and the European Energy Poverty Index (EEPI). 2. RELATED LITERATURE The first attempts to establish EP also attempted to set the criteria
for EP detection. This threshold value for energy expenditure
is approximately 12 percent of household income, according to
Isherwood and Hancock, who are the first names we come across
in the literature (Emre, 2020), (Isherwood, 1979), (Liddell et al.,
2012). The 10% figure, which will unfailingly reach the same result The EU Energy Poverty Observatory (EPOV), which will operate
under the leadership of Thomson and Bouzarowski in 2016, has
officially been put into operation. Not only does this center have
access to data on EP metrics, but it also shares recent literature
(Emre, 2020). Thomson et al. (Thomson, 2017) identified three
main methodologies of measurement: 402 International Journal of Energy Economics and Policy | Vol 12 • Issue 3 • 2022 Emre and Sozen: Energy Poverty Clustering by Using Power-cut Job Order Data of the Electricity Distribution Companie 2013), (Eke, 2018). The World Bank’s 2015 report shows that
energy poverty can exist in any consumer category. The percentage
share of household electricity expenditures was divided into
approximate categories for each consumption category in this
study, and consumers were grouped according to monthly
electricity consumption amounts. In this study, it was stated that
“electricity expenditure in the lowest income group, which is the
lowest income group for 100 kWh per month, which is accepted
as the lowest consumption level, is 11% of the total household
income. For the 150 kWh consumption group, this ratio is over
15% and some note that these values were 12% and 18% in the
previous years” (World Bank, 2015). The proposal to measure the
threshold in terms of kWh electricity consumption was included
in Tennakoon’s report in 2008. In this study, the energy poverty
threshold was found to be 120 kWh per month (Tennakoon,
2008), (Eke, 2018). Eke and Ayrancı devised an approach based
on the provinces’ average monthly consumption (Eke, 2018). The
threshold for energy poverty was set at 100 kWh in this method,
and six provinces in the east and southeast Anatolia were listed
as being energy poor. Selçuk et al. 1
Turkish social support covers electricity invoices according to a cap since
2019 March. This 2020 results can reflect the fluctuated EP population and
the absolute EP population with over-use. 2. RELATED LITERATURE The
collection time of many evaluation criteria used from the In Turkish literature, Bağdadioğlu analyzed the Household Budget
Survey data in his report (Bağdadioğlu, 2009). He proposed a 10%
investment criterion for electricity and gas, a 3–5% criterion for
water, and a 25% criterion for overall energy poverty, with no
electricity poverty value calculated outside of the Southeastern
Anatolia Region (Erdoğdu, 2020). Kaygusuz stated that rural
areas are the more vulnerable comparing economical conditions
(Kaygusuz, 2010). Ozcan et al. offered a poverty cluster varying
according to monthly income (Moore’s perspective) (Özcan, 403 Emre and Sozen: Energy Poverty Clustering by Using Power-cut Job Order Data of the Electricity Distribution Companie in the World. The boards of trustees founded in the provinces
create the poor family list in the state registries, which is then
created by local executive councils after on-site determinations
(Republic of Turkey Ministry of Family and Social Policy,
2017). The problem is all the personal information is secure even
though the information allows bodies to support the families in
possible needs. This means it is not possible to access data such as
identity information, wage information, health information, asset
information (title deed, rental income), expenditure information,
debt information, credit information, bank records, which can
be collected separately, within the same date range. Even if this
data of all household members can be accessed, unregistered data
are quite high. Non-public companies (distribution companies –
DisCo) do not disclose certain types of information, especially
trade secrets. Consumer payment habits, in their opinion, are an
indication of a company’s valuation process, which is very safe for
an individual. We need to simplify the methodology and generate
some policy-making activities in order to make the energy poverty
cluster simpler, more basic, and comparable. The EPOV study
emphasizes this condition, stating that debt bills are a significant
source (EU Energy Poverty Observatory, 2020). relevant institutions and organizations is different and causes
inconsistencies. Thomson and Herrero stated that many data subject to be collected
are not systematically evaluated. Moreover, “composite indices
are hard to institutionalize both due to questions around how
to assign weight-age and because the European Union (EU)
policy-making environment prefers simplification of metrics”
(Sareen, 2020), (Espeland, 1998), (Sebastian and Bauler, 2013). 2.1. Poverty - Energy Poverty Relation 2.1. Poverty - Energy Poverty Relation
In literature, there are observations about the association between
the general state of poverty and energy expenditure affordability,
and these clusters are not the same. Poverty describes a lower
socio-economic situation than energy poverty in the context. “Energy Poverty” might represent absolute or periodic inability
for each consumer. In EP circumstances, households reduce their
consumption for making economy, and this may affect their health
since the living room temperature reduces in the cold winter days
and nights. It should be understood that this state is long-lasting in
cases of absolute poverty and absolute energy deprivation and that
this situation periodically exits in the fluctuating state. Aside from
the Law on protecting personal data, poor people do not want to be
grouped psychologically. As a result, there are difficulties in reaching
the list of the absolute poor. Since household income and expense
statistics cannot be accessed simply, easily, or specifically due to
secrecy concerns, and in order to avoid violations, absolute poverty
reports are typically created by field visits and information gathering. The most obvious of these shreds of evidence are the debt bills. Official statistical institutes prepare the socio-economical index of
the regions. These indexes are useful for expressing poverty and
EP for methodological purposes (EU-SILC is one of them (Sareen,
2020). In the Turkish case, the Turkish Ministry of Industry and
Technology has released a study report with the name “2017 in
the Provinces and Regions of the Socio-Economic Development
Ranking Study-SEDI” (Regional Indicators of socioeconomic
well-being) (Acar, 2019). The Strong Principle Components
Analysis approach was used to perform detailed analyses on 52
variables in the social, housing, education, health, competitive
and creative capability, financial, accessibility, and quality of life
clusters, and the provinces’ growth indices were presented. This
report is used in related studies as the most up-to-date and scientific
source. According to this report, the development index of the
provinces is between 4.051 points and –1.788 points. 2. RELATED LITERATURE Mahoney (2020) stated that for EP level adjustment, which is
tried to be determined over many indices, data collection is almost
impossible and the accuracy of the collected data is questionable
(Mahoney, 2020). The most basic question is “what is the number
of households?” Even the answer to the question is based on the
figures or approaches of the official statistical institutions of the
countries and is assumed correct. Kose (2019) offered that poverty-health relationship id mostly
related with the individuals economical conditions rather than
regional conditions. The situation that has to be noticed above and above both of these
issues is “utility bill arrears,” which is the official indication of
inability. The direct use of this data is a more simple and precise
method of calculating the EP since it is based on energy demand,
which is considered one of the most important human needs
(Himachal Pradesh High Court, 2018). Confirming the “problem of developed countries” approach
(Bouzarovski, 2018) for EP, other problems may arise in
distribution regions, which have heterogeneous socio-economic
structures. In these regions, illegal electricity consumption (non-
technical loss & theft) is a problem. Psychologically, the inability
afford to the energy used can turn into abuse and, the abuse can
turn into habits and disrupt payment habits. This is how the proper
payment habit turns into illegal electricity usage. The customer
who cannot pay the electricity bill at the 1st time will make
economy from consumption for a while, then accept that he cannot
pay and switch to normal use, if he encounters an opportunity,
he will start illegal use and eventually will come to the point of
making illegal consumption more than he needs and wasting. This
situation is a serious obstacle to determining the real need level. While no official federal agency can say how many households
exist in a region, the number of customers and invoices produced
by all infrastructure firms, whether public or private, is certainly
on a monthly basis. In case it is not used for heating, the invoice
amount for electricity consumption does not make up the largest
percentage of household expenses. Therefore, a debt for electricity
and a power cut from debt is a sign of absolute poverty. 2.1. Poverty - Energy Poverty Relation International Journal of Energy Economics and Policy | Vol 12 • Issue 3 • 2022 3.1. Data Available for this Research Study In this study, we observed the trends of the arrears on utility bills
through the 7-year power-cut data of 8 DisCo’s’ which includes
payment characteristics of 33 provinces. Since this information
is very secure, study was conducted by closing all data including
the name of the cities and the companies. Figure 3 shows the DisCo-2 Pareto graph. The kWh consumptions
the region which is subject to power-cut job orders are examined. ol 12 • Issue 3 • 2022
405
-0.002
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0
100
200
300
400
500
normal distribution
# of days for payment
#152k power-cut work order
cancelled in 390 days
σ = 25 days
Figure 2: DisCo-2 payment period for the debt
10,938
23,050
9,558
14,504
64,160
17,422
31,611
8,090
24,634
42,933
-
20,000
40,000
60,000
80,000
100,000
120,000
City-49
City-53
City-64
City-36
City-26
# of notification
cancel
executed
Figure 1: DisCo-2 power-cut execution 10,938
23,050
9,558
14,504
64,160
17,422
31,611
8,090
24,634
42,933
-
20,000
40,000
60,000
80,000
100,000
120,000
City-49
City-53
City-64
City-36
City-26
# of notification
cancel
executed
Figure 1: DisCo-2 power-cut execution 3.3. Power-Cut Job Order 3.3. Power-Cut Job Order It is a job order created for customers whose deposits have not been
made after notice of a power-cut. This job order may also be canceled
without completing a transaction, such as a notification, although the
cancellations in this section are mainly due to reimbursement and
are not contingent on mistakes that could exist in external services. 3. DATA AND METHODOLOGY As mentioned in the previously, the main obstacle of the
identification is data privacy. Almost all of the information
registered as indicator of energy poverty is quite secure
information stored or created by different entities. Even for the
Governmental companies there is no exception for sharing data. In 2016, Italian Regulatory Body Commissioner informed us that
Italy has prepared a model for common file sharing securely in
the same municipality. In this section details of the data available
and the approach is explained in details. In a deeper examination, the normal distribution for the payment
cycle is another important factor to consider when determining the
degree of inability. Figure 2 shows that the average payout cycle is
about 25 days. On the other hand, in the DisCo-2 area, the number
of cancelled power-cuts is double, which can be discussed at the
inability level as well. In another perspective, although there can be a level in each
consumption group (which should be searched deeply), generally
people consume what they need and as much as they can in
inability case. 2.2. Definition of Electricity Arrears on Utility Bills
and “debt” Data The term “debt” for electricity represents the receivable whose
consumption is measured by a registered device, the invoice of
which is made but not paid. The meter of an energy customer is Turkey, with its comprehensive social assistance program, must
be one of the best examples of poverty/energy poverty support International Journal of Energy Economics and Policy | Vol 12 • Issue 3 • 2022 Emre and Sozen: Energy Poverty Clustering by Using Power-cut Job Order Data of the Electricity Distribution Companie that the cancellation was due to a lack of funds. Because of the
problems in the automatic payment systems of the bank, problems in
the communication channels, etc., the customers who make regular
payments and do not have any payment difficulties may receive
power-cut notifications. The cancellation and implementation of the
power-cut, on the other hand, sends a message about affordability
levels. Almost half of the power-cut orders are subject to cancellation,
which may include total and fluctuating EP. read once a month, and an invoice is given. The invoice released
has a 10-day due date. At the due date, the “receivable,” which is
the equivalent of consumption, becomes a “debt.” After this date,
a power-cut notice order is issued for the unpaid debt. According
to the Electricity Distribution Company (DisCo) application, the
notification can be sent using SMS, e-mail, etc. or the hard copy
can be delivered to the address. If the debt has not been paid
10 days after the notification job order is issued, a power-cut job
order is generated. According to this job order, when paying a loan,
an individual whose power has been turned off will be charged an
extra cut/open fee. Even then, if he does not pay his debt, execution
proceedings are started. According to the table; For a general assumption, we can divide the numbers as: 500
thousand electricity bills are generated per month for DisCo-2, 70
thousand notification for power-cut, 10 thousand power-cut orders. C1 investigates the correlation between the development index
with the # of power-cut job orders (issued). There is a reverse
correlation discovered between these variables. However, it is
strong in some specific DisCos. In the region that can be considered being homogeneous and not
behind in the development index; In the region that can be considered being homogeneous and not
behind in the development index; •
10/500 ~ 2% power-cut job order (corresponding to absolute
energy poverty) C2 investigates the correlation between the development index
with the # of power-cut job orders (DONE). There is a reverse
correlation between these variables. •
70/500 ~ 14% notice (energy poverty problem cluster) are
produced. C3 is investigating the correlation of the development index
with the arrears on utility bills subject to power-cut. There is a
reverse correlation discovered between these variables. However,
likewise the C1 and C2, it is strongly correlated in some DisCo’s’. Potentially this variable offers if city is more developed, there
are less arrears on utility bills. However, it is not strong enough
to offer there is much more affordability problem in such cities. These figures, previously with studies related to Turkey
(Bağdadioğlu, 2009), Appendix A (World Bank, 2015), (Emec,
2015) Emeç World Bank [21]) are compatible figures. In light of all of these metrics, we came to the conclusion that
electricity debt (as a result of power-cut job orders) can be used
to diagnose energy poverty. A holistic replication of this research
performed across individual companies is needed. In further
analysis, we investigated a relationship between the development
index, # of population, # of consumer, # of power-cut, # of power-
cut (DONE), power-cut performance (PCP), arrears to utility bills
subject to power-cut, non-technical power-loss (NTL), and loss
per consumer (LPC). While the TR-2018 line reflects an analysis
based on media information, the provinces line covers all provinces
participating in the analysis, and the region’s lines only cover the
provinces under their jurisdiction. The data sample represents
approximately 10 million DisCo customers from 33 Turkish cities. C4 examines the relationship of the development index with LPC. This test offers how developed city; that less NTL related with the
loss & theft. 3.2. Tools and Methodology This study is intended to assess the population in need of resources,
with just the state of electricity debt and the details on the power-
cut job order. Other data that can be compared for this purpose are
the numbers calculated according to the literature and the figures of
the people receiving social assistance. SPSS was used to perform
statistical analysis with big data such as electricity consumption
and bills, normal distribution, correlation, tests were performed. In this study, analyzes were carried out for customers who could
not pay their bills and were subject to power cuts in the DisCo-2
region (City-26, City-36, City-53, City-64, City-49) and DisCo-4
region (City-42, City-44, City-59, City-71). The study was carried
out by scanning a 7-years data set as 2013,14,15,16,17,18,19 and
reflects the payment habits after the privatization period. In this
analysis, only residential customers were considered. Periodic
tariff values for kWh conversions are determined using an average
estimate since the data is on a TL basis. -0.002
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0
100
200
300
400
500
normal distribution
# of days for payment
#152k power-cut work order
cancelled in 390 days
σ = 25 days
Figure 2: DisCo-2 payment period for the debt Figure 2: DisCo-2 payment period for the debt Many structural characteristics of the region, such as economic,
demographic, and cultural factors, as well as DisCo practices, can
influence the numbers related to power-cut notifications, job orders,
and execution procedures. Each region’s invoice rate is different from
one another. For example, in the DisCo-2 area in 2018, the number of
things canceled after a power-cut was about 50% (Figure 1). It would
be incorrect to interpret this data based solely on the presumption 405 International Journal of Energy Economics and Policy | Vol 12 • Issue 3 • 2022 Emre and Sozen: Energy Poverty Clustering by Using Power-cut Job Order Data of the Electricity Distribution Companie Most people who are “in debt” are those who use 100–200 kWh
per day. On the other hand, each consumption category, as might
be predicted, has some affordability issues due to the fragility of
all consumption. Most people who are “in debt” are those who use 100–200 kWh
per day. On the other hand, each consumption category, as might
be predicted, has some affordability issues due to the fragility of
all consumption. 3.2. Tools and Methodology results between the cities having negative development index. By
these four calculations, we try to understand the strong drivers of
the correlation also in different clusters. According to the table; Expecting a negative correlation, there is a reverse
correlation in negatife for the non-Homogenous DisCo’s which
is not that high. On the other hands, some DisCo’s have more
negative correlation scores in this manner. C5 means the relationship between the development index and the
consumer density (number of customers per population). Since
only one registration for each consumption point, we expect a
certain value due to the average number of households in the
countries. However, according to the climate, households have
more than one provinces to live in different seasons. Although
homogenous samples are not correlated, we observe a weak
correlation in total together with some strong correlation results
of the cities’. 4. RESULTS An average of 53 thousand people create a debt of
approximately 8.5 million, and there are energy-poor people in this
section. It turns out that 63% of the debt is carried out by the 18%
installation. In this section, we understand that there are cases of
abuse. In contrast to many installations with less debt, there are
a few installations with high debt. It showed the ratios in terms
of number and amount of debt in the graph in the stratification of
the sections below and above the average to 2 times, 3 times, and
4 times. While the overall volume of debt is small, the percentage
of borrowers consuming less than half of the average valuation is
significantly high. This is a price-effective indicator. The results of
the analysis are unique in terms of their socio-economic, cultural,
and psychological characteristics, but also in accordance with
Disco 4. The NTLs are also different between these two regions. The assessment of the APAPI has the potential to present the
solvency problem more clearly according to these results. C7 symbolizes a more fundamental analysis of the previous
indicator. The correlation of energy lost, which is the difference
between purchased energy and invoiced energy by the utility, and
development index has been found to be positively correlated
weakly. The amount of energy lost increases as the degree of
growth rises. C8 and C10 searches a correlation between # of power-cut job
orders and the # of consumer which is highly correlated for all
provinces. C9 offers a relationship between # of power-cut and LPC. It is not
related even for the non-homogenous cities. The outrageous amount of debt in developed regions shows that
the power-cut order can be used in detecting energy poverty in
developed regions. Another question is whether the affordability
level is stable. In the analysis made for this, the consumption
quantities subject to the power-cut order were divided into groups
and analyzed the changes. The number of consumers facing power-cuts due to debt is not
as high as predicted. The amount of debt varies according to
the consumption behavior of the household, which means the
lifestyle. In 2018 figures, 500 consumers made up 95% of the
total debt among two hundred thousand consumption points
(DisCo-2). The Average Power-cut Amount Per Installation
(APAPI) was calculated over the years (Figure 4). 4. RESULTS Table 1 represents the correlation test results for the variables. In the first raw we search the general statistical data with the all
sample city data. On “Samples” row, we searched the correlation
among all 33 cities. “H-Samples” raw represents us the correlation
results of the cities which have positive development index which
is the ideal case. “nH-Samples” raw represents the correlation test C6 offers a general relationship between the development index
and # of people in the cities. It’s a common proportion. In C6 offers a general relationship between the development index
and # of people in the cities. It’s a common proportion. In Table 1: Correlation analysis results
Correlation Analysis Results
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
A/H
A/B
A/C
A/D
A/E
A/K
A/F
B/G
B/D
H/G
TR‑2018
−0.6991
−0.5826
−0.6991
−0.5677
0.7170
0.3312
0.2662
0.9995
−0.0523
0.9985
Samples
−0.6972
−0.5229
−0.6798
−0.5804
0.7665
0.6298
−0.0686
0.8396
−0.0018
0.9270
H‑Samples
−0.7158
−0.6877
−0.7243
0.3653
0.1631
0.6186
0.6471
0.8566
0.0640
0.9420
nH‑Samples
0.2203
0.1469
0.1909
−0.6900
0.8601
0.3685
−0.5080
0.7877
0.2612
0.7211
DisCo‑2
−0.8228
−0.9084
−0.8163
−0.0884
0.6848
0.7828
0.6973
0.9918
−0.0885
0.9918
DisCo‑4
−0.8740
−0.6147
−0.8288
−0.4118
0.5303
0.6650
0.5234
0.9785
0.3119
0.9795
DisCo‑8
0.0839
0.0512
0.3641
−0.7530
0.8533
0.2058
−0.6376
0.9420
0.2770
0.9420
DisCo‑14
−0.7489
−0.7884
−0.4078
0.1140
0.0096
0.3047
0.3771
0.9894
−0.1977
0.9894
DisCo‑18
DisCo‑1
−0.8816
−0.7473
0.6110
−0.9043
−0.2605
0.9890
0.9865
0.8175
−0.9487
0.8175
DisCo‑13
−0.5929
−0.5922
−0.8738
0.0530
−0.8572
0.6692
0.7621
0.8998
−0.1258
0.9532
DisCo‑20
0.3570
0.3669
−0.2408
−0.2956
0.5132
0.4053
0.2500
0.7599
−0.8103
0.8654 Table 1: Correlation analysis results 406 Emre and Sozen: Energy Poverty Clustering by Using Power-cut Job Order Data of the Electricity Distribution Companie points to the consumer, which has broken the payment habit and
has taken an attitude towards wasting. industrialized provinces, we can assume that the population is
larger. There is a weak correlation. In the analysis, we examined whether the total amount of debt
and the number of consumers represent significant clusters of the
half, a third, a fourth, 2 times, 3 times, and 4 times the APAPI
value. Figure 4 shows the outputs for the DisCo-2 and DisCo-4
regions. As per our analysis, we see that the <APAPI section
represents a group of 45% in number and 37% in the amount
over the years. 4. RESULTS According to
the analysis, evaluation should be over an average APAPI value
in order to minimize the socio-economic, political, cultural, and
psychological effects. This value is a virtual threshold. The result for two different regions gave a similar result almost
superimposed (Figure 5). At the point of absolute energy poverty
visible in blue, there are dwellings that cannot pay their bills
despite low consumption. This case is not greatly influenced by ol 12 • Issue 3 • 2022
407
160
170
201
226
241
260
308
267
201
156
171
235
291
271
-
50
100
150
200
250
300
350
2013
2014
2015
2016
2017
2018
2019
APAPI (TL)
APAPI DisCo-2
APAPI DisCo-4
Figure 4: APAPI amounts consumption pareto of power-cuts in
DisCo-2
0
10000
20000
30000
40000
50000
60000
2013
2014
2015
2016
2017
2018
2019
# of home subject to power-cut
<APAPI/4
<APAPI/3
<APAPI/2
Figure 5: APAPI cluster trends in DisCo-2 160
170
201
226
241
260
308
267
201
156
171
235
291
271
-
50
100
150
200
250
300
350
2013
2014
2015
2016
2017
2018
2019
APAPI (TL)
APAPI DisCo-2
APAPI DisCo-4
Figure 4: APAPI amounts consumption pareto of power-cuts in
DisCo-2
Figure 5: APAPI cluster trends in DisCo-2 Figure 4: APAPI amounts consumption pareto of power-cuts in
DisCo-2 In Figure 4, APAPI is increasing continuously in DisCo-2. However, APAPI shows greater variation over the years in
DisCo-4. Because the development index of DisCo-4 is smaller
than DisCo-2, We arrive at the conclusion that the amount of debt
held by each borrower can be a useful measure of affordability. The
decrease in average debt indicates that the number of consumers
who are under indebted has risen. The increase will not show the
opposite of this discourse, however; this may show that the amount
of debt has increased too much, that the amount of repeated or
one-off debt has increased (increase in fraud). From this point of
view, APAPI should be accepted as a threshold value showing
those whose payment habit is disrupted. The part above this debt International Journal of Energy Economics and Policy | Vol 12 • Issue 3 • 2022
407
of debt has increased too much, that the amount of repeated or
one-off debt has increased (increase in fraud). From this point of
view, APAPI should be accepted as a threshold value showing
those whose payment habit is disrupted. 4. RESULTS The part above this debt
2013
2014
2015
2016
2017
2018
2019
Figure 3: Consumption pareto of power-cuts in DisCo-2
0
10000
20000
30000
40000
50000
60000
2013
2014
2015
2016
2017
2018
2019
# of home subject to power-cut
<APAPI/4
<APAPI/3
<APAPI/2
Figure 5: APAPI cluster trends in DisCo-2 Figure 5: APAPI cluster trends in DisCo-2 0
10000
20000
30000
40000
50000
60000
2013
2014
2015
2016
2017
2018
2019
# of home subject to power-cut
<APAPI/4
<APAPI/3
<APAPI/2
Figure 5: APAPI cluster trends in DisCo 2 Figure 3: Consumption pareto of power-cuts in DisCo-2 Figure 3: Consumption pareto of power-cuts in DisCo-2 Emre and Sozen: Energy Poverty Clustering by Using Power-cut Job Order Data of the Electricity Distribution Companie the circumstances. The red and green lines represent the fluctuating
value of energy poverty at different levels. Accordingly, to the
Figure 5, we understand that while the number of consumers in
the APAPI/3 cluster remained very constant, it in APAPI/2 and
APAPI/3 cluster increased over the years. Here, APAPI/3 shows
the absolute affordability level, while APAPI/2 level is the variable
affordability level. According to correlation-test results, cities with less development
indexes have more arrears on utility bills in number and amount. Less developed characteristics are in a weak correlation with
NTL (loss & theft). Another interesting outcome is the relation
between the development index and the number of the customer. Separating the consumption levels of arrears, it is found that 63%
of total non-payment is depending on 18% of consumers. Trend
analysis confirmed that every energy consumption level has the
absolute and fluctuated component inside. All these numbers
are in parallel with the coherence with literature and national/
international approaches. According to the findings in Figure 5, we might build a hypothesis
that the ratio of debt at each stage to the number of borrowers
affected by a power-cut is a constant that does not vary from region
to region. According to the results, the APAPI/2 consumption
level corresponds to a monthly consumption of 110 kWh,
while the APAPI/3 consumption level corresponds to a monthly
consumption of 80 kWh. In order to develop a social tariff, to make applications related
to energy efficiency, and to plan strategies, it is necessary to
identify the consumer suffering from energy poverty. For this
determination, special data such as household income, expenditure
items must be provided in a suitable period for analysis. REFERENCES Acar, S. (2019), İllerin ve Bölgelerin Sosyo-ekonomik Gelişmişlik
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the most serious criterion that reveals the amount of debt, payment
patterns, and willingness to afford is energy use. The study found
that an analysis based on power-cut job order records can reveal
the EP cluster without the need for additional data. The certainty
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Immediate loading over lower prosthesis in edentulous mandibles: comparison between two types of prosthetic connection
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RGO - Revista Gaúcha de Odontologia
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1 Universidade Estadual Paulista Júlio de Mesquita Filho, Faculdade de Odontologia de Araraquara. Rua Humaitá, 1680, Centro, 14801-903, Araraqua
ra, SP, Brasil. Correspondência para / Correspondence to: MR OLIVEIRA. Email: <arinareis89@hotmail.com>.
2 Aarhus University, Section of Oral Radiology. Aarhus, Denmark. http://dx.doi.org/10.1590/1981-863720170002000043369 http://dx.doi.org/10.1590/1981-863720170002000043369 Conclusão Conclusão
A carga imediata sobre protocolos inferiores é uma opção viável para o tratamento de mandíbulas edêntulas e a conexão hexágono externo
ou cone Morse não interferiram no sucesso dos implantes em um período curto de avaliação. carga imediata sobre protocolos inferiores é uma opção viável para o tratamento de mandíbulas edêntulas e a
cone Morse não interferiram no sucesso dos implantes em um período curto de avaliação. Termos de indexação: Implantação dentária. Prótese dentária. Arcada edêntula. Termos de indexação: Implantação dentária. Prótese dentária. Arcada edêntula. Conclusion Conclusion
Immediate loading of the lower prosthesis is a viable option for the treatment of edentulous mandibles and that the external hexagon
or Morse cone connections do not interfere with the success of the implants in a short-term evaluation. ower prosthesis is a viable option for the treatment of edentulous mandibles and that the external hexago
do not interfere with the success of the implants in a short-term evaluation. Indexing terms: Dental implantation. Dental prosthesis. Jaw edentulous. Results
l Results
Implants with Morse cone connections showed smaller, statistically significant probing depth values for all periods (0.68/1.19/1.31), when
compared to the external hexagon connections (1.08/1.52/1.64). A statistically significant difference was observed between baseline, 3 months
(p<0.01 for Morse cone; p<0.001 for external hexagon) and 6 months (p<0.001 for both connections). When periods were considered there
was a statistically significant difference in Implant Stability Quotient ISQ values between baseline and 6 months for both prosthetic connections. Immediate loading over lower prosthesis in edentulous mandibles:
comparison between two types of prosthetic connection Carga imediata sobre protocolos inferiores: comparação entre dois tipos de conexões protéticas Marina Reis OLIVEIRA1
Ariane de Souza OLIVEIRA1
Vitor Augusto LEITE1
Marisa Aparecida Cabrini GABRIELLI1
Oriana Elara Barelli PAGANELLI1
Rubens SPIN-NETO2
Valfrido Antonio PEREIRA-FILHO1 Methods Methods
Seventy-two implants placed in 18 patients were analyzed. The parameters evaluated included probing depth, stability of implants and
perimplant bone loss, which were measured immediately when installing the prosthesis and after 3 and 6 months. All data underwent
statistical analysis (T-Test and ANOVA, 5% significance level). Métodos
S Métodos
Setenta e dois implantes instalados em 18 pacientes foram analisados. Os parâmetros avaliados incluíram: profundidade de sondagem,
estabilidade dos implantes e perda óssea periimplantar, os quais foram mensurados imediatamente após a instalação das próteses e depois
de 3 e 6 meses pós-operatórios. Todos os dados foram submetidos à análise estatística (Teste T e ANOVA, ao nível de 5% de significância). l
d Resultados Resultados
Implantes com conexão cone Morse mostraram profundidade de sondagem estatisticamente menor em todos os períodos (0,68/1,19/1,31),
quando comparado com a conexão hexágono externo (1,08/1,52/1,64). Diferença estatisticamente significante foi observada entre o pré-
operatório imediato, 3 meses (p<0,01 para cone Morse; p<0,001 para hexágono externo) e 6 meses (p<0,001 para ambas as conexões). Quando os períodos foram considerados houve uma diferença estatisticamente significante no quociente de estabilidade do implante (ISQ)
entre o pré-operatório imediata e de 6 meses para ambas as conexões protéticas. RESUMO Objetivo
Avaliar dois sistemas de conexões protéticas sobre carga imediata em próteses tipo protocolo em mandíbulas edêntulas. is sistemas de conexões protéticas sobre carga imediata em próteses tipo protocolo em mandíbulas edêntulas. j
Avaliar dois sistemas de conexões protéticas sobre carga imediata em próteses tipo protocolo em mandíbulas ABSTRACT Objective
This study evaluated two implant-abutment connection systems under immediate loading of lower prosthesis in edentulous mandibles. INTRODUCTION with the maintenance of the perimplant bone tissue, such with the maintenance of the perimplant bone tissue, such
as: surgical trauma, load conditions, degree of precision as: surgical trauma, load conditions, degree of precision Dental implants have been widely used due to
their high success rates. However, several factors interfere
g
g
and adjustment between components, resistance and
stability of the implant/abutment interface when subjected Dental implants have been widely used due to
their high success rates. However, several factors interfere RGO, Rev Gaúch Odontol, Porto Alegre, v.65, n.4, p. 308-314, out./dez., 2017 Comparison between two types of prosthetic connection Comparison between two types of prosthetic connection to loads1-3. In regard to the load conditions, it is necessary a
period of 5-6 months for osseointegration of the implants
in the maxilla and 3 months in the mandible. However,
osseointegration with early loading is also a possibility, as
shown in recent studies2-4. Initially immediate loading was
mostly recommended for lower prosthesis in edentulous
mandibles. It is presently used in various types of implant-
supported rehabilitation, as long as there is primary stability
of the implants5-6. However, the connection type and load
conditions directly affect the success of implant-supported
rehabilitation4,7-8. The patients underwent clinical and imaging
examination through panoramic radiographs. The patients
who met the following criteria were selected: a) total
lower edentulism; b) good bone availability of residual
ridge (highest height of 11 mm and thickness greater than
5 mm); c) type I and II13 bone tissue; and d) no systemic
contraindications. Patients who followed the criteria
previously established by Chiapasco et al.14 were excluded
from the study. Each patient was treated with four implants of
regular diameter (3.75 x 11 mm) for lower protocol-
type rehabilitation with functional immediate loading in
the immediate postoperative period. The implants were
distributed between the mental foramens, so that the two
implants on the left side of each patient had Morse cone
connections, while the two implants on the right side had
external hexagon prosthetic connections, following a split
mouth design (Figure 1). In this respect, many studies have evaluated
different types of implant/abutment connections and their
effect on the perimplant tissues9-10. The main advantages
of the conventional external hexagon connection are easy
installation of the prosthesis, avoidance of anti-rotational
movements and compatibility among different implant
systems. INTRODUCTION However, this type of connection shows limited
efficacy when oblique forces are applied, due to its
reduced height, which can cause micro-movements, screw
loosening and even fracture of the connection system2,11. Figure 1. Dental implants installed between mentuals foramen. In regard to the internal connections, it show
higher flexural strength and better distribution of forces
in relation to external systems, thus being biomechanically
more favorable1,8,12. According to Streckbein et al.7,
internal connections present smaller microgaps between
the implant and the abutment in relation to the external
connections, which is highly desirable because the higher
the space between implant and abutment, the greater the
risk of biological and mechanical problems, such as failure
by fatigue or perimplantitis. Morse cone connections
present advantages over the previously mentioned
connections by promoting higher sealing and providing
greater stability. That is so because this type of connection
provides close contact at the implant/abutment interface,
providing greater stability3,12. The objective of this study
was to compare the behavior external hexagon and
Morse cone implant/abutment connection systems under
immediate loading on the same biological system. Figure 1. Dental implants installed between mentuals foramen. Surgical and prosthetic procedures All surgical procedures were performed under
local anesthesia by the same surgeon. The implants
were placed following a standardized surgical protocol
and using previously prepared surgical guides. After
installation of the implants, mini-abutments were adapted
over them, and then transferred by molding with open
molders (Figure 2). Figure 2. Adaptation of mini-piers on implants and molding with open molders. Evaluation 3. Stability of the implants: Implant stability
was measured by resonance frequency analysis (RFA)
by magnetic transduction, using Osstell® ISQ (Osstell
AB, Göteborg, Sweden). For measuring, except for the
baseline, the prosthesis was removed and SmartPeg A3
was adapted to the prosthetic component. The Osstell®
sensor was positioned perpendicular to the long axis of
the implant in the buccolingual and mesiodistal directions. All measurements were taken in triplicate and an overall
average of both axis was calculated. The parameters evaluated included probing
depth, stability of the implants and perimplant bone
loss, which were measured at the baseline (immediately
after installing the prosthesis) and after 3 and 6 months
postoperatively. All examinations were performed by a
previously trained professional. 1. Probing depth: Evaluation of the probing depth
was based on the previous clinical study by Gerber et al.15
(2009), and it was performed with a plastic millimetric
periodontal probe (Colorvue® Hu-Friedy®, Rotterdam,
Belgium) which was positioned perpendicular to the long
axis of the implant on the buccal, lingual and proximal
surfaces, measuring the distance from the gingival margin
to the base of the sulcus15. For each implant, probing
depth (mm) was calculated based on the average of the 4
values obtained16. For reproducibility and standardization
of probe positions two devices were prepared, one
for the right side and one for the left side, made from
acrylic, which were adapted to the mini-abutments and
contained perforations in the buccal, lingual, mesial and
distal regions. 1. Probing depth: Evaluation of the probing depth
was based on the previous clinical study by Gerber et al.15
(2009), and it was performed with a plastic millimetric
periodontal probe (Colorvue® Hu-Friedy®, Rotterdam,
Belgium) which was positioned perpendicular to the long
axis of the implant on the buccal, lingual and proximal
surfaces, measuring the distance from the gingival margin
to the base of the sulcus15. For each implant, probing
depth (mm) was calculated based on the average of the 4
values obtained16. For reproducibility and standardization
of probe positions two devices were prepared, one
for the right side and one for the left side, made from
acrylic, which were adapted to the mini-abutments and
contained perforations in the buccal, lingual, mesial and
distal regions. The data obtained received T-Test for comparison
between the two groups of prosthetic connections and
analysis of variance (ANOVA) to compare the results of the
periods for each prosthetic connection. Patients The experimental protocol was approved by the
Research Ethics Committee with humans (CEP) of the
Araraquara Dental School, São Paulo State University,
UNESP, under protocol number 72/2009. Figure 2. Adaptation of mini-piers on implants and molding with open molders. The analogs were positioned on the transfer
dies and sent to the laboratory phase. Within 72 hours, 309 RGO, Rev Gaúch Odontol, Porto Alegre, v.65, n.4, p. 308-314, out./dez., 2017 MR OLIVEIRA et al. MR OLIVEIRA et al. in the Vixwin Pro 1.2c software (Gendex-Dentsply) and
enlarged on the monitor screen until they occupied the
largest space possible. The tool "shades of gray" was
selected in order to enable the evaluation of the numerical
values of the pixels around the uppermost spirals of the
implants. Analysis of spirals 1-2 was chosen as standard,
in their mesial and distal surfaces, where the numerical
values of the pixels for the bone tissue immediately
next to each spiral were checked and recorded. The tool
"shades of gray" provides the values of each pixel present
in the images, ranging from 0 to 255. Near zero values
are representative of bone loss areas (radiolucent image)
and values close to 255 are radiopaque and represent the
presence of bone tissue. Therefore, values from 0-40 were
considered representative of areas without bone. protocol-type hybrid prosthesis made with metal and
acrylic superstructure were installed with the appropriate
occlusal adjustment. All patients received also complete
upper dentures (Figure 3). Figure 3. Postoperative of 2 days, metallic superstructure and hybrid prosthesis type
protocol installed. Figure 3. Postoperative of 2 days, metallic superstructure and hybrid prosthesis type
protocol installed. RESULTS The sample consisted of 18 patients (12 female
and 6 male), totaling 72 implants inserted (36 with
Morse cone and 36 with external hexagon connection). The average age of patients was 59 years. No implants
were lost and no prosthetic complications were observed
during the study. 2. Perimplant bone levels: Each implant received a
periapical radiograph using the parallelism technique with
modified positioners and with the aid of an individualized
bite block made of condensation silicone. The radiographs
were scanned and the images were imported into the
Adobe Photoshop CS-3 software. Brightness and contrast
were standardized based on the values of the histogram of
each image, which provides uniformity in shades of gray,
making it easier to visualize the contours of the anatomical
structures present. The images were opened one by one Evaluation The significance
level used in both tests was 5%. RGO, Rev Gaúch Odontol, Porto Alegre, v.65, n.4, p. 308-314, out./dez., 2017 Probing depth A total of 876 measurements of probing depth
were obtained, 292 for each evaluation period. In general,
there was an increase in probing depth values for the two
prosthetic connections according to the period, and the
implants with Morse cone connections had statistically
significant lower values of probing depth for all periods
(0.68/1.19/1.31) when compared to implants with external 310 RGO, Rev Gaúch Odontol, Porto Alegre, v.65, n.4, p. 308-314, out./dez., 2017 Comparison between two types of prosthetic connection Comparison between two types of prosthetic connection hexagon connections (1.08/1.52/1.64) (Table 1). around the first and/or second spirals was detected in
15, 29 and 37 evaluations at the baseline, 3-month
and 6-month periods, respectively. In the group
with external hexagon connections bone loss was
detected in 16, 33 and 50 evaluations at the baseline,
3-month and 6-month periods, respectively. However,
when comparing the groups there were no statistical
differences (p<0.61) (table 1). Similarly, it was also not
observed any statistically significant difference in the
perimplant bone loss between the periods considered
for either of the prosthetic connection tupes (table 2). g
(
) (
)
As for the analysis comparing the periods for
the same type of prosthetic connection, a statistically
significant difference among baseline, 3 months (p<0.01
for Morse cone and p<0.001 for external hexagon) and 6
months (p<0.001 for Morse cone and external hexagon)
was observed (Table 2). Table 1. Average of the parameters evaluated according to the type of
prosthetic connection: T-Test at the 5% significance level. Parameters
Prosthetic connection
P
Number of
analyses
ISQ
CM
EH
Baseline
71.94
71.08
0.38
432
3 months
70.73
69.05
0.08
432
6 months
69.0
68.23
0.038
432
PD
MC
EH
Baseline
0.68
1.08
0.0015*
292
3 months
1.19
1.52
< 0.001*
292
6 months
1.31
1.64
< 0.001*
292
PBL
MC
EH
Baseline
with loss
15
16
0.61
272
without loss
120
121
0.61
272
3 months
with loss
29
33
0.61
272
without loss
107
103
0.61
272
6 months
with loss
37
50
0.61
272
without loss
99
86
0.61
272
Note: MC: Morse Cone; EH: External Hexagon; ISQ: Implant Stability
Quotient; PD: Probing Depth; PBL: Peri-implant bone loss; *: this
indicates a statistically significant difference. Table 1. Average of the parameters evaluated according to the type of
prosthetic connection: T-Test at the 5% significance level. DISCUSSION The type of prosthetic connection may interfere
with the results of implant-supported rehabilitation3,12. The different types of connection have their specific
characteristics and it is worth noting that the greater
the gap between the implant and the abutment, the
greater the bacterial colonization, fatigue and risk of
fracture. Those factors directly affect the surrounding
bone tissue, causing resorption and even leading to the
loss of the implant. Some studies show that the internal
connections have a smaller space between the implant
and the abutment and thus lower risk of biological and
mechanical problems7,16-18. As in this study, in order to
determine which implant system causes less damage
to the perimplant tissues, some studies compared the
different types of prosthetic connections7,19. Note: MC: Morse Cone; EH: External Hexagon; ISQ: Implant Stability
Quotient; PD: Probing Depth; PBL: Peri-implant bone loss; *: this
indicates a statistically significant difference. Stability A total of 1296 measurements of implant
stability were obtained, 432 per evaluation period and
216 for each type of connection. Considering the three
study periods, there was a reduction in the ISQ values
for both types of prosthetic connection. No statistical
difference was observed between groups, so that for
implants with Morse cone connections the values were
71.94, 70.73 and 69 for the baseline, 3-month and
6-month periods, respectively. For implants with external
hexagon connection the values were 71.08, 69.05 and
68.23 (Table 1). Note: MC: Morse Cone; EH: External Hexagon; ISQ: Implant Stability
Quotient; PD: Probing Depth; PBL: Peri-implant bone loss; *: this
indicates a statistically significant difference. In the comparative analysis of the study periods
for each prosthetic connection, statistically significant
differences occurred between baseline and 6 months for
both types of prosthetic connection (Table 2). Table 2. Average of the parameters evaluated comparing the periods for
each prosthetic connection: ANOVA at the 5% significance level. Table 2. Average of the parameters evaluated comparing the periods for
each prosthetic connection: ANOVA at the 5% significance level. Parameter
Periods
p
Number of
analyses
ISQ
Baseline 3 months 6 months
MC
71.94a
70.73
69.0b
0.001a,b*
648
EH
71.08a
69.05
68.23b
0.004a,b*
648
PD
MC
0.68a
1.19b
1.31c
0.01a,b*
438
0.001a,c*
EH
1.08a
1.52b
1.64c
0.001a,b*
438
0.001a,c*
PBL
MC
15
29
37
0.051
816
EH
16
33
50
0.07
816
Note: MC: Morse Cone; EH: External Hexagon; ISQ: Implant Stability
Quotient; PD: Probing Depth; PBL: Peri-implant bone loss; *: this
indicates a statistically significant difference. Perimplant marginal bone levels A total of 136 measurements per period for
each one of the prosthetic connections was obtained. In the group with Morse cone connections, bone loss D’ercole et al.19 compared Morse cone and 311 RGO, Rev Gaúch Odontol, Porto Alegre, v.65, n.4, p. 308-314, out./dez., 2017 MR OLIVEIRA et al. radiographs and found significantly higher bone loss
in implants with external connections over those with
internal connections. Galindo-Moreno et al.21 also found
significantly higher marginal bone loss in implants with
external connection (0.714 mm/year) as compared
to implants with internal connection (0.516 mm/
year) installed in the mandible after 6 and 18 months. However, as in the present study, other authors found no
statistical difference in bone loss around implants with
internal and external connections6,9. Thus, it is observed
that the action of different types of connections on the
perimplant bone resorption is a controversial subject in
the literature and requires further studies and longer
follow-ups for better clarification. internal hexagon connections for bacterial infiltration at
the implant/abutment interface and found no statistical
differences between them. However, numerically lower
bacterial infiltration was observed in the Morse cone
group19. In the present study, statistical difference was
only observed between the groups for the parameter
probing depth, with lower values for the Morse cone
connection. For the other parameters assessed no
statistical differences were observed between the two
types of prosthetic connections. The internal connections, especially the Morse
cone, have shown better performance over the external
connections in some experimental studies1,3,10,17-18. Goiato et al.3, showed better distribution of loads on
implants with Morse cone connections when compared
to external hexagon connections. The superior results
of internal over external connections may be primarily
due to the lower vertical force transmitted from the
bottom of the implant to the abutment, distribution
of the lateral load into the implant and large contact
surface between the implant and the abutment present
in the internal connections3. In accordance, the present
results showed lower probing values for implants with
Morse cone connection when compared to the external
hexagon. With regard to the load conditions, according to
Ghanavati et al.22, success rate of implants under early
loading is approximately 93.7%. Li et al.6, followed for
12 months implants installed in fully edentulous jaws
subjected to immediate loading and found a higher
success rate (98.7%). Perimplant marginal bone levels In this study, we observed a
100% success rate for the short follow-up, since no
loss or failure was observed. However, in the systematic
literature review by Sanz-Sánchez et al.5, the authors
reported a higher chance of failure (p<0.036), higher
bone loss (p<0.000) and lower increase in the ISQ values
(p<0.001) for implants with immediate loading over
implants with conventional loading. g
Maintenance of perimplant bone crest height is
very important for the success of dental implants9-10. For
Albrektsson et al.20, a bone loss of up to 1.5 mm in the
first year followed by 0.2 mm every year is acceptable
for a successful implant. Assuming that the type of
prosthetic connection interferes with the dispersion
of loads around the implant, it also influences the
perimplant bone loss. Therefore, several authors evaluate
the perimplant bone loss in different types of prosthetic
connections1,3,7-8,17-18. In this study, the largest amount of
bone loss was observed for the external hexagon group
in comparison to the Morse cone group, but without
statistically
significant
difference
between
them. Similarly, Lin et al.9, evaluated the marginal bone loss on
radiographs of implants with external hexagon, internal
hexagon and Morse cone prosthetic connections and no
statistically significant difference was observed. These
results indicate that there seems to be no difference
in marginal bone loss around implants with different
prosthetic connections in a short period of follow-up. In contrast, Rismanchian et al.4, evaluated the
stability quotient (ISQ), bleeding on probing, pocket
depth and histomorphometric assessment after 3 months
of follow-up of implants inserted into dog mandibles. Regarding the amount of bone in contact with the
implant, they observed larger areas for implants with
early loading (46.17% ± 12.89%), without statistical
difference when compared to implants with late loading
(44.4% ± 10.45%). With regard to ISQ values, statistical
difference was observed between implants with early
loading (71 ± 6.35) compared to late loading (66.75
± 11.86), and the highest values were observed for
the implants with early loading. Rocci et al.23, in turn,
compared the bone/implant contact in implants with
immediate loading and early loading. The bone area in
the immediate loading group (92.9%) was higher than
in the early loading group (81.4%), but no statistical
difference was observed. Similarly, Pontes et al.24,
compared the bone area in contact with implants placed
in different positions undergoing early or late loading
and observed no significant differences. RGO, Rev Gaúch Odontol, Porto Alegre, v.65, n.4, p. 308-314, out./dez., 2017 REFERENCES 9. Lin MI, Shen YW, Huang HL, Hsu JT, Fuh LJ. A retrospective
study of implant-abutment connections on crestal bone
level. J Dent Res. 2013;92(12 Suppl):202S-207S. doi:
10.1177/0022034513510322 1. Tonella BP, Pellizzer EP, Ferraço R, Falcón-Antenucci RM,
Carvalho PSP, Goiato, MC. Photoelastic analysis of cemented or
screwed implant-supported prosthesis with different prothetic
connections. J Oral Implantol. 2011;37:401-10. doi: 10.1563/
AAID-JOI-D-12-00200 10. Koo KT, Lee EJ, Kim JY, Seol YJ, Han JS, Kim TI, et al. The effect of
internal versus external abutment connection modes on crestal
bone changes around dental implants: a radiographic analysis. J
Periodontol. 2012;82(9):1104-9. doi: 10.1902/jop.2011.110456 2. Takahashi JMFK, Dayrell AC, Consani RLX, Nóbilo MAA,
Henriques GE, Mesquita MF. Stress evaluation of implant-
abutment connections under different loading conditions: a
3D finite element study. J Oral Implantol. 2015;41:133-7. doi:
10.1563/AAID-JOI-D-11-00205 11. Almeida DAF, Pellizzer EP, Verri FR, Santiago-Jr JF, Carvalho PS. Influence of tapered and external hexagon connections on
bone stresses around tilted dental implants: three-dimensional
finite element method with statistical analysis. J Periodontol. 2014;85(2):261-9. doi: 10.1902/jop.2013.120713 3. Goiato MC, Pesqueira AA, Falcón-Antenucci RM, Santos DM,
Haddad MF, Bannwart LC, et al. Stress distribution in implant-
supported prosthesis with external and internal implant-
abutment connections. Acta Odontol Scand. 2013;71:283-288. doi: 10.3109/00016357.2012.672823 12. Schwarz F, Hegewald A, Becker J. Impact of implant-abutment
connection and positioning of the machined collar/microgap
on crestal bone level: a systematic review. Clin Oral Impl Res. 2014;25(4):417-25. doi: 10.1111/clr.12215 4. Rismanchian M, Bajoghli F, Gholamreza T, Razavi M. Dental
implants: early versus standard two-stage loading (Animal
Study). J Oral Implantol. 2014;40(1):85-93. doi: 10.1563/AAID-
JOI-D-10-00202 13. Lekholm U, Zarb G. Tissue integrated protheses. Chicago:
Quitenssence; 1985. 5. Sanz-Sánchez I, Sanz-Martín I, Figuero E, Sanz M. Clinical
efficacy of immediated implant loading protocols compared to
conventional loading depending on the type of the restoration:
a systematic review. Clin Oral Impl Res. 2015;26(8):964-82. doi:
10.1111/clr.12428 14. Chiapasco M, Gatti C, Rossi E, Haefliger W, Markwalder TH. Implant retained mandibular overdentures with imeediate
loading. A retrospective multicenter study on 226 consecutive
cases. Clin Oral Impl Res. 1997;8(1):48-57. doi: 10.1111/j.1600-
0501.1997.tb00007.x 6. Li W, Chow J, Hui E, Lee PK, Chow R. Retrospective study on
immediated functional loading of edentulous maxillas and
mandibles with 690 implants up to 71 months of follow-up. J Oral Maxillofac Surg. 2009;67(12):2653-62. doi: 10.1016/j. joms.2009.07.015 15. Gerber JA, Tan WC, Balmer TE, Salvi GE, Lang NP. Perimplant marginal bone levels In the present In contrast, Koo et al.10 evaluated the linear
bone change, dimensional bone change and the angle
between the implant and the adjacent bone through 312 RGO, Rev Gaúch Odontol, Porto Alegre, v.65, n.4, p. 308-314, out./dez., 2017 Comparison between two types of prosthetic connection Collaborators study, the implants were subjected to immediate loading
and the two types of connection presented similar ISQ
values. A statistical difference in ISQ values was only
found when comparing baseline and 6-month periods
in both groups The nature and level of contribution where
each of the authors was involved were as follows. MR
OLIVEIRA and AS OLIVEIRA: collect of data; literature
review, writing and submission of the manuscript. MAC GABRIELLI and R SPIN NETO, statistical analisys
and assisted in the preparation of the manuscript. VA
LEITE, assisted in the surgical procedures and of the
documentation of the cases. OEB PAGANELLI, critical
review of the writing of the manuscript and of the
English version. VA PEREIRA FILHO, surgical procedure
and the documentation of the cases. CONCLUSIONS The results suggest that immediate loading
is a viable option for rehabilitation of edentulous
mandibles and the type of connection did not interfere
with the success of the implants in the short-tem
follow-up. Impl Res. 2012;23(suppl 6):202-16. doi: 10.1111/j.1600-
0501.2012.02556.x REFERENCES Bleeding
on probing and pocket probing depth in relation to probing
pressure and mucosal health around oral implants. Clin Oral Impl
Res. 2009;20(1):75-8. doi: 10.1111/j.1600-0501.2008.01601.x 16. Heckmann SM, Schrott A, Graef F, Wichmann MG, Weber HP. Mandibular two implants telescopic overdentures. 10-year clinical
and radiographical results. Clin Oral Impl Res. 2004;15(5):560-9. doi: 10.1111/j.1600-0501.2004.01064.x 7. Streckbein P, Streckbein RG, Wilbrand JF, Malik CY, Schaaf H,
Howaldt HP, et al. Non-linear 3D evaluation of different oral
implant-abutment connections. J Dent Res. 2012;91(12):1184-
9. doi: 10.1177/0022034512463396 17. Gehke SA, Viana MSS, Dedavid BA. Influence of bone
insersion level of the implant on the fracture strength of
different connection designs: an in vitro study. Clin Oral Invest. 2014;18(3):715-20. doi: 10.1007/s00784-013-1039-7 8. Gracis S, Michalakis K, Vigolo P, Vult von Steyern P,
Zwahlen M, Sailer I. Internal vs external connections for
abutments reconstructions: a systematic review. Clin Oral 313 313 RGO, Rev Gaúch Odontol, Porto Alegre, v.65, n.4, p. 308-314, out./dez., 2017 MR OLIVEIRA et al. and
histomorphometric
study
in
dogs. J
Periodontol. 2006;77(10):1701-7. doi: 10.1902/jop.2006.050436 18. Gehke AS. Importance of crown height rations in dental implants
on the fracture strenght of different connection designs: an in
vitro study. Clin Impl Relat Res. 2015;17(4):790-7. doi: 10.1111/
cid.12165 and
histomorphometric
study
in
dogs. J
Periodontol. 2006;77(10):1701-7. doi: 10.1902/jop.2006.050436 23. Rocci A, Martignoni M, Burgos PM, Gottlow J, Sennerby L. Histology of retrieved immediately and early loaded oxidized
implants: light microscopic observations after 5 to 9 months of
loading in the posterior mandible. Clin Implant Dent Relat Res. 2003;5(1):88-98. doi: 10.1111/j.1708-8208.2003.tb00020.x 19. D’ercole S, Scarano A, Perrotti V, Mulatinho J, Piattelli A, Iezzi
G, et al. Implants with internal hexagon and conical implant-
abutment connections: an in vitro study of the bacterial
contamination. J Implantol. 2014;40(1):31-6. doi: 10.1563/
AAID-JOI-D-11-00121 24. Pontes AEF, Ribeiro FS, Iezzi G, Pires JR, Zuza EP, Piattelli A, et
al. Bone implant contact around crestal and subcrestal dental
implants submitted to immediated and conventional loading. Sci
World J. 2014;2014(12):1-5. doi: 10.1155/2014/606947 20. Albrektsson T, Zarb G, Worthington P, Eriksson AR. The long-
term efficacy of currently used dental implants: a review and
proposed criteria of success. Int J Oral Maxillofac Implants. 1986;18(8):11-25. doi: 10.9790/0853-1608021323 21. Galindo-Moreno P, Fernández-Jiménez A, Ó Valle F, Monje A,
Silvestre FJ, Juodzbalys G, et al. Influence of the crowm-implant
connection on the preservation of the peri-implant bone. a
retrospective multifactorial analysis. Int J Maxillofac Implants. 2015;30(2):384-90. Received on: 22/2/2017
Final version resubmitted on: 16/5/2017
Approved on: 22/8/2017 REFERENCES doi: 10.11607/jomi.3804 22. Ghanavati F, Shayegh SS, Rahimi H, Sharifi D, Ghanavati F,
Khalesseh N, et al. The effects of loading time on osseointegration
and new bone formation around dental implants: a histologic 314 RGO, Rev Gaúch Odontol, Porto Alegre, v.65, n.4, p. 308-314, out./dez., 2017 RGO, Rev Gaúch Odontol, Porto Alegre, v.65, n.4, p. 308-314, out./dez., 2017
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https://openalex.org/W2314914423
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https://zenodo.org/records/2388340/files/article.pdf
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German
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Eklampsie
|
Klinische Wochenschrift
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public-domain
| 5,199
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KLINISCHE
WOCHENSCH
RIFT.
i. JAHRGANG.
Nr. 48 Doch sind diese chronisch werdenden F~lle sicher die
Ausnahmen, fiir gew6hnlich bilden sich auch die Nieren-
vergnderungen so v611ig zuriick, dab das Organ wieder yell
Iunktionsfghig wird. Kehren wir noch einmal zu der wichtigsten Frage nach der
Ursaehe der Eklampsie zurfick, so m6chte ich daran festhalten,
dab in der Schwangerschaft entstehende toxisch wirkende
Stofiwechselprodukte, die in den Kreislauf der Mutter ge-
langen, die Ursache darstellen. Leider bleibt die Frage nach
Herkunft und genauer Definition dieses fraglichen Toxins
immer noch often. Die histologischen Untersuchungen ge-
statten wobi, gewisse oben chaxakterisierte Eigenschaffen
des Gifts aus seinen Wirkungen zu erkennen, aber nicht es
chemisch zu definieren, es ist das ia leider eine Frage~ die
fiber die Grenzen der morphologischen Forschung, in der sieh
meine Studien lediglich bewegten, binausgeht. g
,
p
,
Auch die Entstehung der Muskelkr/~mpfe stelle ich mir etwas
anders vet als die beiden genannten Autoren. DaB die Muskel-
krfixnpfe veto Gehirn her ausgel6st werden, seheint aueh mir sicher;
manche Autoren denken dabei an eine direkte toxische Beeinflus-
sung der Hirnsubstanz, doch halte ich es wie ~7OLHARD
-
-
bei dem
an/allsweisen Auftreten der Muskelkr/tmpfe -- f/it sehr wahrschein-
lich, dab bier der ausl6sende Faktor in der Hauptsache in Kr/tmpfen
der HHirngef/tBe zu suchen ist, nut glaube ieh, dab der springende
Punkt weiterhin in der Erndhrungsstdrung des Gebirns, nicht abet
in dem Odem zu sehen ist. Der Gei/tBkrampI Ifihrt zur Hirnangmie
und damit zum Muskelkrampf, str6mt das Blut nach L6sung des
Gef/tBkrampfes wieder in die Hirngef/~ge ein, so kann es bei ent-
sprechender Schgdigung der Capillaren zum Austritt ser6ser Flfissig-
keit zum 0dem, gelegenflieh bei noeh sehwererer Sch/idigung zu den
Blutungen kommen, die wit ja nicht selten im Gehirn Eklamptischer
finden; beide Ereignisse k6nnen abet auch bei kfirzerer Krampf-
dauer, bei geringerer Gef/tBsch/tdigung ausbleiben. Unter Zugrunde-
legung dieser Annahme k6nnen wit sehr gut die weehselnden Be/unde
erklfixen, die wit am Gehirn nach den ausdrficklichen Angaben so
namhafter Autoren, wie OLSHAUSEN U. a. beobachten. Bald ist
nach diesen Aussagen das Gehirn anfixnisch, bald hyper/tmisch,
h/iufig besteht 0dem, manchmal abet auch bestimmt nicht, wie mir
auch Herr FRAENKEL Ifir rnanche yon ihm sezierteu F/~lle, bei denen
gerade auI diesen Punkt genau geachtet wurde, versichert. Mifunter
sehen wir endlich Blutungen, die unter Umst/tnden grebe Ausdeh-
nung erlangen k6nneu. Die entyciekelte Vorstelluug deckt sieh mit
der von PAL. Klinische Beobachtungen und Untersuchungen.
Von
Prof. Th. HEYNE~AN~.
us der U~iversit~ts-Frauenklinik zu Hamburg. Eppendorfer Krankenhaus. Von
Prof. Th. HEYNE~AN~. Prof. Th. HEYNE~AN~. Aus der U~iversit~ts-Frauenklinik zu Hamburg. Eppendorfer Krankenhaus. Die iolgenden Ausffihrungen stellen das Ergebnis Mini~
scher Beobachtungen und Untersuchungen am Krankenbett
dar. Die Anwendung neuer und die Verfeinerung /ilterer Unter-
suchungsmethoden haben Tatsachen zutage gef6rdert, die fiir die
Auffassung uud dm Klinik der Eklampsie nicht ohne Belang sind. Die Anwendung neuer und die Verfeinerung /ilterer Unter-
suchungsmethoden haben Tatsachen zutage gef6rdert, die fiir die
Auffassung uud dm Klinik der Eklampsie nicht ohne Belang sind. Die Deutung
und Yerwertung dieser Feststellungen st6Bt
allerdings noch vielfach auf Schwierigkeiten. Sichere Schlfisse
sind noch nicht immer m6glich. Ich habe mir daher in diesem
Punkte weitgehende Beschr/inkung auferlegt. Die Deutung
und Yerwertung dieser Feststellungen st6Bt
allerdings noch vielfach auf Schwierigkeiten. Sichere Schlfisse
sind noch nicht immer m6glich. Ich habe mir daher in diesem
Punkte weitgehende Beschr/inkung auferlegt. Die I~r~ahrung, dab fast jede neue Untersuchungsmethode
gerade auch bei der Eklampsie zu neuen 13efunden ifihrt, hat
sich besonders Wieder bei der Capillarraikroskopie best~tigt. Ich will indes keineswegs leugnen, dab es im Verlauf der Schwan-
gerschaftstoxikose sehr h/tufig zum Hirn6dem kommt, uud ich
m6chte in diesem Zusammenhang daxauf hinweisen, dab ich nicht
selten bei Kindern, die im Verlauf you Infektionskrankheiten
Krfimpfe bekamen, als einzige Ver/tnderung im Zeniralnerven-
system 0dem der weichen Hirnh~ute und der Hirnsubsfanz gefunden
habe. Dutch sie haben HINSELMANN an der Bonnet und NEVER-
MANN an unserer Klinik bei der Eklampsie und ihren Vorstadien
Ver/tnderungen an den Hautcapillaren festgestellt, die sich in erster
Linie auf die Str6mung, daneben aber auch auf die Form erstrecken. Es zeigt sich eine ausgesprochene Verz6gerung der Capillar-
str6mung, die zu v611igem Stillstand, zur Stase ifihrt. Meist wechseln
Stase und Str6mung ziemlich sehnell miteinander ab, in anderen
F/tUen bestehen die Stasen 1/tngere Zeit, es kommt zu sog. Dauer-
stasen Dutch sie haben HINSELMANN an der Bonnet und NEVER-
MANN an unserer Klinik bei der Eklampsie und ihren Vorstadien
Ver/tnderungen an den Hautcapillaren festgestellt, die sich in erster
Linie auf die Str6mung, daneben aber auch auf die Form erstrecken. Einen Punkt m6chte ich noch kurz streifen, das ist das
Chronisehwerden der eklamptischen OrganverS.nderungen, yon
denen meine Untersuehungen ja urspriinglich ihren Ausgang
genomrnen haben. 13bet das Chronischwerden der Leber-
ver/inderungen ist bis jetzt nichts bekannt, dagegen scheint
es nach den in der Literatur niedergelegten Angaben, auf die
ieh bier im einzelnen nicht eingehen will, immerbin wahr-
scheinlieh, dab sich ein chronisches Nierenleiden an die EMamp-
sie ansehlieBen kann. KLINISCHE
WOCHENSCH
RIFT.
i. JAHRGANG.
Nr. 48 KLINISCHE
WOCHENSCH
RIFT. i. JAHRGANG. Nr. 48 2363 25. NOVEMBER I922 wirkende Sch/~dlichkeit bier nicht in Betracht kommen, und
wir mfissen uns die Sache anders vorstellen. wirkende Sch/~dlichkeit bier nicht in Betracht kommen, und
wir mfissen uns die Sache anders vorstellen. seMidigen auch gerinnungserrege~ute, hiimo~ytische und va~okon-
striktorische Eigenscha]ten zuschreiben. Ich m6chte glauben, dab der weitere Gang der Ereignisse
sich bier in der Weise abspielt wie bei der Glomerulonephritis,
die, wie wit sagen, mit Defekt ausgeheilt ist und infolgedessen
in ein chronisches Stadium iibergeht. Hier wie deft ist das
Gift, das ursprfinglich die Erkrankung hervorrief, nicht mehr
wirksam, daffir k6nnen aber in dem durch hinreichende Glo-
merulussch~digung
,,empfindlich" gewordenen Organe die
zur Ausseheidung bestimmten Sebiacken als entzfindungs-
erregendes Agens wirken (s. meine diesbezfiglichen Ausfiih-
rungen im Handbuch yon HENCE und LUBARSCH). In dem
gesch/~digten Organ spielen, wie LUBARSCH sagt, die nor-
malen Stoffwechselvorg/inge die Rolle pathologischer Reize
und es kann auf diese Weise eine chronische Entziindung an
die akute -- auch degenerative -- Sch~digung sich anschliegen. DaB bei der Eldampsie ein Toxin im K6rper kreist, hat man von
jeher angenommen, ZWEIFEL bezeictmet diesen Satz als eine ,,Binsen-
wahrheit", neuerdings sind aber ~egen diese Annahme yon maB-
gebender Seite yon VOLHARD und ZANGEMEISTER sehr ernst zu-
nehmende Einw~nde erhoben worden. Beide Autoren stehen der
toxischen Tbeorie sehr skepfisch gegenfiber. VOLHARB will alles
auf Gef/tBkr/tmpfe, die er in der Hauptsache ffir reflektorisch be-
dingt h/tit, zuriickzuffihren, f/it ZANGEMEISTER ist das SVesentliche
bei der Eklampsie das 0dem der Schwangeren, das er dutch Sauer-
stoffmangel infolge Eiwachstums mit anschlieBender Capillar-
sch/tdigung erkl/tren Will. Einig sind sich beide Autoren in der An-
nakme, dab das ausl6sende Moment ffir die Muskelkr/tmpfe, die ja
das Minische Bild durchaus beherrschen, in einem ~dem des Ge-
hirns zu suchen ist. Ich bin mit VOLHARn darin einig, dab bei der EMampsie Gef/tg-
spasmen eine sehr erhebliche Rolle beim Zustandekommen der
bier zu beobachtenden anatomischen Ver/tnderungen spielen, doch
kann ich diesen Spasmen nicht die g/tnzlich dominierende Rolle
zuerkennen, wie WOLHARD dies getan hat, auch bin ich der Meinung0
dag sie nicht reflektorisch, sondern toxisch bedingt sind. Ebenso-
wenig kann ich zugeben, dab die Annahme eines prim/tren 0dems
imstande ist, all die mannigfaltigen, im einzelnen oben kurz erw/thn-
ten, histologischen Ver/~nderungen an den parenchymat6sen Or-
ganen, speziell Leber und Niere, zu erldfi~ren. KLINISCHE
WOCHENSCH
RIFT.
i. JAHRGANG.
Nr. 48 PAL meint, der Gef/tBkrampI der cerebralen Arterien
erzeuge nur unter Umstdinden bei besonderer Intensit/tt die eMamp-
tischen Anf/tlle mit oder ohne Gehirnblutungen, es sei jedoch
nicht unbedingt notwendi~, dab die pressorische Gef/tBkrise diese
Folgeerscheinungen ausl6se. ortrag, gehalten im ~rztlicheu Verein zu Hamburg am 23. Mai 1922. *) Vortrag, gehalten im ~rztlicheu Verein zu Hamburg am 23. Mai 1922. *) Vortrag, gehalten im ~rztlicheu Verein zu Hamburg am 23. Mai 1922.
! 54" Klinische Beobachtungen und Untersuchungen.
Von
Prof. Th. HEYNE~AN~.
us der U~iversit~ts-Frauenklinik zu Hamburg. Eppendorfer Krankenhaus. Diese Auffassullg mllB ich, soweit es sich um die Eldampsie
handelt, zurfickweisell, weil bei Schwangeren und Eklamptischen
BesChlellnigung der Blutsenkungsgeschwindigkeit und St6rllngen
in der Str6mung in keiller Weise iibereinstimmen nnd weil wit
sehr starke Beschlenlligung der Blutsenkungsgeschwilldigkeit fill-
dell, ohne dab allsgesprochene St6rungen in der Capillarstr6mung
anftreten. An und ffir sich w~en auch Zirklllafionsst6rullgell, wie wir
sie all dell Hautcapillarell finden, sofern sie stark und ausgesprochen
sind, geeignet, ein so empfindliches Gewebe wie das Gehirn zu
sch~digen. \Vir linden sie abet. wie bereits erw~ihllt, auch bei
Zustgnden, die ohne schwere Hirnerscheinullgell verlaufell, ulld auch
bei der Eklampsie und ihren Vorstadiell Iehlt jede Ubereinstimmllng
zwischell dera Grade der geschilderten Zirkulatiollsst6rungen und
der Schwere der Hirnerscheillungell. iBei unseren Caiserschnittell hat NEVERMANN festgestellt, dab
sich die gleicheI1 SirSmungsver/inderllngen wie an dell Hautcapil-
laren auch an den feinsten Peritoneal- und oberfl/ichlichen Uterus-
gefgBen abspielen, obwohl starke Gef/il3injektion bei der Operation
ulld histologische Befunde gezeigt haben, dab Peritoneal- ulld Ute-
rusgefSBe elltsPrechend dem Bestehell der Schwangerschaft sehr
stark, die Hautgef/iBe abet nur wenig mit Blllt geffillt sind. So bleibt die Anschauung yon der Entstehung der Hirn-
erscheinungen durch Gef/igkriimpfe auch heute noch lediglich
eine Hypothese, immerhin eine, fiir die sich auch klinisch
manches anfiihren 15A3t. Ausgesprochene vasomotorische St6rungen sind bei der Eklamp-
sie vorhandell. Auch bei anderen Zust/indell, bei dellen die Capillar-
verSmderungen ebenso stark sind ulld gleichzeitig Blutdrucksteige-
rungen gefunden werden, kommen Hirnerscheillllngen nicht allzu-
selten vor. Dutch den AderlaB werden einerseits besonders die
geschildertell Zirkulationsst6rungen, zum anderen gerade die Hirn-
erscheinungen gfillstig beeinfluBt. Umgekehrt begfinstigt die Wehell-
t/itigkeit einerseits das Auftreten der MllskelkrS~mpfe ulld verstgrkt
andererseits Blutdrllcksteigerullg und Capillarvergnderungell. End-
lich spricht auch das so pl6tzliche und unvermutete Auftreten. der tlirnerscheinllngen ffir eine solche Auffassung. Darnach miissen wir annehmen, dab die g!eichen vaso-
m0torigchen St6rungen wie an der Haut auch noch in anderen
Capillargebieten des K6rpers vorhanden sind, auch wenn sie
nicht unmittelbar beobachtet werden k6nnen. Die geschildertell Kapillarver~nderungen werden bei der
Eklampsie niemals vermiBt, sie sind aber in keiner Weise auf
diese Erkrankung beschr/inkt. Die gleichen Erscheinungen,
abet nur gallZ gering, eben angedeutet finden wir sehon beim
gesuilden Menschen, starker,' aber ebenfalls llur in geringerem
Grade haben wit sie bei den verschiedensten Zust/inden, auch
in der normalen Schwallgerschaft, ebenso stark abet und yon
vielen Eklampsien nicht zu unterscheiden, z. B. Klinische Beobachtungen und Untersuchungen.
Von
Prof. Th. HEYNE~AN~.
us der U~iversit~ts-Frauenklinik zu Hamburg. Eppendorfer Krankenhaus. In der Regel gehen ja mit Beendigung
der Schwangerschaft einschlieglich der ersten Wochenbetts-
rage alle Ver/inderungen im K6rper zuriick, da jetzt die Quelle
ffir die Produktion der iragliehen Sch~dlichkeit wegf/illt;
wenn in der Niere zter Verlani yon dieser kRegel abweicht,
wenn bier ein chronischer Zustand sich an die akute Sch~di-
gung w/ihrend
der Schwangerschaft
anschlieBt,
so kann
natfiflich die ursprfingliehe w/~hrend der Schwangerschaft Es zeigt sich eine ausgesprochene Verz6gerung der Capillar-
str6mung, die zu v611igem Stillstand, zur Stase ifihrt. Meist wechseln
Stase und Str6mung ziemlich sehnell miteinander ab, in anderen
F/tUen bestehen die Stasen 1/tngere Zeit, es kommt zu sog. Dauer-
stasen. Aul3erdem sind die Capillarschlingen verl/tngert, ihre venSsen
Schenkel h/tufig mehr oder weniger erweitert. Bei vielen Eklampsien 1/iBt sich beobachten, wie der arterielle
Schenkel einer Capillare pl6tzlich verschwindet und diese Erschei-
hung dann, wie in einer peristaltischen Welle, auf den venSsen
Schenkel iibergreift. Allem Anschein nach handelt es sich bier um
Spasmen, eine Feststellung, die ffir die Deutung und Erkl/ixung
auch der iibrigen Capillaxver/tnderungen bei der Eklampsie wichfig
ist. Auch das Uberwiegen der Str6mungsver/tnderungen gegen/iber
den Formver/inderungen weist darauf hin, dab es sieh urn vaso-
motorische St6rungen handelt. ! 54" 2364 KLINISCHE
YVOCHENSCHRIFT. I. JAHRGANG. Nr. 48 25. NOVEMBER ~92e 25. NOVEMBER ~92e Da man ferner die Str6mungsver/~nderullgen kfinstlich durch
Abdrosselullg der Blutzllfuhr hervorrnfen kann, liegt es nahe, sie
auI Spasmen in den prgcapillaren Geffil3en zurfickzuf/ihrell. Jedoch
genfigt diese Erkl~rung nicht ftir alle F/ille. Besonders wenn die
ven6sen Schenkel sehr stark erweitert sind, kommt mall nicht
olme die Annahme ihrer Dilatation oder eines Hindernisses in den
abfiihrellden Bllltwegen aus. des Arteriellkalibers, aber ohne delltliche Beziehung zum eklamp-
tischen Anfall festgestellt, w~ihrend die Vellen meist eher erweitert
xvaren, Es ist zuzugeben, dab die Hirngef/~13e sich durchaus anders ver-
haltell k6nnen, als die Haut- und Perit0nealgef/il3e. Es spricht
Vieles daffir, dab manche GefiiBbezirke eille ganz auffallende
Selbst/indigkeit ill bezllg allf vasomotorische Vorg~nge haben. Man kann aber bisher keinen sicheren Anhalt dafiir gewinllen,
dab die Hirngefgl3e sich bei der Eklampsie nun gerade im Sinne
CARL SCHROEDERS und PALS verhalten. g
Es ist versllcht, die St6rllllgell ill der Capillarstr6mung auf
eine vergnderte Blutbeschaffenheit zllrfickzuffihren, wie sie in tier
BeschleunigUng der Blutsenkungsgeschwindigkeit zum Ausdruck
kommt. Klinische Beobachtungen und Untersuchungen.
Von
Prof. Th. HEYNE~AN~.
us der U~iversit~ts-Frauenklinik zu Hamburg. Eppendorfer Krankenhaus. bei einzelnen
FS~llen yon puerperaler Infektion und bei manchen Nieren-
erkrankungen, bei G10merulollephritis, besonders wenn sie
mit
pr~e!damptischen Erscheinungen
einhergeht,
und
bei
S!derosen. Das Zweite, woffir sich klinisch Zeichell gewinnell lassen, isf ein
gesteigerter intercerebraler Druck. Es ist bekannt, dab ihml und
zwar gerade einem Hirn6dem ZANGEM~ISTER eine allsschlaggebende,
Bedeutung ffir die Entstehung der Hirnerscheinungen zllerkennt. Es ~bt abet Eklampsien, bei denell llicht die Spur eines 0dems
zu erkenllen ist. ~rasserretention besteht auch bei ihllell. Auch
bei Sektionen EMamptischer wird sehr h/~ufig jegl~ches Hirll6dem
vermil3t. Es besteht bei der Eklampsie noch eine weitere Erschei-
nung, die mit einer vasomot0rischen St6rung in urs~chlichen
Zusammenhang gebracht wird. Das ist die Blu tdr}teksteigerung. Der Lllmbaldruck ist jedoch erh6ht. Untersuchungell ilach
RXlCHARDT fiber Hirnschwellung bei unseren ad exitllm gekom-
menen Eklampsien werden yon HELLMUTI~ allgestellt. Gegen die
Methode sind schwere Bedenkei1 zu erheben~ auch sind llnsere
Ulltersuchungen noch nicht zum AbschluB gebracht,
jedenfalls
spricht das bisherig e Ergebnis eher fiir wie gegen eine I-Iirnsehwel-
lung, sie scheint abet nicht sehr erheblich zu seill, in diesem Sinne
spricht ferller, dab V~rOHLWlLL ulld SIOLI Amoiboidose des Gehirns
festgestellt haben, aber sehr wechseinden Grades llnd nicht der
Schwere der Hirnerscheinungen entsprechelld. Endlieh besteht
der h/iufigste und typische t3efund'de s Augellhintergrundes bei der
Eklampsie in Odem ulld Verschleierung der Papille und der llm-
gebenden Netzhaut,
Eille ausgesprochene Stauullgspapille ist
noch llicht beobachtet. Um Elltzfilldung handelt es sich nicht;
~ie auf meine Bitte vonder
AugenMinik durchgeffihrte Unter-
suchung allf Dunkeladaption hat stets normalen Befund ergeben. l i h
b
d
G
d d
b ll
hild
d ,~Sie ~indell wir ebenfalls bei der Glomerlllonephritis und der
Sklerose, nicht aber bel dell anderen eben erw~hnten Zust/inden. Abet auch bei den Eklampsien und ihren Vorstadien sfimmell
Blutdrucksteigerullg und Ver/~nderungen all dell Halltcapillaren
in keiner Weise miteillalider fiberein. Ein urs/ichlicher Zusammen-
hang zwischen diesen beiden Erscheillungen ist sicher nicht vor-
handen. Besteht tr0tzdem die Annahme, dab es sich hei beiden
um eine Folge yon vasomotorischell St6rllngen handelt, zu Recht,
so mfissell diese St6rllngen verschiedener Natllr sein nnd sich bei
der Blutdrucksteigerllng wohl auBerhalb der Hauteapillaren ab-
spielen. Die eben gemachte Feststellung mahnt zur Vorsicht in der
Beurteilung, illwieweit alldere Krankheitserscheinungen der
E!dai~psie aui die geschilderten Vorg/inge an den Capillaren
zudickgeffihrt werdell k6nnen. Klinische Beobachtungen und Untersuchungen.
Von
Prof. Th. HEYNE~AN~.
us der U~iversit~ts-Frauenklinik zu Hamburg. Eppendorfer Krankenhaus. Alle aiideren I\![ethoden zur Beurteilung der Leber bei der
EMampsie nnd ihren Vorstadien haben praktische Bedeutung bisher
nicht gewonnen. Selbst bei sehr schweren Eldampsien fallen die
sonst iiblichen Funktionsprfifungen der Leber sehr hg~ufig normal
aus. p
g
Man kann sich nach den Erfahrungen bet intraven6sen Injek-
tionen sehr gut vorstellen, dab zur Wirkung der toxischeii Sub-
stanz die erw/ihnten vasomotorischen oder andere noch unbekannte
St6rungen erforderlich stud. Wenn die Hirnerscheinungen bet der Eklampsie auch
hgufig sehr sehnell und unerwartet auftreten, so kann es
doch keinem Zweifel unterliegen, dab sich das Ereignis vorher
1/~ngst angebahnt hat. Ffir alle Ver~nderungen, die bis-
her bet der Eklampsie aufgedeckt stud, besteht eine aug
steigende Linie volt der normalen Schwangerschaft, fiber
Schwangerschaftsst6rungen, fiber Pr/ieklampsie zur Eklamp-
sie, wenn auch einzelne Stadien des Weges schnell durch-
laufen oder anscheinend auch ganz fibersprungen werden
k6nnen. Die Eklampsie stellt lediglich den Ausbruch einer
l/ingst drohenden Katastrophe dar. Da der Nachweis des Hs
spektroskopisch erbracht werden
mul3 uiid die Bilirubinbestimmung im Serum Ubung erfordert, ist
es verstiindlich, weiin diese Methoden in der Praxis bisher kaum
Anwenduiig gefunden haben. Dort weisen lediglich die seltene H/imoglobiiiurie uiid der ge-
w6hnlich erst sp~t auftretende Ikterus auf Blutzerfatl und Leber~
sch~digung bin. Die prognostische Bedeutung der Nie~'enerscheinungeli
sch/itze ich deshalb weniger h0ch ein, well bet ihnen das
WechseIvolle nnd Unvorhergesehene des Eklampsieverlaufs
sehr zum Ausdruck kommt. Eine geringe Harlimenge und viel
EiweiB weisen auf einen schweren, viel Ham und wenig Ei-
weiB alif einen leichten Verlauf hill. Besonders I~LBt eine aus-
gesproehene Vermehrung des EIarns im Verlaufe der Er-
krankung einen guten Ausgang erhoffen. Immer wieder abet
erlebt man es, wie ein pl6tzlicher Wechsel dieser Erscheinungen
uns auch zu einem unerwarteten Wechsel in der Prognosen-
stellung zwingt. Sodanli ist die Art und Natur dieses ,,Schwangerschafts-
giftes" noeh v611ig hypothetisch. Weder serologische noeh biologische noch chemische Unter-
suchuiigen haben bisher einen sieheren "Anhalt daftir erbringen
k6nnen. Das Vorliegen eines spezifischen,
abgestimmten Vorganges
(Vergiftung dutch f6tales oder placentares EiweiB, Anaphylaxie)
halte ich deshalb ftir unwahrscheinlich, well kS trotz uneiidlicher
darauf verwandter Mfihe nicht gelungen ist, spezifische ImrnunitS~ts-
reaktionen mit Sicherheit nachzuweisen. Vereinzelten positiven
stehen zahlreiche negative Ergebnisse gegentiber. Derartige eigene
Untersuchungen (Precipitation,
Komplementbindung,
Anaphy-
laxie) sind stets negativ ausgefatlen. Klinische Beobachtungen und Untersuchungen.
Von
Prof. Th. HEYNE~AN~.
us der U~iversit~ts-Frauenklinik zu Hamburg. Eppendorfer Krankenhaus. an allen anderen Organen. Die Annahme liegt nahe, dab die
Ursache, die die anderen Organe sch~idigt, auch auf das Gehirn
einwirkt. Fiir sehr viele Ver/inderungen wfirde man eine ausreichende
Erkl/irung in der Annahme yon Gefggkr/impfen linden, fiir
manche abet ist bet dem heutigen Stande uliserer Kenntnisse
die Annahme eines unmittelbar toxiseh wirlcenclen Agens sicher
alas Naheliegeride. g
Die vasomotorischen St6rungen,
vielleicht durch
die
gleiche Substanz hervorgerufen, spielen eine wichtige Rolle, Ms
belanglos kann auch die intercerebrale Drueksteigerung liicht
aligesehen werden. Die ausschlaggebenden und wichtigsten Einzelheiten alle{"
dieser Vorg~nge und ihrer Bedeutung ffir die Eklampsie sind
aber noch unbekannt. In der Hinsicht habe ich bereits auf das 0dem hingewiesen. Hier sind welter zu nennen die Zeichen des Blutzerfalles, der
positive Ausfall
des
Stauungsversuches
nach
RVMPEL-
LEEI)n, und die Ver~inderungeli an Leber, Niere und Hirn-
gef/~Ben. Das klare BewuBtsein dieser Tatsache ist insofern yon
praktischer Bedeutung, als das Vertrauen auf bestimmte
Eklampsieerkl~rungeli und Theorien die Behandlung der
Kranken in falsche Bahneli lenken kann. Das schnelle Schwinden der Erscheinungen spricht nicht
gegen eine solche Annahme. Die Sch~idigungen sind zuliXehst
und vor allem bet leichteren F/~Ilen augeliseheinlich schnell
reparabel. Selbst bet einer Frau, die 5 Tage nach dem Auf-
h6ren
der eklamptischen Anf/~lle zugrunde gegangen ist,
sind z. B. an den Glomeruli nur noch vereinzelt nnd ganz
geringe Ver~inderungen zu erkennen gewesen. Diese Gefahr ist deshalb besonders groB, well die Eklampsie
ill bezug auf Sehwere und Verlauf die denkbar wechselvollste
Erkrankung ist. Es st613t daher auf Schwierigkeiten, stati-
stisch den Weft oder Unwert einer Behandlungsmethode zu
erweisen. Die gleicheli Schwierigkeiten findeli wir bet der
Prognosenstellunff im Einzelfalle. Bedenken bestehen abet auch gegen diese Annahme. Ich bin der Ansicht, dab die Untersuchungen auf Erschei-
Iiungen yon Blutzerfall (Hi}~matin, Bilirubin) im Serum einen Fort-
schritt in der Prognosenstellung bet der Eklampsie bedeuten. Uber
die Erfahrungen unserer Klinik hat HELLMUTtI (Berl. ldin. Wochen-
sehr. 1921 , Nr. 25, S. 67o ) berichtet. Zun/ichst hat t3~MN his zu t 1 Blur Eklamptischer an Uterus-
carcinom leidenden Frauen transfundiert, ohne dab Krankheits-
erscheinungen danach aufgetreten stud, im Gegenteil, die Trans-
fusionen habeii, wie es beabsichtigt war, gfinstig gewirkt. Diesem Vorgang fehlt insofern eine Beweiskraft,
als die
Transfusion Iiicht an Schwangeren oder gar an Vorstadien der Ek-
lampsie ausgeftihrt ist. Etwa 5O~ derartiger F~lle kommeii ad exitum, bei der Sektion
werden ausgedehnte Leberver~nderungen gefunden. Klinische Beobachtungen und Untersuchungen.
Von
Prof. Th. HEYNE~AN~.
us der U~iversit~ts-Frauenklinik zu Hamburg. Eppendorfer Krankenhaus. Vergleicht man aber den Grad der ebell geschilderten Vergnde-
rungen mit dem bei anderell Erkrankllngen, die ebenfalls mit
intercerebraler Druekerh6hllng und
Hirnerscheinungen eillher-
gehen, so erhebell sich starke Bedellkell, die schweren lllld charak-
teristischen Hirnerscheinullgen bei der Eklampsie in erster Linie
durch die bei ihr bestehende intercerebrale Drllcksteigerung er-
ldS~rell zu wollen. Allem Anschein naeh kommt ihnen eine Bedeutung Itir
die St~rke und Lokalisation des HautOderrts .zu. Das ist bei
lang dauernden Stasell nnd starken Erweiterungell der ven6-
sen SchenkeI ohne weiteres verstgndlich. Klinische Beob-
achtung und die sehr ghnlichen, oft sogar iibereinstimmenden
Capillarbefunde bei sicheren Stauungs6demen weisen darauf
bin. Tr0tzdem k6nnell sie meiner Ansicht nach die eigentliche
Ursaclle der Schwangerschafts6deme llicht sein, da unter
Umstgnden jegliches erkennbares Haut6dem bei Eklampsie
Iehlt und umgekehrt selbst bei starkem Hydrops gra,ddarum
die Str6mungsver~inderungen an den Capillaren nut gering
sind. ZANG~MEISTER hat daher stets welliger Weft ant den Grad
als allf das schllelle Entstehen der Drucksteigerullg gelegt. Abet auch gegell die se Allffassung lassen sich Einw/illde erheben,
ZONDEK h
F
ll ll
i
S h
h f
i
i Abet auch gegell die se Allffassung lassen sich Einw/illde erheben,
ZONDEK hat Frallell mit Schwangerschaftsniere mit ausge-
sproehener "Wasserretention nnd C)demell groBe Mengen Fltissig-
keit zugefflhrt, ohne dab sich irgendwelthe Hirllerscheinungen
eillgestellt haben. Zahlreiche Berichte fiber die gfinstige VCirkullg voll Kochsalz-
infusionell liege/1 in der Literatur v0r. Selbst wenll es sicll hierbei:
lediglich um das Ausbleiben einer sch/idigenden Wirkung halldelt,
so ist auch schon dieses und ebenso der verh~ltnismS~Big geringe
therapelltische Erfolg der Lumbalpullktion ulld anscheinend auch
der Trepanation schlecht mit einer ausschlaggehenden Bedeutung
des I-Iirn6dems und der intercerebralen Drucksteigerung .vereinbar. C2~RL SCnROnDER und in neuester Zeit neben anderell vor
allem PAL haben die Hirnerscheinungen, die ja das Auffatlendste
bei der Eklampsie sind, dllrch Gef~il3kr/~mpfe mit nachf01gende ~
Hirnall/tmie erkl/iren wollen. ~on All~Lmie finder man bei der Capillarmikroskopie iaichts
und ebenso auch nicht am Allgenhilltergrund, und zwar allch nicht
tmmittelbar vor einem eklampfischen Anfalh Nur hei einzelllen
unserer EMan}pslen hat HENS~N dort eine'gewisse Verengerung Odem nnd intercerebrale Drn~cksteigerung k6nnen auch
lediglich fiir ein e Erkliiruiag der klinischen Hirnerscheinllngen
in Betracht k0mmen, nicht aber ffir die der Ver/~nderungen KLINISCHE
WOCHENSCHRIFT. I. JAHIRGANG. Nr. 48 2365 25. NOVEMBER 1922 wir nicht alle Erseheinungeli der Eklampsie einwandfrei er-
kl/h'eli k61inen, im weselitlich ver~nderteli Stoffwechsel der
Sehwangeren zu suchen. wir nicht alle Erseheinungeli der Eklampsie einwandfrei er-
kl/h'eli k61inen, im weselitlich ver~nderteli Stoffwechsel der
Sehwangeren zu suchen. 1,2LINISCHE
WOCHENSCHRIFT.
~. JAHRGANG.
Nr. ~8 1,2LINISCHE
WOCHENSCHRIFT. ~. JAHRGANG. Nr. ~8 25. NOVEMBER 1922 25. NOVEMBER 1922 sogar umnittelbarer und schneller in Erscheinung treten als der
Nutzen ether Ausschaltung der Sehwangerschaft. I)a der Tod h/~ufig im Anfall auch bet anscheinend nicht beson-
ders schweren F/illen erfolgt, mfissen wir ihr Auftreten durch
Fernhaltung gulBerer Reize und dureh Narkotika noch besonders
bek/impfen. g
g
Die klinische 13eobachtuflg best/~tigt dies, sie zeigt, dal3 der
operative Eingriff bisweilen Schaden und die Wendung zum Schleeh-
ten bringt. Mall mag aber die E!dampsie behandelll, wie man will;
setzt die Behandlung erst nacl~ ihrem Ausbruch ein, so kommt
sie ffir viele F~lle zu Sp/it. Ist die Geburt bereits im Gallge, so muB ihre baldige
]3eendigung trotzdem unser Ziel bleibell, weil nur dies die
Erkrankung elldgfiltig und sicher zum AbschlnB bringt, zur
Ausschaltullg der zweifellos llllgiinstig wirkendell Wehen-
t~tigkeit und well wir immer wieder auch Fglle findell, ill
denen alle anderen Mittel versagell, die Entbindung aber die
Heilllng herbeif/ihrt. p
Wollen wir bier mit den uns zur Verffigung stehenden
Mitteln wetter kommen, so muB die Behandlung wenn irgend
m6glich frfiher einsetzen. g
Tretell bereits pr~Leklamptische Erscheinungen auf, so ist
kein Augenblick zu vers/~umen, da die Eklampsie oft un-
mittelbar folgt. g
Neben einer baldigen mfissen wir aber nach dem Gesagten
auch eine schonende Entbindullg anstreben. g
Der AderlaB lgBt die pr/ieldamptischen Symptome meist
verschwinden, llicht selten aber kehrell sie wieder. Solche
F/ille eriordern aufmerksamste klinische Beobachtullg. Einig sind daher alle Geburtshelfer in der Anwendung der
Zange, sobald sie ohne Schwiefigkeiten durchffihrbar ist. Nicht sehr viel eingreifender ist die Kolpohysterotomia anterior
bet Mehrgebtxenden. Schwilldell die pr/ieklamptischen Symptome (Kopfweh,
Sehst6rung, L?belkeit, Schmerzen ill der Nierengegend und im
Epigastrium) nicht schnell, wiederholen sie sich oder weisen
wit im Serum h/imolytische Prozesse llach, so wird sofort ent-
bunden, da sonar der I. Anfall hgufig der Vorbote des Todes ist. Schwilldell die pr/ieklamptischen Symptome (Kopfweh,
Sehst6rung, L?belkeit, Schmerzen ill der Nierengegend und im
Epigastrium) nicht schnell, wiederholen sie sich oder weisen
wit im Serum h/imolytische Prozesse llach, so wird sofort ent-
bunden, da sonar der I. Anfall hgufig der Vorbote des Todes ist. In der gleichen Weise behandelll wir die Schwanger-
schaftseMampsie. Morphium dient zur Unterdriickung einer
etwa auftretenden Wehent~tigkeit. g
Schwieriger ist die Entscheidung bet Erstgebgrenden. W'enn m6glich, versuchen wit hier mit der abwartenden Be-
handlung auszukommen. ORIGINALIEN. L/~Bt man aus ether mit einem Reservoir versehenen
Pipette ein bestimmtes Fliissigkeitsvolumen austropfen, so
ist die Gr6Be und damit die Zahl der Tropien abhiing]g yon der
Oberfl/ichenspannullg der Flfissigkeit. Tributyrin erniedrigt
die Oberf!Xchenspannllng des Wassers, wie alle Fette, bedeu-
tend. Wird das Tributyrin aber durch Lipase aufgespalten,
so erh6ht sich die Oberfl/ichenspannung wieder, da die Spalt-
produkte dieselbe llicht beeinflussen. Man kann somit aus der
Tropfenzahl eines bestimmten Volumens Tributyrin]6sung
die Konzentration dieser bestimmen ulld gegebenellfalls die
fermentative Aufspaltung des Fettes verfolgen. 1111 einzehllell
gestalter sich die Versuchsanordnung folgendermagen: Eille
ges/ittigte Tributyrinl6sung wird hergestellt durch mehr-
stiindiges Schfitteln yon 4--5 Tropfen Tributyrin ill I 1 Aqua
dest. Die L6sllllg wird filtriert, um die ungel6sten Fettreste
zu entfernell. Zu je 3 ccm Serum werden 3 ccm einer Puffer-
mischung (ein Tell priln/~res m/3 Natrillmphosphat + 14 Teile
seknnd/tres m/8 Natriumphosphat, PH ~- 7,6) gegeben und das
eille mit I ecm eiller o,2 proz. L6sung yon Chillin. hydrochlor.,
das andere mit i ccm Aqua dest. versetzt. Letzteres dient
als Kontrolle. Die angegebene Menge Chinin, 2 rag, genfigt
zur v611igell Vergiftung der Serumlipase, w/ihrend Leberlipase
davoll gar nicht beeinfluBt wird. Nach etwa 1/2 Stunde gieBt
man diese Mischullg zu 5 ~ ccm der ges~ttigten Tributyrin-
16sung, ~otiert die Zeit llnd bestimmt die Tropfenzahl sofort
und in gewissen Zeitabst/illdell nach 3 o, 6o, 9o Milluten wieder. War nut die normMe Lipase im Serum vorhallden, so dart
keine ~nderung der Tropfenzahl eintreten, da die Chinillmenge
gelliigte, um das Ferment v611ig zu hemmen. Andererseits 1,2LINISCHE
WOCHENSCHRIFT.
~. JAHRGANG.
Nr. ~8 Nur wenn ein deutlicher Erfolg nicht
zutage tritt nlld die Geburt voraussichtlicti noch sehr lange dauern
~vird, machell wit den cervicalen Kaiserschnitt. g
g
In der gleichen Weise behandelll wir die Schwanger-
schaftseMampsie. Morphium dient zur Unterdriickung einer
etwa auftretenden Wehent~tigkeit. Bet der abwartenden Behandlung steht der Aderlag ganz im
Vordergrulld, bet dem richtige Dosierung (etwa 5oo ccm) und recht-
zeitige Wiederholung yon grol3er Bedeutung stud. g
Aueh bet der 13ehandlung der Schwallgerschaftsniere muB
die Prophylaxe gegeniiber der E!dampsie in den Vordergrund
treten. Es ist eine bekanllte Tatsache, dab das Auftreten eir/er
Eldampsie bet ether sachgem/~B behandelten Schwangerschafts-
niere verh/iltnism/iBig selten ist. Das Wichtigste ist auch hier die genaue Beobachtung der IZrank-
heitserscheinungen. Daneben versuchen wir aus capillarmikro-
skopischen Befunden und t3estimmungen yon Blutdruck und Hgmo-
globin den richtigen Weg Zu linden. .~ Die Erfahrungell der Kriegsjahre haben dazu geffihrt,
diese Prophylaxe auch auf die gesunde Schwangere auszu-
dehnen. Es ist ihnen bisweilen vegetarische Di/it vorgeschrie-
ben. Ich halte aber die Verhinderung ether fibermgBigen Ei-
weiBerllghrung ffir ausreichend. Was die Lumbalpunktion anbelangt, so kommt eine giinstige
Beeinflussung der ttirnerseheinungen dabei vor, man erlebt aber
auch das Gegenteit, es sind geradezu Todesf/tlle dabei beobachtet. Da der AderlaB mindestens das gleiche, vielmehr sogar Besseres
leistet, abet nieht schadet, so muB ieh reich auf das bestimmteste
gegen die Lumbalpunkfion ausspreehen. g
g
Die gr613te Schwierigkeit liegt darin, dab die Frauen
leiehten St6rungen der Schwangerschaft keine Beachtung
schenken ulld sich deshalb nicht immer all eillen Arzt wellden. Intraven6se Injektionen silld bet der Igklampsie in der ver-
schiedensten Form zur Anwendnng gekommen (Kochsalz-, iRinger-
und Zuckerl6sung, Enzytol usw.), ttier m6ehte ich aber zu aller-
gr6Bter Vorsicht mahnen. Eine m6glichst frfihzeifige t3ehandlullg ist abet yon be-
sollderer Bedeutung. Tretell Blutdrucksteigerungen oder
pr/ieMamptische Symptome au{, so ist die sofortige uber-
fiihrung ill eine Klinik unbedingt erforderlieh. Das Gef/iBsystem befindet sich angellscheinlich in einem Zustand
h6chster Reizbarkeit und Labilitgt, dab mit pl6tzlicher~Sehoek-
wirkung bis zum Exitus gereehnet werden muB. Klinische Beobachtungen und Untersuchungen.
Von
Prof. Th. HEYNE~AN~.
us der U~iversit~ts-Frauenklinik zu Hamburg. Eppendorfer Krankenhaus. Die Bedeutung der Nierelierscheinungen liegt: i n erster
Linie ~uf diagn0stischem Gebiet~ (Differentialdiagr~0se gegen-
~fiber anderen Krampfzustiiliden) undin der Anzeige tfir pro-
phylakt~sche MaBnahmen vor Ausbruch der Eklampsie: Es liegt in dem wechselvollen Verlauf und der Unsicherheit
der Prognosenstellung begrfindet, wenn die Zahl der thera-
peutischen Vorschliige gerade bet der Eklampsie so ungeheuer
groB ist. Der gfinstige Verlauf einer kleineren Anzahl F/file
beweist bier nicht das geringste f fir den Wert einer Behand-
lungsmethode. In neuester Zeit wird besonderer Wert auf das Bestehen einer
Acidosis und auf koIloidchemische Vorg~inge gelegt. Eine deutliche Acidosis ist vorhanden. Wit findeii sie abet
ebenso stark und st~irker bet anderen Erkrankuiigen, ohne dab
eklamptische Erscheinungen uiid Veriinderungen auftreten. Es besteht ein vermehrtes Auftreten mancher intermedi~irer
Abbauprodukte des EiweiBstoffwechsels. Abet aueh sie sind bisher
lediglich in einem Grade und eiller Form nachgewiesen, dab daraus
noch nicht eine Erkl~rung der Eldampsieerscheinungen erfolgen
kann. Von den vielen Vorschl/igen steheii 2 im Vordergruiid, die
s0fortige operative Schiielleiitbindung und die abwartende Be~
handlung niit AderlaB und Narkotika (ZwEIFEI.-STROGANOW). Zwischen beiden steht die von ENGELMANN SO genannte Therapie
der mittleren Linie, bet der zun&chst abgewartet, beim Ausbleiben
elnes Erfolges abet operiert wird. Eklampsie ist keine echte UrXmie, es besteht nur elite geringe
Steigerung des Rest-N und auch nicht in allen F/ilien. Auch bet der Gestations- und der sog. eklamptischen Ur/imie
der Nierenerkrankungen muB es sich letzten Endes um verschiedene
Prozesse handeln, wenn aueh elite Sehr weitgehende Ubereinstim-
mung der kllnischeii Erscheiniingen vorhanden ist. Sie l~13t sich
vieUeicht z. T. durch alas Hervortreten vasomotorischer Vorg~inge
und intercerebraler Druckerh6hung bet beiden ZiistSmden erM~ren. Da die letzte Ursache der Eklampsie in der Schwaiigerschaft
liegen muB, ist die Ausschaltung der Schwangerschaft sehr n~/he-
Iiegend. g
Schon das Vorkommen der VVochenbettseklampsie zeigt abet,
dag diese Ausschaltung nicht sofort und Iiicht sichere Heiluiig
bringen kanii. Das Wesen der Eklampsie stellt auch heute noch ein un-
gel6stes Problem dar. Bet einem so schweren Krankheitszustande kann auch ein
schwergr operatlver Eingriff, wie ihn die sofortige Entbindung
h~ufig darstellt, und eine laiige Narkose Iiieht gleichgiiltig seth. Die schSdigende Schockwirkung des Eingriffes wird untef UmstS.nden 13el dem heutigen Stande unserer Kenntnisse ist es das
Naheliegendste, die toxische Substanz, ohne deren Annahme 2366 (Ein Beitrag zur Diagnose yon Organerkrankungen.)
V Prof. P. RONA, Dr. H. P~.TOW, H. SC~R~IBER. Au$ der chem Abtlg des patholog Inst u d I reed Klinikder CharitY Prof. P. RONA, Dr. H. P~.TOW, H. SC~R~IBER. Au$ der chem.Abtlg.des patholog.Inst. u. d. I. reed.Klinikder CharitY, Prof. P. RONA, Dr. H. P~.TOW, H. SC~R~IBER. Au$ der chem. Abtlg. des patholog. Inst. u. d. I. reed. Klinik der CharitY, Berlin u
fiber Fermeutvergiftung ergaben, dab sich ver-
schiedene Fermente gegen einzelne Gifte verschieden ver-
halter/, z. 13. Invertase gegen Chinin anders als die Lipase des
normalen Serums 1). 13esollders bemerkenswert aber war es,
dab auch Fermente, die ant dasselbe Substrat wirken, so z. B. Lipasen verschiedener Herkunft, durch Chillill ganz ver~
sehieden beeinfluBt werden. Die Lipase des normalen
Menschenserums ist gegen Chillin auBerordelltlich viel emp-
findlicher als die eines Tierserums, z. ]3. I4atze, Kallinchen
uud iVIeerschweinchen, und die Lipase, die aus der Leber zu
extrahieren ist, verhNt sich gegen Chinill v611ig refraktgr 2). Man kann also dutch die Reaktion des Fermelltes auf Chinin
die verschiede~ell Lipasen nach ihrer I-Ierkunft llnterscheiden. Es lag null der Gedanke llahe, dab bet Leberaffektiollen,
bet denen Gallellbestandteile ins Blur fibertreten, auch Leber-
lipase im Serum Vorhalldell sein k6llnte, die dureh ihre Chinin-
llnempfindlichkeit roll der llormalen Serumlipase zu llllter-
scheidell sein miil3te. Von diesem Gesichtspunkte aus haben
wir eille gr6Bere Allzahl Leberkranker ulltersneht und haben
nllsere Annahme vollauf best/itigt gefunden. Als Untersllchungsmethode diellte das yon RONA ulld
iV~ICI-IAELIS angegebene stalagmometrische Verfahren.
|
https://openalex.org/W2519371443
|
https://periodicos.utfpr.edu.br/rebrapa/article/download/3518/pdf_1
|
Portuguese
| null |
Using of flour mesocarp of babassu (Orbignya sp.) in food preparation of fresh pasta noodles type
|
Brazilian Journal of Food Research
| 2,016
|
cc-by
| 4,382
| ERROR: type should be string, got "https://periodicos.utfpr.edu.br/rebrapa Palavras-chave: Macarrão, Farinha, Babaçu. Adeval Alexandre Cavalcante Neto \nadeval@ifma.edu.br \nInstituto Federal de Educação, Ciência e \nTecnologia do Maranhão- Campus Codó Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. Página | 105 INTRODUÇÃO As massas alimentícias estão definitivamente incorporadas à dieta brasileira \ne com alto índice de aceitabilidade, servindo como prato principal ou \ncomplemento, em muitas combinações (MENEGASSI e LEONEL, 2006). O macarrão é um alimento popular e bastante consumido a nível mundial, \nprincipalmente por populações de baixa renda (NICOLETTI et al., 2008). A \ncomposição centesimal do macarrão a base de trigo é de aproximadamente 1,3 g \nde lipídio, 10,0 g de proteína, 77,9 g de carboidrato e 2,9 g de fibra alimentar. Em \ntermos de valor nutricional, apresenta-se com deficiência, pois é destaque com \nrelação a riqueza de carboidratos simples em detrimento a quantidade e \nqualidade de proteínas, além de um baixo valor de fibras (NEPA/UNICAMP, 2011; \nREIS, 2013). Essas deficiências das massas alimentícias podem ser compensadas pela \nadição de outros ingredientes de baixo custo e torná-las um item importante na \nalimentação (GARIB, 2002). Assim, é de fundamental importância que sejam \ndesenvolvidas pesquisas com matérias primas alternativas que substituam \nparcialmente a farinha de trigo na formulação de macarrões e que acrescentem \nao produto melhor qualidade nutricional. Nesse contexto uma alternativa \ninteressante, encontra-se na farinha de mesocarpo do babaçu (Orbignya sp.). O babaçu é uma palmeira encontrada na forma de conjunto denominada \nbabaçuais, sendo o Maranhão o estado brasileiro que apresenta a maior \nconcentração dessa palmeira, ocupando uma área de mais de 10 milhões de \nhectares, onde mensalmente são extraídas em torno de 140.000 toneladas de \namêndoas ((VINHAL, 2011; SANTOS; PASTORI JR., 2003; LIMA et al., 2007). O fruto, denominado coco babaçu, é formado por quatro partes: epicarpo, \nmesocarpo, endocarpo e amêndoas. O mesocarpo é a camada marrom-clara que \nse localiza depois do epicarpo, de natureza amilácea e corresponde a 20% do \npeso do fruto seco, sendo constituído basicamente de água, carboidratos (amido \ne celulose), proteínas, lipídios e sais minerais (SANTOS; PASTORI JR., 2003). É a \npartir do mesocarpo que é produzida a denominada farinha de mesocarpo do \nbabaçu. O objetivo desta pesquisa foi desenvolver tecnologia para a produção de \nmacarrão com a substituição parcial da farinha de trigo pela farinha de \nmesocarpo de babaçu, visando seu enriquecimento nutricional. RESUMO Adeval Alexandre Cavalcante Neto \nadeval@ifma.edu.br \nInstituto Federal de Educação, Ciência e \nTecnologia do Maranhão- Campus Codó \nJoão da Paixão Soares \njpsoares@ifma.edu.br \nInstituto Federal de Educação, Ciência e \nTecnologia do Maranhão- Campus Caxias \nCecília Teresa Muniz Pereira \nceciteresa@ifma.edu.br \nInstituto Federal de Educação, Ciência e \nTecnologia do Maranhão- Campus Codó \nMaron Stanley Silva Oliveira Gomes \nmaron@ifma.edu.br \nInstituto Federal de Educação, Ciência e \nTecnologia do Maranhão- Campus Bacabal \nArmando Ubirajara Oliveira Sabaa-\nSrur \nsabaasrur@gmail.com \nUniversidade Federal do Rio de Janeiro Adeval Alexandre Cavalcante Neto \nadeval@ifma.edu.br \nInstituto Federal de Educação, Ciência e \nTecnologia do Maranhão- Campus Codó O macarrão é um alimento consumido mundialmente com alto índice de aceitabilidade \nem todas as classes sociais. Por ser constituído basicamente por farinha de trigo (FT) é \nconsiderado um alimento energético, mas pobre em nutrientes e de alto índice glicêmico. Assim, é de fundamental importância que sejam desenvolvidas pesquisas com matérias \nprimas alternativas que substituam parcialmente a farinha de trigo na formulação desses \nprodutos, com vista a melhorar a sua qualidade nutricional no tocante ao teor de fibras \nalimentares. O objetivo desta pesquisa foi desenvolver tecnologia à produção de macarrão \ncom a substituição parcial da farinha de trigo pela farinha de mesocarpo de babaçu (FMB), \nvisando o seu enriquecimento nutricional. Foram preparadas quatro formulações de \nmacarrão: F0: controle (100% FT); F1: 90% FT + 10% FMB; F2: 85% FT+ 15% FMB; F3: 80% \nFT + 20% FMB. Os resultados mostraram que as formulações suplementadas com 10%, \n15% e 20% de FMB apresentaram maiores teores proteicos (5,36±0,02%, 5,89±0,02% e \n6,31±0,01%, respectivamente), do que o controle (4,16±0,07%), além de maior quantidade \nde fibras. A formulação F3 acrescentou em 127 ± 0,3% o teor desse nutriente no macarrão \nde trigo tradicional. Verificou-se ainda que os macarrões suplementados com FMB \nreduziram a perda de sólidos durante o cozimento das massas. As massas alimentícias \nelaboradas com FMB apresentaram um maior potencial nutritivo do que a massa \ntradicional e podem ser utilizadas como mais uma alternativa nutricional e de renda para \nas comunidades que vivem do extrativismo do coco babaçu. João da Paixão Soares \njpsoares@ifma.edu.br \nInstituto Federal de Educação, Ciência e \nTecnologia do Maranhão- Campus Caxias Cecília Teresa Muniz Pereira \nceciteresa@ifma.edu.br \nInstituto Federal de Educação, Ciência e \nTecnologia do Maranhão- Campus Codó Página | 105 Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. Página | 106 MATERIAL As matérias primas utilizadas na elaboração das massas alimentícias frescas \ntipo talharim (farinha de trigo especial (FT) e farinha de mesocarpo de babaçu \n(FMB) foram adquiridas no comércio local do município de Codó-MA. Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. Preparo das formulações Para elaboração dos macarrões foram feitas quatro formulações: um \ncontrole e as outras três, com proporção (FT: FMB) de 100:0, 90:10; 85:15 e \n80:20, conforme visto na Tabela 1. Tabela 1 - Preparo das formulações. Processamento \nF0 \n100% FT* \n(controle) \nF1 \n90% FT + \n10% FMB** \nF2 \n85% FT + \n15% FMB \nF3 \n80% FT + \n20% FMB \nHomogeneização \n12 minutos \n15 minutos \n16 minutos \n18 minutos \n*FT = farinha de trigo \n**FMB = farinha de mesocarpo de babaçu **FMB = farinha de mesocarpo de babaçu A diferença nos tempos de homogeneização deve-se ao fato de que a adição \nde uma maior quantidade de FMB necessita de um tempo maior de mistura, para \nobtenção de uma massa homogênea. Para tanto controlou-se a velocidade do \nequipamento e o tempo de amassamento. As farinhas foram misturadas em uma batedeira industrial, durante 5 \nminutos, enquanto ocorria a mistura, a porção de água era acrescentada aos \npoucos. O amassamento foi realizado com auxílio de uma amassadeira, semi-\nrápida, própria para massas. As etapas de moldagem ou laminação e corte foram realizadas em máquina \nde macarrão manual, na qual a massa foi prensada em cilindros, que reduziram a \nespessura até a formação de lâmina. Para isso, utilizou-se a metodologia \nadaptada de Costa e Soares Júnior (2001), onde a massa foi cilindrada por quatro \nvezes na espessura de 7 mm e ao sair dos rolos, foi esticada sobre uma mesa e \nlevemente polvilhada com farinha de trigo. Esse procedimento foi repetido nas \nespessuras de 6, 5 e 4 mm (espessura final da massa) até que a mesma \napresentasse aparência lisa, uniforme e não quebradiça. Em seguida a massa foi \nmoldada na forma de talharim e cortada manualmente para que os macarrões \nadquirissem o comprimento de 25 cm. Os macarrões foram empacotados em sacos plásticos de polietileno em \nquantidades de 100 gramas cada, sendo fechado em seladora manual. Depois \nforam armazenados sob refrigeração até a realização das avaliações. Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. ANÁLISES FÍSICO-QUÍMICAS Os teores de umidade, cinzas e lipídeos foram determinados através de \nmetodologias descritas pela Official Methods of Analysis (AACC, 1995). O teor de \nproteína foi determinado pelo método nº 14.136 da AOAC que é baseado na \nmetodologia de Kjedhal, para o cálculo do teor de proteína bruta ou total \nutilizou-se o fator de conversão de 5,70 para a farinha de trigo e 6,25 para farinha Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. de babaçu (AOAC, 2005). A determinação de carboidratos foi realizada pelo \nmétodo da diferença (AOAC, 1997). de babaçu (AOAC, 2005). A determinação de carboidratos foi realizada pelo \nmétodo da diferença (AOAC, 1997). de babaçu (AOAC, 2005). A determinação de carboidratos foi realizada pelo \nmétodo da diferença (AOAC, 1997). As determinações das frações de fibra alimentar, pelo método enzimático-\ngravimétrico, assim como as de pH e acidez, seguiram as recomendações do \nInstituto Adolfo Lutz (IAL, 2008). TESTES DE COZIMENTO Os testes de cozimento foram realizados segundo o método 16-50 da \nAmerican Association Cereal Chemists ( AACCC, 1995). O tempo de cozimento e o \naumento de massa foram obtidos conforme Paucar-Menacho et al. (2008), o \nprimeiro foi determinado pela cocção de 10 g de amostra em 140 mL de água \ndestilada em ebulição, até atingir a qualidade visual adequada em consequência \nda gelatinização do amido em toda a seção da massa. Esse ponto foi determinado \npela compressão de amostras do produto cozido, a cada 30 segundos, \nmanualmente, entre duas lâminas de vidro até o desaparecimento do eixo \ncentral. O aumento de massa foi determinado pela pesagem da amostra antes e \napós a cocção, utilizando-se o tempo de cozimento ideal de cada amostra. O \nvalor do aumento da massa foi definido como a razão entre a massa da pasta \ncozida e a massa da pasta crua (10 g), expresso em porcentagem (%). Determinou-se o aumento de volume das massas antes e após o cozimento. As amostras foram imersas em 140 mL de hexano; mediu-se então o volume de \nhexano deslocado pela massa crua e cozida, conforme Chang e Martínez flores \n(2004). Os resultados foram expressos em número de vezes que a massa \naumentou de volume após a cocção. A quantidade de sólidos na água de cozimento foi determinada pela \nevaporação de 25 mL da suspensão da água de cocção da amostra em estufa a \n105°C até peso constante, conforme Paucar-Menacho et al. (2008). ANÁLISES ESTATÍSTICAS As análises foram realizadas em triplicata e avaliou-se os resultados pelos \nmétodos de análise de variância (ANOVA) com comparação das médias pelo teste \nde Tukey com 95% de confiança, utilizando-se o software XLSTAT. Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. ANÁLISES FÍSICO-QUÍMICAS Os resultados das análises físico-químicas das massas frescas tipo talharim \nelaboradas neste experimento encontram-se aprestados na Tabela 2. Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. Tabela 2 - Resultados das análises físico-químicas das massas frescas tipo talharim \nelaboradas com diferentes concentrações de FMB. Análises \nF0 \nControle \nF1 \n10% de FMB* \nF2 \n15% de FMB \nF3 \n20% de FMB \nUmidade (%) \n32,90 ± 0,10c \n33,34 ± 0,46c \n34,11 ± 0,14b \n34,92 ± 0,06a \nCinzas (%) \n0,27 ± 0,00c \n0,28 ± 0,01bc \n0,29 ± 0,01b \n0,34 ± 0,01a \nProteínas (%) \n4,16 ± 0,07d \n5,36 ± 0,02c \n5,89 ± 0,02b \n6,31 ± 0,01a \nLipídeos (%) \n1,41 ± 0,11c \n2,13 ± 0,01b \n2,17 ± 0,01b \n2,85 ± 0,02a \nCarboidratos (%) \n61,26 ± 0,14a \n58,89 ± 0,46b \n57,54 ± 0,14c \n55,59 ± 0,15d \nFibra Solúvel (%) \n2,22 ± 0,12d \n3,44 ± 0,01c \n4,33 ± 0,40b \n5,41 ± 0,31a \nFibra Insolúvel (%) \n0,64 ± 0,18b \n0,68 ± 0,17b \n0,95 ± 0,17ab \n1,10 ± 0,32a \npH \n4,70 ± 0,00a \n5,16 ± 0,05b \n5,30 ± 0,00c \n5,77 ± 0,00d \nAcidez Titulável (mL/100g) \n3,80 ± 0,00a \n3,77 ± 0,17a \n3,93 ± 0,01a \n3,98 ± 0,00a \n*FMB = farinha de mesocarpo de babaçu. Médias seguidas de mesma letra na horizontal não diferem significativamente entre si \npelo teste de Tukey a 5% de probabilidade. Tabela 2 - Resultados das análises físico-químicas das massas frescas tipo talharim \nelaboradas com diferentes concentrações de FMB. Médias seguidas de mesma letra na horizontal não diferem significativamente entre si \npelo teste de Tukey a 5% de probabilidade. O teor de umidade das massas variou entre 32,9% (100% FT) a 34,92% (20% \nde FMB). Este resultado pode ser explicado pelo comportamento diferenciado \ndos dois tipos de farinha com relação à adsorção de água, pois a FMB apresenta \nmaior teor fibras, e por isso maior retenção de água. Todas as massas elaboradas \nse encontram dentro dos limites recomendados pela RDC nº 93/2000 da ANVISA, \nque estabelece limites máximos de 35% de umidade para as massas frescas \n(BRASIL, 2000). As amostras de macarrão com maiores quantidades de farinha de mesocarpo \nem suas composições (15% e 20%) apresentaram teores de cinzas \nsignificativamente superiores ao macarrão controle. Esses resultados são \ncoerentes, já que Silva et al. Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. Página | 109 ANÁLISES FÍSICO-QUÍMICAS (2007) analisando os minerais da farinha de \nmesocarpo, afirmaram que essa matriz é uma boa fonte de Ca, Mg, K e Fe, apesar \nde não terem enfatizado a biodisponibilidade desse minerais. Todos os macarrões adicionados de FMB diferiram estatisticamente quanto \nao teor de proteínas do macarrão controle, sendo superiores em quantidade. Os \nníveis de proteína dos macarrões aumentaram à medida que ocorreu a \nsubstituição parcial da farinha de trigo pela farinha de mesocarpo de babaçu. O \nmacarrão com adição de 10% da FMB, quando comparado com o controle, \nproporcionou acréscimo no valor proteico de 28,85%, já o adicionado de 15% de \nFMB elevou em 41,6% o teor de proteína e o macarrão com 20% de FMB \nacrescentou à massa, 51,44% o teor desse nutriente. Portanto, essa \nsuplementação proporcionou melhor incremento proteico das massas em função \nda adição da FMB. Apesar deste trabalho não revelar o perfil de aminoácidos da \nporção proteica da FMB, sabe-se que as proteínas são responsáveis pelo \ncrescimento, conservação e reparação dos nossos órgãos, tecidos e células, e \npodem ser utilizadas como fonte energética caso haja deficiências de outras \nfontes de energia. Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. Após análise constatou-se que as frações lipídicas dos macarrões adicionados \nda farinha de mesocarpo de babaçu apresentaram maiores teores desse \nnutriente, mostrando diferença significativa, em comparação ao produzido \napenas com farinha de trigo. Isso é importante do ponto de vista nutricional e \ntambém tecnológico, já que os lipídeos possuem grande importância no que diz \nrespeito à maciez do produto (MENEGASSI e LEONEL, 2006). Apesar da diferença em \nrelação ao controle estas formulações são consideradas como alimentos com \nbaixo teor lipídico. A Resolução nº 54/2012 da ANVISA, que trata do Regulamento Técnico \nreferente à Informação Nutricional Complementar, enfatiza que um alimento é \nconsiderado com baixo teor de lipídeo se contém um máximo de 3g/100g de \nmassa (BRASIL, 2012). Apesar de as formulações contendo FMB conterem maior \nteor de lipídios em comparação a controle, todas as massas alimentícias \napresentaram-se com baixo teor lipídico, podendo ser consideradas como \nalimentos saudáveis. A análise do teor de carboidratos mostrou maior quantidade no macarrão \ncontrole (61,26%) do que nos macarrões adicionados de 10, 15 e 20% de FMB, \nque obtiveram percentuais de 58,89%, 57,54% e 55,59%, respectivamente. ANÁLISES FÍSICO-QUÍMICAS Embora tenha ocorrido diferença significativa entre o macarrão controle e os \ndemais, pode-se dizer que os macarrões adicionados de farinha de mesocarpo \nmantiveram as características de um alimento energético. A quantidade mínima de carboidrato necessária para promover glicose às \ncélulas cerebrais é estimada em 130g/dia para adultos com idade superior a 19 \nanos (OMS, 2003). Sendo assim, o consumo de 100 g de macarrão suplementado \ncom 10%, 15% e 20% de FMB forneceria 76,16%, 74,80% e 72,26% das \nnecessidades diárias, respectivamente. Os teores de fibra alimentar que é constituída das porções solúveis e \ninsolúveis foi maior nos macarrões suplementados com FMB do que no controle \n(Tabela 2). A suplementação em 10% de FMB proporcionou teor de fibra \nalimentar de 4,12%, a de 15% de FMB 5,18% e a com 20% de FMB 6,51%, ao \npasso que a formulação controle apresentou teor de fibras de 2,86%. No Brasil, de acordo com a Resolução nº 54/2012 da ANVISA, um alimento \nsólido é considerado fonte de fibras alimentares se contiver em sua composição \nno mínimo de 3g de fibras/100g e se possuir 6g ou mais quantidades de \nfibras/100g é considerado alimento com alto conteúdo de fibra alimentar (BRASIL, \n2012). Assim sendo, com base nesses teores, pode-se afirmar que os macarrões \nacionados de 10% e 15% de FMB são fontes de fibra alimentar e o macarrão com \nadição de 20% de FMB é um alimento com alto teor de fibras, o que é de extrema \nimportância, pois estes produtos podem ampliar as opções dos consumidores \ncomo potenciais fontes desse elemento. TESTES DE COZIMENTO Os resultados obtidos nas análises de cozimento das massas alimentícias \ncontrole e das mistas de farinha de trigo e de mesocarpo de babaçu encontram-\nse na Tabela 3. Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. Tabela 3 - Resultados dos testes de cozimentos das massas frescas massas frescas tipo \ntalharim elaboradas com diferentes concentrações de FMB. Parâmetros \nMacarrão \nControle \n10% de FMB* \n15% de FMB \n20% de FMB \nTempo de Cozimento (min) \n8 ± 0,00b \n9 ± 0,00a \n9 ± 0,00a \n9,17 ± 0,28a \nAumento da massa (%) \n88,7 ± 0,06a \n81,3 ± 0,05b \n80,7 ± 0,05b \n80 ± 0,00b \nAumento do volume (%) \n2 ± 0,35a \n1,87 ± 0,30ab \n1,79 ± 0,30b \n1,75 ± 0,24b \nPerda de sól. solúveis (%) \n8,79 ± 0,22a \n4,44 ± 0,11b \n4,14 ± 0,23c \n3,73 ± 0,36d \n*FMB= farinha de mesocarpo de babaçu. Médias seguidas de mesma letra na horizontal não diferença significativamente entre si \npelo teste de Tukey a 5% de probabilidade. Tabela 3 - Resultados dos testes de cozimentos das massas frescas massas frescas tipo \ntalharim elaboradas com diferentes concentrações de FMB. Médias seguidas de mesma letra na horizontal não diferença significativamente entre si \npelo teste de Tukey a 5% de probabilidade. Médias seguidas de mesma letra na horizontal não diferença significativamente entre si \npelo teste de Tukey a 5% de probabilidade. Houve diferença estatística significativa com relação ao aumento de massa \ndo produto cozido, tanto para o controle, como para os demais macarrões. O aumento de massa variou de 1,75 a 2,0 sendo que o macarrão controle \napresentou a maior média. Estes resultados encontram-se de acordo com o \ndescrito por Menegassi e Leonel (2006) e Ormenese et al. (2001), segundo esses \nautores, o aumento está relacionado com outros tipos de farinha e/ou \ningredientes em sua composição, uma vez que além da farinha de trigo, quanto \nmaior a porcentagem dessas outras farinhas, menor será o aumento de volume. Os valores para a perda de sólidos variaram entre 3,73% e 8,79% para os \ndiversos tipos de macarrão elaborados. A maior perda foi da massa controle e a \nmenor do macarrão com substituição de 20% de FMB. TESTES DE COZIMENTO Este resultado é bastante \nsatisfatório e coerente, pois de acordo com Embrapa (1994) pode-se obter êxito \nna produção de massas com farinhas mistas, mas é preciso considerar as \ncaracterísticas das farinhas sucedâneas para reduzir ao máximo os efeitos dessa \nsubstituição, entre esses, a baixa perda de sólidos durante o cozimento. Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. CONCLUSÕES A substituição parcial da farinha de trigo pela FMB permitiu a obtenção de \num macarrão mais nutritivo, com maior teor proteico e principalmente, de fibras \ndo que o macarrão de trigo tradicional, podendo-se tornar em mais uma \nalternativa nutricional e de renda para as comunidades que vivem do \nextrativismo do coco babaçu. A adição da farinha de mesocarpo babaçu reduziu consideravelmente a \nperda de sólidos dos macarrões durante o cozimento, característica bastante \ndesejável do ponto de vista tecnológico. Página | 111 KEWORDS: Pasta, Flour, Babassu. Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. ABSTRACT The pasta is a consumed food a globally with high levels of acceptability in all social \nclasses. As basically consisting of wheat flour is considered an energy food but low in \nnutrients and high glycemic index. Thus, it is vitally important that research be developed \nwith alternative raw materials to replace partially the flour in the formulation of these \nproducts to improve their nutritional quality regarding the content of food fiber. The \nobjective of this study was to develop technology for the production of noodles with the \npartial replacement of wheat flour by flour babassu mesocarp (FBM), aiming at its \nnutritional enrichment. Were prepared four formulations of pasta: F1: control (100% \nwheat flour), F2: 90% flour + 10% flour mesocarp babassu oil, F3: 85% flour + 15% flour \nmesocarp babassu oil, F4: 80% flour + 20% of babassu mesocarp flour. The results \nshowed that the formulations supplemented with 10%, 15% and 20% showed higher \nprotein content (5.36%, 5.89% and 6.31% respectively), than the control (4.16%) addition \nof larger amount of fibers. The formulation F3 added in 127% of this nutrient in \ntraditional wheat pasta. It was further found that the FBM pasta supplemented with \nreduced loss of solids during the cooking of pasta. The pasta products prepared with FBM \nshowed a greater nutritional potential than traditional pasta and can be used as one \nmore nutrition alternative and income for communities who live of extraction of babassu \ncoconut. Página | 112 Página | 112 Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. 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Avaliação físico-química da farinha de mesocarpo de babaçu \n(Orbignya spp. Mart.), comercializada em municípios do Estado do Maranhão. \nHigiêne Alimentar, v. 21, n. 157, p. 86-89, 2007. Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. Página | 115 ORGANIZAÇÃO MUNDIAL DE SAÚDE. Manual das necessidades nutricionais \nhumanas. São Paulo: Atheneu, 2003 ORGANIZAÇÃO MUNDIAL DE SAÚDE. Manual das necessidades nutricionais \nhumanas. São Paulo: Atheneu, 2003 ORMENESE, R. et al. Massas alimentícias não-convencionais à base de arroz–\nperfil sensorial e aceitação pelo consumidor. Brazilian Journal of Food \nTechnology, v. 4, p. 67-74, 2001. Disponível em: < \nhttp://bj.ital.sp.gov.br/artigos/brazilianjournal/2001/p0160.pdf >. PAUCAR-MENACHO, L. M. et al. Desenvolvimento de massa alimentícia fresca \nfuncional com a adição de isolado protéico de soja e polidextrose utilizando \npáprica como corante. Ciência e Tecnologia de Alimentos, v. 28, n. 4, p. 767-\n778, 2008. Disponível em: < http://www.scielo.br/pdf/cta/v28n4/a02v28n4 >. REIS, T. A. Caracterização de macarrão massa seca enriquecido com farinha de \npolpa de pescado. 83 f. (Mestrado) Universidade Federal de Lavras. Programa de \nPós-Graduação em Ciência dos Alimentos, Lavras, 2013. SANTOS, V. E.; PASTORE JÚNIOR, F. Projeto ITTO PD 31/ 99 Revi. 3 (1). Produção não madeireira e desenvolvimento Sustentável na Amazônia. Objetivo \nEspecífico N°. 1, Resultado 1.5 Análise crítica tecnológica: Babaçu. ITTO - \nOrganização Inter- nacional de Madeiras Tropicais, UnB, LATEQ, FEPAD. Brasília. jun. 2003. 11 p. SILVA, L. G. et al. Avaliação físico-química da farinha de mesocarpo de babaçu \n(Orbignya spp. Mart.), comercializada em municípios do Estado do Maranhão. Higiêne Alimentar, v. 21, n. 157, p. 86-89, 2007. Brazilian Journal of Food Research, Campo Mourão, v. 7, n. 1, p. 105-115, jan./abr. 2016. NEPA/UNICAMP. Tabela brasileira de composição de alimentos – TACO. 4 \ned. rev e amp. Campinas: NEPA/UNICAMP, 2011. VINHAL, Jonas Oliveira. Estudo da pirólise do óleo e da amêndoa de babaçu \n(Orbignya phalerata). 76 f. (Mestrado) Universidade Federal de Viçosa, Programa \nde Pós-Graduação em Agroquímica, Viçosa-MG, 2011. Recebido: 29 jan. 2015. Aprovado: 26 fev. 2016. DOI: 10.14685/rebrapa.v7n1.3518 \nComo citar: \nCAVALCANTE NETO, A. A.; SOARES, J. P.; PEREIRA, C. T. M.; GOMES, M. S. S. O.; SABAA-SRUR, A. U. O. Utilização de Farinha de Mesocarpo de Babaçu (Orbignya Sp.) no Preparo de Massa Alimentícia Fresca \nTipo Talharim. Brazilian Journal of Food Research, Campo Mourão, v. 7, n.1, p. 105-115, jan./abr. 2016. Disponível em: <https://periodicos.utfpr.edu.br/rebrapa> \nCorrespondência: \nAdeval Alexandre Cavalcante Neto \nInstituto Federal de Educação, Ciência e Tecnologia do Maranhão- Campus Codó, Codó-MA, Brasil. Direito autoral: Este artigo está licenciado sob os termos da Licença Creative Commons-Atribuição 4.0 \nInternacional. Recebido: 29 jan. 2015. Aprovado: 26 fev. 2016. DOI: 10.14685/rebrapa.v7n1.3518 \nComo citar: \nCAVALCANTE NETO, A. A.; SOARES, J. P.; PEREIRA, C. T. M.; GOMES, M. S. S. O.; SABAA-SRUR, A. U. O. ORGANIZAÇÃO MUNDIAL DE SAÚDE. Manual das necessidades nutricionais \nhumanas. São Paulo: Atheneu, 2003 Utilização de Farinha de Mesocarpo de Babaçu (Orbignya Sp.) no Preparo de Massa Alimentícia Fresca \nTipo Talharim. Brazilian Journal of Food Research, Campo Mourão, v. 7, n.1, p. 105-115, jan./abr. 2016. Disponível em: <https://periodicos.utfpr.edu.br/rebrapa> \nCorrespondência: \nAdeval Alexandre Cavalcante Neto \nInstituto Federal de Educação, Ciência e Tecnologia do Maranhão- Campus Codó, Codó-MA, Brasil. Direito autoral: Este artigo está licenciado sob os termos da Licença Creative Commons-Atribuição 4.0 \nInternacional. Página | 115 Página | 115"
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Measurement of the track reconstruction efficiency at LHCb
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Journal of instrumentation
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DOI:
10.1088/1748-0221/10/02/P02007 DOI:
10.1088/1748-0221/10/02/P02007 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 Document Version
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Publisher's PDF, also known as Version of record Link to publication in University of Groningen/UMCG research database Citation for published version (APA):
Aaij, R., Adeva, B., Adinolfi, M., Affolder, A., Ajaltouni, Z., Akar, S., Albrecht, J., Alessio, F., Alexander, M.,
Ali, S., Alkhazov, G., Alvarez Cartelle, P., Alves, A. A., Amato, S., Amerio, S., Amhis, Y., An, L., Anderlini,
L., Anderson, J., ... LHCb Collaboration (2015). Measurement of the track reconstruction efficiency at
LHCb. Journal of Instrumentation, 10, Article 02007. https://doi.org/10.1088/1748-0221/10/02/P02007 Citation for published version (APA):
Aaij, R., Adeva, B., Adinolfi, M., Affolder, A., Ajaltouni, Z., Akar, S., Albrecht, J., Alessio, F., Alexander, M.,
Ali, S., Alkhazov, G., Alvarez Cartelle, P., Alves, A. A., Amato, S., Amerio, S., Amhis, Y., An, L., Anderlini,
L., Anderson, J., ... LHCb Collaboration (2015). Measurement of the track reconstruction efficiency at
LHCb. Journal of Instrumentation, 10, Article 02007. https://doi.org/10.1088/1748-0221/10/02/P02007 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). University of Groningen University of Groningen University of Groningen Measurement of the track reconstruction efficiency at LHCb Measurement of the track reconstruction efficiency at LHCb Aaij, R.; Adeva, B.; Adinolfi, M.; Affolder, A.; Ajaltouni, Z.; Akar, S.; Albrecht, J.; Alessio, F.;
Alexander, M.; Ali, S. Aaij, R.; Adeva, B.; Adinolfi, M.; Affolder, A.; Ajaltouni, Z.; Akar, S.; Albrecht, J.; Alessio, F.;
Alexander, M.; Ali, S. Published in: Published in:
Journal of Instrumentation PUBLISHED BY IOP PUBLISHING FOR SISSA MEDIALAB PUBLISHED BY IOP PUBLISHING FOR SISSA MEDIALAB RECEIVED: August 7, 2014
REVISED: December 6, 2014
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PUBLISHED: February 12, 2015 RECEIVED: August 7, 2014
REVISED: December 6, 2014
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PUBLISHED: February 12, 2015 2015 JINST 10 P020 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-
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number of authors shown on this cover page is limited to 10 maximum. Download date: 24-10-2024 PUBLISHED BY IOP PUBLISHING FOR SISSA MEDIALAB c
⃝CERN 2015 for the benefit of the LHCb collaboration, published under the terms
of the Creative Commons Attribution 3.0 License by IOP Publishing Ltd and Sissa
Medialab srl. Any further distribution of this work must maintain attribution to the author(s) and the
published article’s title, journal citation and DOI. doi:10.1088/1748-0221/10/02/P02007 Measurement of the track reconstruction efficiency
at LHCb JINST 10 P02007 The LHCb collaboration doi:10.1088/1748-0221/10/02/P02007 doi:10.1088/1748-0221/10/02/P02007 Contents
1
Introduction
1
2
Detector and software description
2
3
Track reconstruction at LHCb
3
4
Tag-and-probe methods
4
4.1
VELO method
4
4.2
T-station method
5
4.3
Long method
6
5
Trigger and selection requirements
6
5.1
Mass resolution
7
6
Calculation of efficiency
8
7
Efficiency dependencies
9
7.1
Comparison of magnetic field polarities
10
7.2
Dependencies of track reconstruction efficiency
10
7.3
Efficiency ratios
10
8
Systematic uncertainties
15
9
Hadronic interactions
15
10 Conclusion
16
The LHCb collaboration
19 Contents
1
Introduction
2
Detector and software description
3
Track reconstruction at LHCb
4
Tag-and-probe methods
4.1
VELO method
4.2
T-station method
4.3
Long method
5
Trigger and selection requirements
5.1
Mass resolution
6
Calculation of efficiency
7
Efficiency dependencies
7.1
Comparison of magnetic field pola
7.2
Dependencies of track reconstructi
7.3
Efficiency ratios
8
Systematic uncertainties
9
Hadronic interactions
10 Conclusion
The LHCb collaboration Contents
1
Introduction
1
2
Detector and software description
2
3
Track reconstruction at LHCb
3
4
Tag-and-probe methods
4
4.1
VELO method
4
4.2
T-station method
5
4.3
Long method
6
5
Trigger and selection requirements
6
5.1
Mass resolution
7
6
Calculation of efficiency
8
7
Efficiency dependencies
9
7.1
Comparison of magnetic field polarities
10
7.2
Dependencies of track reconstruction efficiency
10
7.3
Efficiency ratios
10
8
Systematic uncertainties
15
9
Hadronic interactions
15
10 Conclusion
16
The LHCb collaboration
19 2015 JINST 10 P020 5 JINST 10 P02007 The LHCb collaboration 19 The LHCb collaboration E-mail: michel.de.cian@cern.ch ABSTRACT: The determination of track reconstruction efficiencies at LHCb using J/ψ →µ+µ−
decays is presented. Efficiencies above 95% are found for the data taking periods in 2010, 2011,
and 2012. The ratio of the track reconstruction efficiency of muons in data and simulation is com-
patible with unity and measured with an uncertainty of 0.8% for data taking in 2010, and at a
precision of 0.4% for data taking in 2011 and 2012. For hadrons an additional 1.4% uncertainty
due to material interactions is assumed. This result is crucial for accurate cross section and branch-
ing fraction measurements in LHCb. KEYWORDS: Large detector-systems performance; Pattern recognition, cluster finding, calibration
and fitting methods; Performance of High Energy Physics Detectors ARXIV EPRINT: 1408.1251 c
⃝CERN 2015 for the benefit of the LHCb collaboration, published under the terms
of the Creative Commons Attribution 3.0 License by IOP Publishing Ltd and Sissa
Medialab srl. Any further distribution of this work must maintain attribution to the author(s) and the
published article’s title, journal citation and DOI. 1
Introduction The track reconstruction efficiency is an important quantity in many physics analyses, especially
those that aim at measuring a production cross section or a branching fraction. The uncertainty on
the track reconstruction efficiency was a source of large systematic uncertainties with early LHCb
data [1]. The method presented in this paper has significantly reduced this uncertainty for recent
measurements [2]. In physics analysis, the track reconstruction efficiency is usually estimated with simulated
events. To take possible differences between simulation and data into account, a data-driven cor-
rection procedure is applied. A clean sample of J/ψ →µ+µ−decays is selected in data with a
tag-and-probe approach. J/ψ →µ+µ−decays are ideal candidates for efficiency measurements as – 1 – they are abundant, clean, and the decay products cover the momentum spectrum needed in most
physics analyses in LHCb. The purity of the sample is enhanced by selecting J/ψ from b-hadron
decays. The tag track is fully reconstructed and is well identified as a muon. The probe track is
only partially reconstructed, not using information from at least one subdetector which is probed. The track reconstruction efficiency is determined by checking for the existence of a fully recon-
structed track corresponding to the probe track as this allows to determine the efficiency of the
subdetector that is not used in the reconstruction of the probe track. It is calculated as a function of
the momentum of the probe track, p, its pseudorapidity, η, and the track multiplicity of the event,
Ntrack. These are chosen because the efficiency is most affected by them. No strong dependence on
the polar angle φ is observed. The main result of this paper is the track reconstruction efficiency
ratio between data and simulation for prompt tracks and tracks from B and D mesons. This ratio
is used in physics analyses to correct the track reconstruction efficiency in simulated events and to
determine its uncertainty. The measurement is performed on several data samples to meet the re-
quirements of the analyses performed at LHCb. 1
Introduction In this paper, the results are presented for the three
data samples from run I, corresponding to different running conditions, proton-proton (pp) centre-
of-mass energies and integrated luminosities: data taken in 2010 at √s = 7 TeV corresponding to
29 pb−1, data taken in 2011 at √s = 7 TeV corresponding to 1 fb−1, and data taken in 2012 at √s =
8 TeV corresponding to 2 fb−1. The 2010 results are valid for the full 2010 data set, corresponding
to a luminosity of 37 pb−1, since the same running conditions and track reconstruction were used
throughout this period. 2015 JINST 10 P02 5 JINST 10 P02007 2
Detector and software description The LHCb detector [3] is a single-arm forward spectrometer covering the pseudorapidity range
2 < η < 5, designed for the study of particles containing b or c quarks. The detector includes a
high-precision tracking system consisting of a silicon-strip vertex detector, VELO [4], surround-
ing the pp interaction region; a large-area silicon-strip detector, TT [5], located upstream of a
dipole magnet with a bending power of about 4Tm; and three stations of silicon-strip detectors
(Inner Tracker) [6] and straw drift tubes (Outer Tracker) [7] placed downstream of the magnet,
called T stations. The tracking system provides a measurement of momentum, p, with a rela-
tive uncertainty that varies from 0.4% at low momentum to 0.6% at 100GeV/c. The minimum
distance of a track to a primary vertex (PV), the impact parameter (IP), is measured with a res-
olution of (15 + 29/pT)µm, where pT is the component of p transverse to the beam, in GeV/c. The polarity of the dipole magnet is reversed periodically throughout data taking. The config-
uration with the magnetic field vertically upwards (downwards), bends positively (negatively)
charged particles in the horizontal plane towards the centre of the LHC. Different types of charged
hadrons are distinguished using information from two ring-imaging Cherenkov detectors, RICH1
and RICH2. Photon, electron, and hadron candidates are identified by a calorimeter system con-
sisting of scintillating-pad and preshower detectors, an electromagnetic calorimeter, and a hadronic
calorimeter. Muons are identified by a system composed of alternating layers of iron and multi-
wire proportional chambers [8]. The trigger [9] consists of a hardware stage, based on informa-
tion from the calorimeter and muon systems, followed by a software stage, which applies a full
event reconstruction. – 2 – Figure 1. Tracking detectors and track types reconstructed by the track finding algorithms at LHCb. 2015 JINST 10 P02 Figure 1. Tracking detectors and track types reconstructed by the track finding algorithms at LHCb. In the simulation, pp collisions are generated using PYTHIA 6.4 [10] with a specific LHCb
configuration [11]. Decays of hadronic particles are described by EVTGEN [12], in which final
state radiation is generated using PHOTOS [13]. The interaction of the generated particles with
the detector and its response are implemented using the GEANT4 toolkit [14, 15] as described in
ref. [16]. Hit inefficiencies, e.g. due to dead channels, are typically in the range 1-2% and are in-
cluded in the simulation. 2
Detector and software description Differences in the positioning of the sensors between data and simulation
are at the level of 0.5mm. Both effects have a negligible impact on the tracking efficiency. The
simulated events used in this study are required to contain at least one J/ψ →µ+µ−decay. 5 JINST 10 P02007 Differences in the response of the detectors in simulation and data could potentially lead to
a different behaviour of the track reconstruction. The hit efficiencies have been measured in data
using tracks. For the different subdetectors, they range from 98-100%. Dead channels are included
in the simulation, using an average over the data taking period. From simulations it is known that
the (high) hit efficiency does not have any impact on the track reconstruction, as the algorithms have
been written to be robust against small hit inefficiencies. The size of the sensitive detector elements
are known very accurately and the positioning of the sensitive elements in the simulation is accurate
at the level of 0.5mm. Compared to the overall size of the tracking system, any inaccuracy at this
level has negligible impact on the acceptance of the detector. 3
Track reconstruction at LHCb Owing to the design of the LHCb detector, which consists of tracking detectors mainly outside the
magnetic field, charged particle tracks are in approximation straight line segments in the upstream
part (VELO and TT) and in the downstream part (T stations). Figure 1 shows an overview of the
different track types defined in the LHCb reconstruction: VELO tracks, which have hits in the
VELO; upstream tracks, which have hits in the two upstream trackers; T tracks, which have hits in
the T stations; downstream tracks, which have hits in TT and the T stations; and long tracks, which
have hits in the VELO and the T stations. The latter tracks can additionally have hits in TT. If a particle is reconstructed more than once, as different track types, only the track best suited
for analysis purposes is kept. Hereby, long tracks are preferred over any other track type, upstream
tracks are preferred over VELO tracks, and downstream tracks are preferred over T tracks. The – 3 – number of unique tracks in an event, Ntrack, is used in this study as a measure for the event multi-
plicity; it is strongly correlated with the number of hits in the tracking detectors. The number of
tracks is chosen over the number of hits in a tracker to give a balanced measure of the upstream
and the downstream occupancy. The reconstruction of long tracks starts with a search for VELO tracks [17, 18]. VELO tracks
are reconstructed exploiting the fact that tracks form straight lines due to the absence of a magnetic
field in the VELO. Two algorithms promote these VELO tracks to long tracks. The first algorithm,
called forward tracking [19], combines VELO tracks with hits in the three T stations. For a given
VELO track and a single hit in one of the T stations the momentum is fixed, enabling the algorithm
to project hits in the T stations along the trajectory. Hits which form clusters in the projection are
used to define the final long track. In the second algorithm, called track matching [20, 21], long
tracks are made combining VELO tracks with T tracks, which are found by a standalone track
finding algorithm [22]. 2015 JINST 10 P02 If hits compatible with the long track trajectory are found in TT, they are added to the track
to improve the momentum resolution and as discrimination against fake tracks. 3
Track reconstruction at LHCb This procedure is
identical for the forward tracking and the track matching. JINST 10 P02007 Most analyses use long tracks because they provide the best momentum and spatial resolution
among all track types. Unless otherwise stated, track reconstruction at LHCb refers to the recon-
struction of long tracks. In a typical signal triggered event in 2011 or 2012, around 60 long tracks
are reconstructed. Other track types, such as downstream tracks [23], are used for the reconstruc-
tion of decay products of long-lived particles such as K0
S mesons, or for internal alignment of the
tracking detectors. They are reconstructed from T tracks, which are propagated back through the
magnetic field to find corresponding hits in the TT stations. The efficiency to reconstruct charged particles as long tracks is determined in two approaches. The first approach measures the track reconstruction efficiency in the VELO and in the T stations
individually and combines these efficiencies to a single measurement. The second approach deter-
mines the efficiency to reconstruct a long track directly. 4
Tag-and-probe methods The tag-and-probe method uses two-prong decays, where one of the decay products, the “tag”, is
fully reconstructed as a long track, while the other particle, the “probe”, is only partially recon-
structed. The probe should carry enough momentum information that the invariant mass of the
parent particle can be reconstructed with a sufficiently high resolution. The invariant mass of the
two-prong decay allows for a discrimination against background. The track reconstruction effi-
ciency for long tracks is then obtained by matching the partially reconstructed probe track to a long
track. If a match is found, the probe track is defined as efficient. The three methods described
below all use J/ψ →µ+µ−decays, as the daughter particles have information in the muon system
which can be exploited in the reconstruction of the probe track. The approaches, however, use
different combinations of tracking detectors for the partial reconstruction of the probe track. 4.1
VELO method The track reconstruction efficiency in the VELO is measured using downstream tracks as probes, as
illustrated in figure 2(a). A downstream track and a long track of the same muon do not necessarily – 4 – (a)
(b)
(c)
Figure 2. Illustration of the three tag-and-probe methods: (a) the VELO method, (b) the T-station method,
and (c) the long method. The VELO (black rectangle), the two TT layers (short bold lines), the magnet coil,
the three T stations (long bold lines), and the five muon stations (thin lines) are shown in all three subfigures. The upper solid blue line indicates the tag track, the lower line indicates the probe with red dots where hits
are required and dashes where a detector is probed. (a)
(b)
(c) (a) 2015 JINST 10 P020 (b) JINST 10 P02007 Figure 2. Illustration of the three tag-and-probe methods: (a) the VELO method, (b) the T-station method,
and (c) the long method. The VELO (black rectangle), the two TT layers (short bold lines), the magnet coil,
the three T stations (long bold lines), and the five muon stations (thin lines) are shown in all three subfigures. The upper solid blue line indicates the tag track, the lower line indicates the probe with red dots where hits
are required and dashes where a detector is probed. share all hits in the T stations. Therefore, a probe track is considered to be found as a long track
if there is a long track with at least 50% common hits in the T stations. In simulated events the
fraction of 50% common hits is found to be an appropriate and stable matching criterion. 4.2
T-station method The measurement of the track reconstruction efficiency in the T stations for particles that have
VELO and muon segments is illustrated in figure 2(b). A dedicated algorithm reconstructs muons
as straight tracks starting from hits in the last muon station, see for example refs. [24, 25]. These
are subsequently matched to VELO tracks. A long track is considered to be matched to a probe track if two requirements are met. Firstly,
the probe track and the long track have to be reconstructed from the same VELO seed. Secondly, at – 5 – Table 1. Settings of the software trigger selection as a function of data taking period. Only the tag muon
required to pass the selection. For more information see refs. [9, 26–28]. Table 1. Settings of the software trigger selection as a function of data taking period. Only the tag muon is
required to pass the selection. For more information see refs. [9, 26–28]. required to pass the selection. For more information see refs. [9, 26 28]. 2010
2011
2012
2012
(first 0.7 fb−1)
(remaining 1.3 fb−1)
IP > 0.11mm
IP > 0.5mm
IP > 0.5mm
IP > 0.5mm
χ2
IP > 16
χ2
IP > 200
χ2
IP > 200
χ2
IP > 200
p > 8.0GeV/c
p > 8.0GeV/c
p > 8.0GeV/c
p > 3.0GeV/c
pT > 1.0GeV/c
pT > 1.3GeV/c
pT > 1.3GeV/c
pT > 1.3GeV/c
χ2/ndf(track) < 2
χ2/ndf(track) < 2
χ2/ndf(track) < 2.5
χ2/ndf(track) < 2.5 2015 JINST 10 P02 least two hits on the probe track in the muon stations have to be compatible with the extrapolation
of the long track into the muon stations. It is found in simulated events that requiring two common
hits in the muon stations is sufficient to ensure compatible trajectories of the long track and the
VELO-muon probe track. JINST 10 P02007 4.3
Long method The long method uses probe tracks that have hits in the TT and in the muon stations as illustrated
in figure 2(c). This method measures the efficiency to reconstruct long tracks because the long-
track-finding algorithms do not require the presence of TT hits. Therefore, the efficiency to find
a long track is, to first order, independent of the efficiency to find such a TT-muon track. These
(TT-muon) tracks are found by a dedicated reconstruction of tracks in the muon stations, which
are subsequently matched to TT hits. A TT-muon track is considered to be reconstructed as a long
track in case more than 70% of the hits in the muon stations are compatible with the extrapolation
of the long track into the muon stations. In case the long track has TT hits, it needs to share at least
60% of the TT hits as well. These fractions have been optimised in simulation and the results are
stable with respect to small differences in data and simulation. 5
Trigger and selection requirements VELO
T-station
Long
method
method
method
Tag
Long track
used in single muon trigger
DLLµπ > 2
DLLµπ > 2
χ2/ndf(track) < 5
χ2/ndf(track) < 3
χ2/ndf(track) < 2
p > 5.0GeV/c
p > 7.0GeV/c
p > 10GeV/c
pT > 0.7GeV/c
pT > 0.5GeV/c
pT > 1.3GeV/c
IP > 0.5mm
Probe
Downstream track
VELO-muon track
TT-muon track
p > 5.0GeV/c
p > 5.0GeV/c
p > 5.0GeV/c
pT > 0.7GeV/c
pT > 0.5GeV/c
pT > 0.1GeV/c
J/ψ
Mµµ ∈[2.9,3.3]GeV/c2
Mµµ ∈[2.7,3.5]GeV/c2
Mµµ ∈[2.6,3.6]GeV/c2
χ2/ndf(vertex) < 5
χ2/ndf(vertex) < 5
χ2/ndf(vertex) < 5
NJ/ψ = 1
NJ/ψ = 1
NJ/ψ = 1
p > 7.0GeV/c
IP < 0.8mm
software trigger in order to avoid any bias on the track reconstruction efficiency, caused by fully
reconstructing the two-prong decay with two long tracks. Table 2. Selection requirements on the tag and probe tracks and on the combination into a J/ψ candidate for
the three different methods. 2015 JINST 10 P02 JINST 10 P02007 software trigger in order to avoid any bias on the track reconstruction efficiency, caused by fully
reconstructing the two-prong decay with two long tracks. Further selection criteria are applied as listed in table 2: the χ2/ndf from the track fit of
the tag tracks must be small to reduce the number of fake tracks. Tag tracks have to fulfil the
standard muon selection, which requires hits in the muon stations in a search window around the
track extrapolation as explained in ref. [29]. Both the tag and probe tracks have minimal p and
pT requirements to remove badly reconstructed tracks and combinatorial background. In order to
remove contamination from hadrons, the particle identification system is used. The differences
between the logarithm of the likelihood of the tag to be a muon and to be a pion, DLLµπ, is
computed and only tag tracks with a high DLLµπ are used. The range of the invariant mass of
the µ+µ−combination, Mµµ, is chosen sufficiently large to estimate the background contribution
from the mass sidebands. Finally, the χ2/ndf from the vertex fit of the tag- and the probe-track
has to be small, in order to remove combinatorial background; and the number of J/ψ decays
per event (NJ/ψ ) must be one, to simplify the association procedure described in the preceding
subsections. 5
Trigger and selection requirements Additionally, the T-station method only considers J/ψ candidates with a momentum
greater than 7GeV/c, and the long method only J/ψ candidates with an IP smaller than 0.8mm, as
both selections are effective in reducing background contamination without biasing the efficiency
determination. After the full selection chain the sample amounts to about 6000 decays for 2010
for the long and the T method, while for the VELO method 12000 decays are selected. The
2011 and 2012 data samples comprise more than 300000 decays in total for all methods and data
taking periods. 5
Trigger and selection requirements The candidate decays are first required to pass a hardware trigger, which selects muons in the muon
system with a transverse momentum, pT > 1.48GeV/c, or dimuons where the product of the two
transverse momenta is greater than pT1 × pT2 > (1.296GeV/c)2. In 2012 these thresholds have
been raised to pT > 1.76GeV/c and pT1 × pT2 > (1.6GeV/c)2, respectively. The reconstruction
of both muons in the hardware trigger does not bias the determination of the track reconstruction
efficiency since it does not use information from the tracking system (VELO, TT, and T stations). The subsequent software stage reconstructs the tag muon in the entire tracking system and in
the muon system. The tag muon is required to have high pT, high p, large IP and χ2
IP with respect
to all PVs in the event, where χ2
IP is defined as the difference in χ2 of a given PV reconstructed
with and without the considered track. Furthermore, a good χ2 per degree of freedom (χ2/ndf) of
the trigger track fit is required. Different selection criteria are used during data taking as listed in
table 1 to fit different data taking conditions. The IP and χ2
IP requirements restrict the sample to
J/ψ originating from b hadron decays. Only the tag muon is required to be reconstructed in the – 6 – Table 2. Selection requirements on the tag and probe tracks and on the combination into a J/ψ candidate for
the three different methods. VELO
T-station
Long
method
method
method
Tag
Long track
used in single muon trigger
DLLµπ > 2
DLLµπ > 2
χ2/ndf(track) < 5
χ2/ndf(track) < 3
χ2/ndf(track) < 2
p > 5.0GeV/c
p > 7.0GeV/c
p > 10GeV/c
pT > 0.7GeV/c
pT > 0.5GeV/c
pT > 1.3GeV/c
IP > 0.5mm
Probe
Downstream track
VELO-muon track
TT-muon track
p > 5.0GeV/c
p > 5.0GeV/c
p > 5.0GeV/c
pT > 0.7GeV/c
pT > 0.5GeV/c
pT > 0.1GeV/c
J/ψ
Mµµ ∈[2.9,3.3]GeV/c2
Mµµ ∈[2.7,3.5]GeV/c2
Mµµ ∈[2.6,3.6]GeV/c2
χ2/ndf(vertex) < 5
χ2/ndf(vertex) < 5
χ2/ndf(vertex) < 5
NJ/ψ = 1
NJ/ψ = 1
NJ/ψ = 1
p > 7.0GeV/c
IP < 0.8mm
software trigger in order to avoid any bias on the track reconstruction efficiency, caused by fully
reconstructing the two-prong decay with two long tracks. Table 2. Selection requirements on the tag and probe tracks and on the combination into a J/ψ candidate for
the three different methods. 5.1
Mass resolution To illustrate the mass resolutions that can be achieved, the dimuon invariant mass distributions
from J/ψ candidates in the three methods are shown in figure 3 using the 2011 data sample. The – 7 – ]
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Figure 3. Invariant mass distributions for reconstructed J/ψ candidates from the 2011 dataset. The solid
line shows the fitted distribution for signal and background, the dotted line is the signal component. The
subfigures are (a) the VELO method, (b) the T-station method, (c) the long method. For comparison of
resolution and signal purity (d) shows the invariant mass distribution of J/ψ candidates obtained with the
standard reconstruction at LHCb. ]
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Long method 5 JINST 10 P02007 Figure 3. Invariant mass distributions for reconstructed J/ψ candidates from the 2011 dataset. The solid
line shows the fitted distribution for signal and background, the dotted line is the signal component. The
subfigures are (a) the VELO method, (b) the T-station method, (c) the long method. 5.1
Mass resolution For comparison of
resolution and signal purity (d) shows the invariant mass distribution of J/ψ candidates obtained with the
standard reconstruction at LHCb. difference in the visible ranges in figure 3(a) compared with the other distributions in figure 3
is a consequence of the different dimuon invariant mass cuts as listed in table 2. The invariant
mass distribution using two long tracks is shown in figure 3(d) for comparison. The signal peak
is fitted with the sum of two Gaussian functions for this illustration. The effective mass resolution
is about 24MeV/c2 for the VELO method, 57MeV/c2 for the T-station method and for the long
method. This is to be compared to the standard reconstruction with two long tracks that achieves a
resolution of 16MeV/c2. 6
Calculation of efficiency The track reconstruction efficiency is calculated as the fraction of reconstructed J/ψ decays where
the probe track can be matched to a long track. To estimate the number of J/ψ decays, an un-
binned extended maximum likelihood fit is performed to the mass distributions. For the VELO and
T-station methods the mass distributions are described by a single Gaussian function for the signal
and an exponential function for the combinatorial background. This model is preferred over the
aforementioned sum of two Gaussian functions to improve the fit stability when measuring the de-
pendence of the track reconstruction efficiency on kinematic variables and other event parameters. For the long method, a Crystal Ball function [30] is used for the signal, to take the tail on the left-
hand side of the mass peak into account. Since the number of decays in the 2010 data is relatively – 8 – low, in this case a simple sideband subtraction is applied for the VELO and T-station methods. All
shape parameters were allowed to vary in the fit for the denominator of the efficiency; they were
constrained to the found values for the numerator of the efficiency. This procedure was performed
to stabilise the fit, as no difference in the shape of the numerator and denominator could be ob-
served. It has been checked that the choice of the model for the mass distribution has a negligible
effect on the efficiency determination. The efficiencies obtained from the VELO and T-station methods are assumed to be uncorre-
lated, aside from effects due to dependencies on the track kinematics and the event multiplicity. The data sample is binned in kinematic variables and Ntrack to combine the VELO and T-station
efficiencies. The efficiencies obtained with the VELO and T-station methods can be multiplied in
each bin to obtain the efficiency for finding long tracks. This combined efficiency can be compared
with the efficiency found by the long method, giving two independent methods to probe the long
track reconstruction efficiency. 2015 JINST 10 P02 There are, however, small differences between these two approaches. The long method mea-
sures the efficiency for tracks that pass through TT. In the combined method, only the VELO
method requires this. Furthermore, both the VELO method and T-station method include the ef-
ficiency that, given that both the VELO and the T-station segment tracks are reconstructed, the
corresponding long track is found. 6
Calculation of efficiency Therefore, in the combined efficiency, this so-called matching
efficiency is counted twice. All these effects can lead to small differences in the measured long-
track efficiency. For this reason, the ratio between the efficiencies in data and simulation is used to
compare the methods, as these uncertainties are common for simulated and real decays and cancel
when the ratio of efficiencies is formed. JINST 10 P02007 On simulated events the track reconstruction efficiency is commonly defined as the fraction of
simulated charged particles with sufficient hits in the VELO and T stations that can be associated
to a track that shares at least 70% of the hits in each participating subdetector with this particle. For
all methods, this so-called hit-based efficiency in simulation agrees within 1% with the efficiency
measured with the tag-and-probe methods. Furthermore the matching efficiency was determined
to be very close to 100%. The very small matching inefficiency does not affect the agreement
between the hit-based efficiency and the tag-and-probe based efficiency in simulation. By taking
the ratio between the efficiencies on data and simulation, these discrepancies are reduced to a
negligible level. 7
Efficiency dependencies Using the momentum spectrum of the J/ψ decay products obtained with the VELO method from
data as a benchmark, the average track reconstruction efficiency for long tracks is measured to
be (95.4 ± 0.7)% for 2010 data, (97.78 ± 0.07)% for 2011 data and (96.99 ± 0.05)% for 2012
data. All results confirm the good performance of the LHCb tracking system. The uncertainties
on these numbers are statistical only; they are binomial errors with additional terms to account
for the statistical uncertainty on the number of background events. Systematic uncertainties are
discussed in section 8. The difference in the efficiencies between the three years is a consequence of
changes in the track reconstruction and the higher centre-of-mass energy, leading to a higher track
multiplicity and hence lower reconstruction efficiency for the 2012 running period. Dependencies – 9 – Table 3. Track reconstruction efficiencies in % for the individual running periods using the long method for
positive and negative muons and different magnetic field polarities (statistical uncertainties only). Magnet up
Magnet down
Data
Positive
Negative
Positive
Negative
2010
94.1±1.3
96.0±1.3
99.3+0.7
−1.8
98.4+1.6
−1.7
2011
97.0±0.3
97.3±0.3
97.2±0.3
97.4±0.3
2012
96.2±0.2
96.2±0.2
96.2±0.2
96.3±0.2 2015 JINST 10 P02 2015 JINST 10 P02007 on the polarity of the dipole magnet, the charge of the muons, and kinematic properties as well as
the agreement with the simulation are investigated in further detail in the following subsections. 7.2
Dependencies of track reconstruction efficiency The efficiency to reconstruct long tracks mainly depends on the particle kinematics and the number
of charged particles in an event. As a parametrisation p, η and Ntrack are chosen, as the track
reconstruction efficiency shows the largest dependence on these three observables. The simulated
events are weighted according to the Ntrack distribution observed in data. The track reconstruction
efficiencies for the combination of the VELO and T-station methods and for the long method are
shown for the different data-taking periods in figures 4–6 as a function of p, η, Ntrack, and as a
function of the number of reconstructed primary vertices, NPV. The efficiency coming from the
combination of the VELO and the T-station method is calculated by multiplying the individual
efficiencies. Overall, a reasonable agreement is found between simulated and real data for all data-
taking periods. As the agreement between the tag-and-probe based track reconstruction efficiency
and the true track reconstruction efficiency (based on hit information) is within 1%, the results
shown in figures 4–6 give an accurate description of the efficiency in simulation. 7.1
Comparison of magnetic field polarities The track reconstruction efficiencies determined from the long method are split up into positively
and negatively charged muons and into the two different magnetic field polarities (named up and
down). The results are summarised in table 3. They show compatible numbers for magnetic field
up and down and for positive and negative muons. JINST 10 P02007 For data from 2011 and 2012 there is no difference between positive and negative muons or be-
tween the different magnet polarities. In 2010 data, a 2.3σ difference between the different magnet
polarities is observed for positive muons. No unambiguous source of the difference is found. 7.3
Efficiency ratios The efficiency ratio is defined as the efficiency measured in data divided by the efficiency measured
in simulation, The efficiency ratio is defined as the efficiency measured in data divided by the efficiency measured
in simulation,
ε ratio = εdata
εsim
. (7.1) ratio = εdata
εsim
. (7.1) The efficiency dependence versus Ntrack and NPV is reasonably well described in the simulation, see
figures 4–6: when fitting a first-order polynomial to the efficiency distributions in simulation and
real data, the slopes agree with each other within 2 standard deviations, except for the efficiency The efficiency dependence versus Ntrack and NPV is reasonably well described in the simulation, see
figures 4–6: when fitting a first-order polynomial to the efficiency distributions in simulation and
real data, the slopes agree with each other within 2 standard deviations, except for the efficiency – 10 – ]c
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Figure 4. Track reconstruction efficiencies for the 2010 data and for weighted simulation. The left-ha
column shows the results of the combined method while the right-hand column shows the results of the lo
method. The efficiency is shown as a function of p (first row), η (second row), Ntrack (third row), and N
(fourth row). The error bars indicate the statistical uncertainties. 7.3
Efficiency ratios ]c
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VELO+T efficiency Figure 4. Track reconstruction efficiencies for the 2010 data and for weighted simulation. The left-hand
column shows the results of the combined method while the right-hand column shows the results of the long
method. The efficiency is shown as a function of p (first row), η (second row), Ntrack (third row), and NPV
(fourth row). The error bars indicate the statistical uncertainties. 7.3
Efficiency ratios – 11 – ]c
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Figure 5. Track reconstruction efficiencies for the 2011 data and for weighted simulation. The left-ha
column shows the results of the combined method while the right-hand column shows the results of the lo
method. The efficiency is shown as a function of p (first row), η (second row), Ntrack (third row), and N
(fourth row). The error bars indicate the statistical uncertainties. 7.3
Efficiency ratios ]c
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VELO+T efficiency Figure 5. Track reconstruction efficiencies for the 2011 data and for weighted simulation. The left-hand
column shows the results of the combined method while the right-hand column shows the results of the long
method. The efficiency is shown as a function of p (first row), η (second row), Ntrack (third row), and NPV
(fourth row). The error bars indicate the statistical uncertainties. 7.3
Efficiency ratios – 12 – ]c
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Long efficiency
Figure 6. Track reconstruction efficiencies for the 2012 data and for weighted simulation. The left-han
column shows the results of the combined method while the right-hand column shows the results of the lon
method. The efficiency is shown as a function of p (first row), η (second row), Ntrack (third row), and NP
(fourth row). The error bars indicate the statistical uncertainties. 7.3
Efficiency ratios ]c
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VELO+T efficiency Figure 6. Track reconstruction efficiencies for the 2012 data and for weighted simulation. The left-hand
column shows the results of the combined method while the right-hand column shows the results of the long
method. The efficiency is shown as a function of p (first row), η (second row), Ntrack (third row), and NPV
(fourth row). The error bars indicate the statistical uncertainties. – 13 – ]c
[GeV/
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Figure 7. Track reconstruction efficiency ratios as a function of p between data and simulation for (left)
2010 data, (right) 2011 data, and (bottom) 2012 data. ]c
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Data/Simulation 2011 Efficiency ratio 5 JINST 10 P02007 10 Figure 7. Track reconstruction efficiency ratios as a function of p between data and simulation for (left)
2010 data, (right) 2011 data, and (bottom) 2012 data. 7.3
Efficiency ratios as a function of the number of tracks in the combination of the VELO and T-station method in
2012. It is therefore sufficient to weight the simulated events to establish agreement in Ntrack while
the efficiency ratio is determined in bins of p and η. The number of bins is chosen to keep the
statistical uncertainty in each bin sufficiently small. For the final result, the weighted average of
the combined and long method is taken in each bin of p and η. Figure 7 shows the efficiency ratio versus p for run I, weighted by the event track multiplicity
observed in data; the data are split into two ranges of η. Overall a good agreement of the track
finding efficiency is found between events in simulation and in data for all data taking periods and
most momenta and pseudorapidity regions. The difference between the track finding efficiencies is
generally smaller than 1% between events from simulation and data and no trend can be observed
for the 2011 and 2012 dataset, with the number of events being too low to draw conclusions from
the 2010 dataset. The agreement is worse for tracks with momentum below 10GeV/c, which might
point to a less accurate modelling of multiple scattering effects in the simulation. The overall efficiency ratio and its uncertainty depend on the particle distribution of the data
in terms of p and η. Using the momentum spectrum of the J/ψ decay products obtained with
the VELO method from data, an average efficiency ratio is found of 0.994 ±0.007 for 2010 data,
0.9983±0.0009 for 2011 data and 1.0053±0.0008 for 2012 data. The uncertainties represent the
statistical uncertainties only. The ratio is close to one in all three cases as different features seen
in the efficiency distributions in simulation and data average out when integrating over the full
momentum spectrum or pseudorapidity range. – 14 – 8
Systematic uncertainties Small differences in the ratio of efficiencies are seen when reweighting the simulated samples in
different parameters such as the number of primary vertices, or the number of hits or tracks in the
different subdetectors. The largest of these differences is taken as a systematic uncertainty and
amounts to 0.4%. No systematic uncertainty is assigned for the agreement of the track reconstruc-
tion efficiency determined by the tag-and-probe method and the hit-based method (which is on the
order of 1%), as the differences cancel when forming the efficiency ratio. Accordingly, no sys-
tematic uncertainties are assigned for the fit model as these cancel when forming the fraction of
reconstructed J/ψ decays where the probe can be matched to a long track. It has been checked
that this is true for a range of fit models, the largest variation being 0.2%. Furthermore, no sys-
tematic uncertainty is assigned to the possible matching of a correctly reconstructed probe track
to a fake long track, as the requirement for a large overlap in the subdetectors ensure that both
reconstructed tracks are either real tracks or fake tracks, where the latter would not peak at the
J/ψ mass. No systematic uncertainty is assigned for the fact that the VELO + T-station method
and the long method show slightly different results in figures 4–6, as both methods probe different
momentum spectra and any residual difference will cancel when forming the ratio with simulation. No systematic uncertainty is assigned for the double-counting of the matching efficiency in the
combined method, as this efficiency is very close to 100%, and any uncertainty would get further
reduced when forming the ratio with simulation. No systematic uncertainty is assigned for the large
difference for the VELO + T efficiency between simulation and data at low momenta in 2011 and
2012, as this is automatically taken into account when forming the ratio of efficiencies. Despite
this difference, the integrated track reconstruction efficiencies between simulation and data are in
agreement due to compensation of this effect for high momenta, where the efficiency is higher in
simulation than in data. 2015 JINST 10 P02 5 JINST 10 P02007 9
Hadronic interactions The methods presented in this paper are based on muons and require that they reach the muon
stations. Thus, these methods are not sensitive to the effects from hadronic interactions and large-
angle scatterings with the detector material. For hadrons, the largest effect is due to hadronic inter-
actions. The cross section depends on the particle type, charge and the momentum. A simulation
of B0 →J/ψ K∗0 decays (where K∗0 →K+π−) shows that about 11% of the kaons (averaged over
positive and negative kaons) and about 14% of the pions cannot be reconstructed due to hadronic
interactions that occur before the last T station. This number depends primarily on the momentum
of the particle. Due to the uncertainty on the material budget and consequently on the interaction
with the detector material, the reconstruction efficiency obtained from simulation has an intrin-
sic uncertainty, which is not accounted for in the track reconstruction efficiencies measured with
muons. When assuming that the total material budget in the simulation has an uncertainty of 10%,
the systematic uncertainty due to hadronic interactions is between 1.1–1.4%. The 10% uncertainty
is used as a conservative upper limit and is composed as follows: for the VELO a calculation in
ref. [4] shows an uncertainty on the material budget of 6%. No direct measurements exist for the
T and TT stations. However, weight measurements for the Inner Tracker for the silicon sensors – 15 – and the detector boxes give an accuracy of 2%, while an agreement of 5% is reached for the ca-
bles and the support structure [31, 32]. The Outer Tracker modules have been weighted and this
measurement is precise to about 1% [33]. Furthermore, the sum of the weights of the individual
components of a module adds up to the total weight of a module within the uncertainties. Taking
into account that some level of detail is missing in the detector description in the simulation, an un-
certainty of 5% is assumed for the outer tracker. Weight measurements for the sensor modules and
the insulation material of TT have been performed. Given the detail of the detector description [34]
an uncertainty of 5% on the material budget is well justified. The beam-pipe was implemented in
the software following the design drawings, where a precision better than 10% for all pieces was
confirmed following measurements after production. 10
Conclusion Track reconstruction efficiencies at LHCb have been measured using a tag-and-probe method with
J/ψ →µ+µ−decays. The average efficiency is better than 95% in the momentum region 5GeV/c <
p < 200GeV/c and in the pseudorapidity region 2 < η < 5, which covers the phase space of LHCb. The uncertainty per track is below 0.5% for muons and below 1.5% for pions and kaons, where
the larger uncertainty takes the uncertainty on hadronic interactions into account. All uncertainties
have been added in quadrature. Furthermore, the ratio of the track reconstruction efficiency of
muons in data and simulation is measured, where an uncertainty of 0.8% for data collected in
2010 and an uncertainty of 0.4% for data collected in 2011 and 2012 is achieved. The integrated
efficiency ratios for all three years of data taking are compatible with unity. This result presents
a significant improvement over the uncertainties determined with previous methods ranging from
3 to 4%. 9
Hadronic interactions The solid radiator (aerogel) and the gas radia-
tor (C4F10) contribute more than two-third of the material budget for the RICH1 detector [35]. The
amount of aerogel is known up to 2% and the differences between 2011 and 2012 are accounted
for in the simulation. The density of the C4F10 was monitored, with the RMS of the distribution
being about 1%. The other components of RICH1 have a smaller contribution to the interaction
length. The overall uncertainty of 10% for the full material budget was then chosen to also take
uncertainties on the GEANT4 cross-sections and additional uncertainties, coming from simplified
descriptions of the detector elements in the simulation, into account. 2015 JINST 10 P02 JINST 10 P02007 Acknowledgments We express our gratitude to our colleagues in the CERN accelerator departments for the excel-
lent performance of the LHC. We thank the technical and administrative staff at the LHCb in-
stitutes. We acknowledge support from CERN and from the national agencies: CAPES, CNPq,
FAPERJ, and FINEP (Brazil); NSFC (China); CNRS/IN2P3 (France); BMBF, DFG, HGF, and
MPG (Germany); SFI (Ireland); INFN (Italy); FOM and NWO (The Netherlands); MNiSW and
NCN (Poland); MEN/IFA (Romania); MinES and FANO (Russia); MinECo (Spain); SNSF and
SER (Switzerland); NASU (Ukraine); STFC (United Kingdom); NSF (U.S.A.). The Tier1 com-
puting centres are supported by IN2P3 (France), KIT and BMBF (Germany), INFN (Italy), NWO
and SURF (The Netherlands), PIC (Spain), GridPP (United Kingdom). We are indebted to the – 16 – communities behind the multiple open source software packages on which we depend. We are
also thankful for the computing resources and the access to software R&D tools provided by Yan-
dex LLC (Russia). Individual groups or members have received support from EPLANET, Marie
Skłodowska-Curie Actions, and ERC (European Union), Conseil g´en´eral de Haute-Savoie, Labex
ENIGMASS, and OCEVU, R´egion Auvergne (France), RFBR (Russia), XuntaGal, and GENCAT
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A. Grecu29, E. Greening55, S. Gregson47, P. Griffith45, L. Grillo11, O. Gr¨unberg62, B. Gui59, E. Gushchin33 A. Grecu29, E. Greening55, S. Gregson47, P. Griffith45, L. Grillo11, O. Gr¨unberg62, B. Gui59, E. Gushchin33, A. Grecu29, E. Greening55, S. Gregson47, P. Griffith45, L. Grillo11, O. Gr¨unberg62, B. Gui59, E. G Yu. Guz35,38, T. Gys38, C. Hadjivasiliou59, G. Haefeli39, C. Haen38, S.C. Haines47, S. Hall53, . Guz35,38, T. Gys38, C. Hadjivasiliou59, G. Haefeli39, C. Haen38, S.C. Haines47, S. Hall53, Hamilton58, T. Hampson46, X. Han11, S. Hansmann-Menzemer11, N. Harnew55, S.T. Harnew46, B. Hamilton58, T. Hampson46, X. Han11, S. The LHCb collaboration R. Aaij41, B. Adeva37, M. Adinolfi46, A. Affolder52, Z. Ajaltouni5, S. Akar6, J. Albrecht9, F. Alessio38,
M. Alexander51, S. Ali41, G. Alkhazov30, P. Alvarez Cartelle37, A.A. Alves Jr25,38, S. Amato2,
S. Amerio22, Y. Amhis7, L. An3, L. Anderlini17,g, J. Anderson40, R. Andreassen57, M. Andreotti16,f ,
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E. Aslanides6, G. Auriemma25,n, M. Baalouch5, S. Bachmann11, J.J. Back48, A. Badalov36, W. Baldini16,
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L. Beaucourt4, J. Beddow51, F. Bedeschi23, I. Bediaga1, S. Belogurov31, K. Belous35, I. Belyaev31,
E. Ben-Haim8, G. Bencivenni18, S. Benson38, J. Benton46, A. Berezhnoy32, R. Bernet40, M.-O. Bettler47,
M. van Beuzekom41, A. Bien11, S. Bifani45, T. Bird54, A. Bizzeti17,i, P.M. Bjørnstad54, T. Blake48,
F. Blanc39, J. Blouw10, S. Blusk59, V. Bocci25, A. Bondar34, N. Bondar30,38, W. Bonivento15,38,
S. Borghi54, A. Borgia59, M. Borsato7, T.J.V. Bowcock52, E. Bowen40, C. Bozzi16, T. Brambach9,
J. van den Brand42, J. Bressieux39, D. Brett54, M. Britsch10, T. Britton59, J. Brodzicka54, N.H. Brook46,
H. Brown52, A. Bursche40, G. Busetto22,r, J. Buytaert38, S. Cadeddu15, R. Calabrese16,f , M. Calvi20,k,
M. Calvo Gomez36,p, P. Campana18,38, D. Campora Perez38, A. Carbone14,d, G. Carboni24,l,
R. Cardinale19,38,j, A. Cardini15, L. Carson50, K. Carvalho Akiba2, G. Casse52, L. Cassina20,
L. Castillo Garcia38, M. Cattaneo38, Ch. Cauet9, R. Cenci58, M. Charles8, Ph. Charpentier38, S. Chen54,
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k53 2015 JINST 10 P020 M. Calvo Gomez36,p, P. Campana18,38, D. Campora Perez38, A. Carbone14,d, G. Carboni24,l, JINST 10 P02007 L. Castillo Garcia38, M. Cattaneo38, Ch. Cauet9, R. Cenci58, M. Charles8, Ph. Charpentier38, S. Chen54 L. Castillo Garcia38, M. Cattaneo38, Ch. Cauet9, R. Cenci58, M. Charles8, Ph. Charpentier38, S. Chen54, P.E.L. Clarke50, M. Clemencic38, H.V. Cliff47, J. Closier38, V. Coco38, J. Cogan6, E. Cogneras5, P.E.L. Clarke50, M. Clemencic38, H.V. Cliff47, J. Closier38, V. Coco38, J. Cogan6, E. Cogneras5, Collins38, A. Comerma-Montells11, A. Contu15, A. Cook46, M. Coombes46, S. Coquereau8, G. Corti38, Corvo16,f , I. Counts56, B. Couturier38, G.A. Cowan50, D.C. Craik48, M. Cruz Torres60, S. Cunliffe53, Currie50, C. D’Ambrosio38, J. Dalseno46, P. David8, P.N.Y. David41, A. Davis57, K. De Bruyn41, S. De Capua54, M. De Cian11, J.M. De Miranda1, L. De Paula2, W. De Silva57, P. De Simone18, De Capua54, M. De Cian11, J.M. De Miranda1, L. De Paula2, W. The LHCb collaboration Hansmann-Menzemer11, N. Harnew55, S.T. Harnew4 Harrison54, J. He38, T. Head38, V. Heijne41, K. Hennessy52, P. Henrard5, L. Henry8, He38, T. Head38, V. Heijne41, K. Hennessy52, P. Henr J. Harrison54, J. He38, T. Head38, V. Heijne41, K. Hennessy52, P. Henrard5, L. Henry8, A. Hernando Morata37, E. van Herwijnen38, M. Heß62, A. Hicheur1, D. Hill55, M. Hoballah5, J.A. Hernando Morata37, E. van Herwijnen38, M. Heß62, A. Hicheur1, D. Hill55, M. Hoballah5, C. Hombach54, W. Hulsbergen41, P. Hunt55, N. Hussain55, D. Hutchcroft52, D. Hynds51, M. Idzik27, Hombach54, W. Hulsbergen41, P. Hunt55, N. Hussain55, D. Hutchcroft52, D. Hynds51, M. Idzik27, P. Ilten56, R. Jacobsson38, A. Jaeger11, J. Jalocha55, E. Jans41, P. Jaton39, A. Jawahery58, F. Jing3, Ilten56, R. Jacobsson38, A. Jaeger11, J. Jalocha55, E. Jans41, P. Jaton39, A. Jawahery58, F. Jing3, M. John55, D. Johnson55, C.R. Jones47, C. Joram38, B. Jost38, N. Jurik59, M. Kaballo9, S. Kandybei4 M. John55, D. Johnson55, C.R. Jones47, C. Joram38, B. Jost38, N. Jurik59, M. Kaballo9, S. Kandybei4 W. Kanso6, M. Karacson38, T.M. Karbach38, S. Karodia51, M. Kelsey59, I.R. Kenyon45, T. Ketel42, nso6, M. Karacson38, T.M. Karbach38, S. Karodia51, M. Kelsey59, I.R. Kenyon45, T. Ketel42, B. Khanji20, C. Khurewathanakul39, S. Klaver54, K. Klimaszewski28, O. Kochebina7, M. Kolpin11, B. Khanji20, C. Khurewathanakul39, S. Klaver54, K. Klimaszewski28, O. Kochebina7, M. Kolpin11, – 19 – Komarov39, R.F. Koopman42, P. Koppenburg41,38, M. Korolev32, A. Kozlinskiy41, L. Kravchuk33, I. Komarov39, R.F. Koopman42, P. Koppenburg41,38, M. Korolev32, A. Kozlinskiy41, L. Kravchuk3 p
pp
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Kreplin11, M. Kreps48, G. Krocker11, P. Krokovny34, F. Kruse9, W. Kucewicz26,o, K. Kreplin11, M. Kreps48, G. Krocker11, P. Krokovny34, F. Kruse9, W. Kucewicz26,o, p
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M. Kucharczyk20,26,38,k, V. Kudryavtsev34, K. Kurek28, T. Kvaratskheliya31, V.N. La Thi39, D. L p
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Kucharczyk20,26,38,k, V. Kudryavtsev34, K. Kurek28, T. Kvaratskheliya31, V.N. La Thi39, D. Lacarrere38, G. Lafferty54, A. Lai15, D. Lambert50, R.W. Lambert42, G. Lanfranchi18, C. Langenbruch48, Lafferty54, A. Lai15, D. Lambert50, R.W. Lambert42, G. Lanfranchi18, C. Langenbruch48, Langhans38, T. Latham48, C. Lazzeroni45, R. Le Gac6, J. van Leerdam41, J.-P. Lees4, R. Lef`evre5, B. Langhans38, T. Latham48, C. Lazzeroni45, R. Le Gac6, J. van Leerdam41, J.-P. Lees4, R. Lef`evr A. Leflat32, J. Lefranc¸ois7, S. Leo23, O. Leroy6, T. Lesiak26, B. Leverington11, Y. Li3, T. Likhomanenko63, Leflat32, J. Lefranc¸ois7, S. Leo23, O. Leroy6, T. Lesiak26, B. Leverington11, Y. Li3, T. Likhomanenko63, M. Liles52, R. Lindner38, C. Linn38, F. Lionetto40, B. Liu15, S. Lohn38, I. Longstaff51, J.H. Lopes Liles52, R. Lindner38, C. The LHCb collaboration Linn38, F. Lionetto40, B. Liu15, S. Lohn38, I. Longstaff51, J.H. Lopes2, N. Lopez-March39, P. Lowdon40, H. Lu3, D. Lucchesi22,r, H. Luo50, A. Lupato22, E. Luppi16,f , N. Lopez-March39, P. Lowdon40, H. Lu3, D. Lucchesi22,r, H. Luo50, A. Lupato22, E. Luppi16,f , G. Mancinelli6, J. Maratas5, J.F. Marchand4, U. Marconi14, C. Marin Benito36, P. Marino23,t, R. G. Mancinelli6, J. Maratas5, J.F. Marchand4, U. Marconi14, C. Marin Benito36, P. Marino23,t, R. M¨arki39, 2015 JINST 10 P020 F. Martinez Vidal64, D. Martins Tostes2, A. Massafferri1, R. Matev38, Z. Mathe38, C. Matteuzzi20, Martinez Vidal64, D. Martins Tostes2, A. Massafferri1, R. Matev38, Z. Mathe38, C. Matteuzzi20, Meier9, M. Meissner11, M. Merk41, D.A. Milanes8, M.-N. Minard4, N. Moggi14, J. Molina Rodriguez60, Monteil5, M. Morandin22, P. Morawski27, A. Mord`a6, M.J. Morello23,t, J. Moron27, A.-B. Morris50, R. Mountain59, F. Muheim50, K. M¨uller40, M. Mussini14, B. Muster39, P. Naik46, T. Nakada39, Mountain59, F. Muheim50, K. M¨uller40, M. Mussini14, B. Muster39, P. Naik46, T. Nakada39, R. Nandakumar49, I. Nasteva2, M. Needham50, N. Neri21, S. Neubert38, N. Neufeld38, M. Neuner Nandakumar49, I. Nasteva2, M. Needham50, N. Neri21, S. Neubert38, N. Neufeld38, M. Neuner11, JINST 10 P02007 A.D. Nguyen39, T.D. Nguyen39, C. Nguyen-Mau39,q, M. Nicol7, V. Niess5, R. Niet9, N. Nikitin32, D. Nguyen39, T.D. Nguyen39, C. Nguyen-Mau39,q, M. Nicol7, V. Niess5, R. Niet9, N. Nikitin32, T. Nikodem11, A. Novoselov35, D.P. O’Hanlon48, A. Oblakowska-Mucha27, V. Obraztsov35, S. Og Nikodem11, A. Novoselov35, D.P. O’Hanlon48, A. Oblakowska-Mucha27, V. Obraztsov35, S. Oggero41, Ogilvy51, O. Okhrimenko44, R. Oldeman15,e, G. Onderwater65, M. Orlandea29, J.M. Otalora Goicochea2, P. Owen53, A. Oyanguren64, B.K. Pal59, A. Palano13,c, F. Palombo21,u, M. Palutan18, J. Panman38, Owen53, A. Oyanguren64, B.K. Pal59, A. Palano13,c, F. Palombo21,u, M. Palutan18, J. Panman38, Papanestis49,38, M. Pappagallo51, L.L. Pappalardo16,f , C. Parkes54, C.J. Parkinson9,45, G. Passaleva17, A. Papanestis49,38, M. Pappagallo51, L.L. Pappalardo16,f , C. Parkes54, C.J. Parkinson9,45, G. Pass G.D. Patel52, M. Patel53, C. Patrignani19,j, A. Pazos Alvarez37, A. Pearce54, A. Pellegrino41, G.D. Patel52, M. Patel53, C. Patrignani19,j, A. Pazos Alvarez37, A. Pearce54, A. Pellegrino4 M. Pepe Altarelli38, S. Perazzini14,d, E. Perez Trigo37, P. Perret5, M. Perrin-Terrin6, L. Pescatore45, Pepe Altarelli38, S. Perazzini14,d, E. Perez Trigo37, P. Perret5, M. Perrin-Terrin6, L. Pescatore45, E. Pesen66, K. Petridis53, A. Petrolini19,j, E. Picatoste Olloqui36, B. Pietrzyk4, T. Pilaˇr48, D. Pinci25, E. Pesen66, K. Petridis53, A. Petrolini19,j, E. Picatoste Olloqui36, B. Pietrzyk4, T. Pilaˇr48, D. Pinci25, D. Popov10, B. Popovici29, C. Potterat2, E. Price46, J. Prisciandaro39, A. Pritchard52, C. Prouve46, D. Popov10, B. The LHCb collaboration Popovici29, C. Potterat2, E. Price46, J. Prisciandaro39, A. Pritchard52, C. Prouve46, Pugatch44, A. Puig Navarro39, G. Punzi23,s, W. Qian4, B. Rachwal26, J.H. Rademacker46, V. Pugatch44, A. Puig Navarro39, G. Punzi23,s, W. Qian4, B. Rachwal26, J.H. Rademacker46, B. Rakotomiaramanana39, M. Rama18, M.S. Rangel2, I. Raniuk43, N. Rauschmayr38, G. Raven4 Rakotomiaramanana39, M. Rama18, M.S. Rangel2, I. Raniuk43, N. Rauschmayr38, G. Raven42, S. Reichert54, M.M. Reid48, A.C. dos Reis1, S. Ricciardi49, S. Richards46, M. Rihl38, K. Rinnert52 Reichert54, M.M. Reid48, A.C. dos Reis1, S. Ricciardi49, S. Richards46, M. Rihl38, K. Rinnert52, Rives Molina36, D.A. Roa Romero5, P. Robbe7, A.B. Rodrigues1, E. Rodrigues54, P. Rodriguez Perez54, Roiser38, V. Romanovsky35, A. Romero Vidal37, M. Rotondo22, J. Rouvinet39, T. Ruf38, F. Ruffini23, H. Ruiz36, P. Ruiz Valls64, J.J. Saborido Silva37, N. Sagidova30, P. Sail51, B. Saitta15,e, Ruiz36, P. Ruiz Valls64, J.J. Saborido Silva37, N. Sagidova30, P. Sail51, B. Saitta15,e, Salustino Guimaraes2, C. Sanchez Mayordomo64, B. Sanmartin Sedes37, R. Santacesaria25, V. Salustino Guimaraes2, C. Sanchez Mayordomo64, B. Sanmartin Sedes37, R. Santacesaria25, C. Santamarina Rios37, E. Santovetti24,l, A. Sarti18,m, C. Satriano25,n, A. Satta24, D.M. Saunders46 Santamarina Rios37, E. Santovetti24,l, A. Sarti18,m, C. Satriano25,n, A. Satta24, D.M. Saunders46, M. Savrie16,f , D. Savrina31,32, M. Schiller42, H. Schindler38, M. Schlupp9, M. Schmelling10, B. Schmidt38 Savrie16,f , D. Savrina31,32, M. Schiller42, H. Schindler38, M. Schlupp9, M. Schmelling10, B. Schmidt38, Schneider39, A. Schopper38, M.-H. Schune7, R. Schwemmer38, B. Sciascia18, A. Sciubba25, M. Seco37, Semennikov31, I. Sepp53, N. Serra40, J. Serrano6, L. Sestini22, P. Seyfert11, M. Shapkin35, Shapoval16,43,f , Y. Shcheglov30, T. Shears52, L. Shekhtman34, V. Shevchenko63, A. Shires9, R. Silva Coutinho48, G. Simi22, M. Sirendi47, N. Skidmore46, T. Skwarnicki59, N.A. Smith52, E. Smith55,49 J. van Tilburg41, V. Tisserand4, M. Tobin39, S. Tolk42, L. Tomassetti16,f , D. Tonelli38, S. Topp-Joergensen55, N. Torr55, E. Tournefier4, S. Tourneur39, M.T. Tran39, M. Tresch40, S. Topp-Joergensen55, N. Torr55, E. Tournefier4, S. Tourneur39, M.T. Tran39, M. Tresch40, – 20 – A. Tsaregorodtsev6, P. Tsopelas41, N. Tuning41, M. Ubeda Garcia38, A. Ukleja28, A. Ustyuzhanin63,
U. Uwer11, V. Vagnoni14, G. Valenti14, A. Vallier7, R. Vazquez Gomez18, P. Vazquez Regueiro37,
C. V´azquez Sierra37, S. Vecchi16, J.J. Velthuis46, M. Veltri17,h, G. Veneziano39, M. Vesterinen11, B. Viaud7,
D. Vieira2, M. Vieites Diaz37, X. Vilasis-Cardona36,p, A. Vollhardt40, D. Volyanskyy10, D. Voong46,
A. Vorobyev30, V. Vorobyev34, C. Voß62, H. Voss10, J.A. de Vries41, R. Waldi62, C. Wallace48,
R. Wallace12, J. Walsh23, S. Wandernoth11, J. Wang59, D.R. Ward47, N.K. Watson45, D. Websdale53,
M. Whitehead48, J. The LHCb collaboration Wicht38, D. Wiedner11, G. Wilkinson55, M.P. Williams45, M. Williams56, F.F. Wilson49,
J. Wimberley58, J. Wishahi9, W. Wislicki28, M. Witek26, G. Wormser7, S.A. Wotton47, S. Wright47, S. Wu3,
K. Wyllie38, Y. Xie61, Z. Xing59, Z. Xu39, Z. Yang3, X. Yuan3, O. Yushchenko35, M. Zangoli14,
M. Zavertyaev10,b, L. Zhang59, W.C. Zhang12, Y. Zhang3, A. Zhelezov11, A. Zhokhov31, L. Zhong3,
A. Zvyagin38 A. Tsaregorodtsev6, P. Tsopelas41, N. Tuning41, M. Ubeda Garcia38, A. Ukleja28, A. Ustyuzhanin63,
U. Uwer11, V. Vagnoni14, G. Valenti14, A. Vallier7, R. Vazquez Gomez18, P. Vazquez Regueiro37,
C. V´azquez Sierra37, S. Vecchi16, J.J. Velthuis46, M. Veltri17,h, G. Veneziano39, M. Vesterinen11, B. Viaud7,
D. Vieira2, M. Vieites Diaz37, X. Vilasis-Cardona36,p, A. Vollhardt40, D. Volyanskyy10, D. Voong46,
A. Vorobyev30, V. Vorobyev34, C. Voß62, H. Voss10, J.A. de Vries41, R. Waldi62, C. Wallace48,
R. Wallace12, J. Walsh23, S. Wandernoth11, J. Wang59, D.R. Ward47, N.K. Watson45, D. Websdale53,
M. Whitehead48, J. Wicht38, D. Wiedner11, G. Wilkinson55, M.P. Williams45, M. Williams56, F.F. Wilson49,
J. Wimberley58, J. Wishahi9, W. Wislicki28, M. Witek26, G. Wormser7, S.A. Wotton47, S. Wright47, S. Wu3,
K. Wyllie38, Y. Xie61, Z. Xing59, Z. Xu39, Z. Yang3, X. Yuan3, O. Yushchenko35, M. Zangoli14,
M. Zavertyaev10,b, L. Zhang59, W.C. Zhang12, Y. Zhang3, A. Zhelezov11, A. Zhokhov31, L. Zhong3,
A. The LHCb collaboration Wills Physics Laboratory, University of Bristol, Bristol, United Kingdom
47 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom
48 Department of Physics, University of Warwick, Coventry, United Kingdom
49 STFC Rutherford Appleton Laboratory, Didcot, United Kingdom
50 School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom
51 School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom
52 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom
53 Imperial College London, London, United Kingdom
54 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom
55 Department of Physics, University of Oxford, Oxford, United Kingdom
56 Massachusetts Institute of Technology, Cambridge, MA, United States
57 University of Cincinnati, Cincinnati, OH, United States
58 University of Maryland, College Park, MD, United States
59 Syracuse University, Syracuse, NY, United States
60 Pontif´ıcia Universidade Cat´olica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil, associated to 2
61 Institute of Particle Physics, Central China Normal University, Wuhan, Hubei, China, associated to 3
62 Institut f¨ur Physik, Universit¨at Rostock, Rostock, Germany, associated to 11
63 National Research Centre Kurchatov Institute, Moscow, Russia, associated to 31
64 Instituto de Fisica Corpuscular (IFIC), Universitat de Valencia-CSIC, Valencia, Spain, associated to 36
65 KVI - University of Groningen, Groningen, The Netherlands, associated to 41
66 Celal Bayar University, Manisa, Turkey, associated to 38
a Universidade Federal do Triˆangulo Mineiro (UFTM), Uberaba-MG, Brazil
b P.N. The LHCb collaboration Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russia
c Universit`a di Bari, Bari, Italy
d Universit`a di Bologna, Bologna, Italy
e Universit`a di Cagliari, Cagliari, Italy
f Universit`a di Ferrara, Ferrara, Italy
g Universit`a di Firenze, Firenze, Italy
h Universit`a di Urbino, Urbino, Italy
i Universit`a di Modena e Reggio Emilia, Modena, Italy
j Universit`a di Genova, Genova, Italy
k Universit`a di Milano Bicocca, Milano, Italy
l Universit`a di Roma Tor Vergata, Roma, Italy
m Universit`a di Roma La Sapienza, Roma, Italy
n Universit`a della Basilicata, Potenza, Italy
o AGH - University of Science and Technology, Faculty of Computer Science, Electronics and
Telecommunications, Krak´ow, Poland
p LIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain
q Hanoi University of Science, Hanoi, Viet Nam
r Universit`a di Padova, Padova, Italy
s Universit`a di Pisa, Pisa, Italy
t Scuola Normale Superiore, Pisa, Italy
u Universit`a degli Studi di Milano, Milano, Italy 39 Ecole Polytechnique F´ed´erale de Lausanne (EPFL), Lausanne, Switzerland 39 Ecole Polytechnique F´ed´erale de Lausanne (EPFL), Lausanne, Switzerland 40 Physik-Institut, Universit¨at Z¨urich, Z¨urich, Switzerland 41 Nikhef National Institute for Subatomic Physics, Amsterdam, The Netherlands 2015 JINST 10 P020 53 Imperial College London, London, United Kingdom 5 JINST 10 P02007 56 Massachusetts Institute of Technology, Cambridge, MA, United States University of Cincinnati, Cincinnati, OH, United States 58 University of Maryland, College Park, MD, United Sta 59 Syracuse University, Syracuse, NY, United States The LHCb collaboration Zvyagin38 2015 JINST 10 P020 1 Centro Brasileiro de Pesquisas F´ısicas (CBPF), Rio de Janeiro, Brazil
2 Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
3 Center for High Energy Physics, Tsinghua University, Beijing, China
4 LAPP, Universit´e de Savoie, CNRS/IN2P3, Annecy-Le-Vieux, France
5 Clermont Universit´e, Universit´e Blaise Pascal, CNRS/IN2P3, LPC, Clermont-Ferrand, France
6 CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France
7 LAL, Universit´e Paris-Sud, CNRS/IN2P3, Orsay, France
8 LPNHE, Universit´e Pierre et Marie Curie, Universit´e Paris Diderot, CNRS/IN2P3, Paris, France
9 Fakult¨at Physik, Technische Universit¨at Dortmund, Dortmund, Germany
10 Max-Planck-Institut f¨ur Kernphysik (MPIK), Heidelberg, Germany
11 Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg, Germany
12 School of Physics, University College Dublin, Dublin, Ireland
13 Sezione INFN di Bari, Bari, Italy
14 Sezione INFN di Bologna, Bologna, Italy
15 Sezione INFN di Cagliari, Cagliari, Italy
16 Sezione INFN di Ferrara, Ferrara, Italy
17 Sezione INFN di Firenze, Firenze, Italy
18 Laboratori Nazionali dell’INFN di Frascati, Frascati, Italy
19 Sezione INFN di Genova, Genova, Italy
20 Sezione INFN di Milano Bicocca, Milano, Italy
21 Sezione INFN di Milano, Milano, Italy
22 Sezione INFN di Padova, Padova, Italy
23 Sezione INFN di Pisa, Pisa, Italy
24 Sezione INFN di Roma Tor Vergata, Roma, Italy
25 Sezione INFN di Roma La Sapienza, Roma, Italy
26 Henryk Niewodniczanski Institute of Nuclear Physics Polish Academy of Sciences, Krak´ow, Poland
27 AGH - University of Science and Technology, Faculty of Physics and Applied Computer Science, Krak´ow,
Poland
28 National Center for Nuclear Research (NCBJ), Warsaw, Poland
29 Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest-Magurele, Romania
30 Petersburg Nuclear Physics Institute (PNPI), Gatchina, Russia
31 Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia
32 Institute of Nuclear Physics, Moscow State University (SINP MSU), Moscow, Russia
33 Institute for Nuclear Research of the Russian Academy of Sciences (INR RAN), Moscow, Russia
34 Budker Institute of Nuclear Physics (SB RAS) and Novosibirsk State University, Novosibirsk, Russia
35 Institute for High Energy Physics (IHEP), Protvino, Russia
36 Universitat de Barcelona, Barcelona, Spain
37 Universidad de Santiago de Compostela, Santiago de Compostela, Spain
38 European Organization for Nuclear Research (CERN), Geneva, Switzerland JINST 10 P02007 13 Sezione INFN di Bari, Bari, Italy 15 Sezione INFN di Cagliari, Cagliari, Italy 16 Sezione INFN di Ferrara, Ferrara, Italy 19 Sezione INFN di Genova, Genova, Italy 20 Sezione INFN di Milano Bicocca, Milano, Italy 21 Sezione INFN di Milano, Milano, Italy 22 Sezione INFN di Padova, Padova, Italy 23 Sezione INFN di Pisa, Pisa, Italy 24 Sezione INFN di Roma Tor Vergata, Roma, Italy 25 Sezione INFN di Roma La Sapienza, Roma, Italy 28 National Center for Nuclear Research (NCBJ), Warsaw, Poland 29 Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest- 30 Petersburg Nuclear Physics Institute (PNPI), Gatchina, Russia 31 Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia 33 Institute for Nuclear Research of the Russian Academy of Sciences (INR RAN), Moscow, Russ 34 Budker Institute of Nuclear Physics (SB RAS) and Novosibirsk State University, Novosibirsk, Russia 35 Institute for High Energy Physics (IHEP), Protvino, Russia 35 Institute for High Energy Physics (IHEP), Protvino, Russia 36 Universitat de Barcelona, Barcelona, Spain 37 Universidad de Santiago de Compostela, Santiago de Compostela, Spain 37 Universidad de Santiago de Compostela, Santiago de Compostela, Spain 38 European Organization for Nuclear Research (CERN), Geneva, Switzerland – 21 – 39 Ecole Polytechnique F´ed´erale de Lausanne (EPFL), Lausanne, Switzerland
40 Physik-Institut, Universit¨at Z¨urich, Z¨urich, Switzerland
41 Nikhef National Institute for Subatomic Physics, Amsterdam, The Netherlands
42 Nikhef National Institute for Subatomic Physics and VU University Amsterdam, Amsterdam, The Netherlan
43 NSC Kharkiv Institute of Physics and Technology (NSC KIPT), Kharkiv, Ukraine
44 Institute for Nuclear Research of the National Academy of Sciences (KINR), Kyiv, Ukraine
45 University of Birmingham, Birmingham, United Kingdom
46 H.H. a Universidade Federal do Triˆangulo Mineiro (UFTM), Uberaba-MG, Brazil b P.N. Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russia c Universit`a di Bari, Bari, Italy d Universit`a di Bologna, Bologna, Italy e Universit`a di Cagliari, Cagliari, Italy f Universit`a di Ferrara, Ferrara, Italy g Universit`a di Firenze, Firenze, Italy h Universit`a di Urbino, Urbino, Italy i Universit`a di Modena e Reggio Emilia, Modena, Italy i Universit`a di Modena e Reggio Emilia, Modena, Italy j Universit`a di Genova, Genova, Italy k Universit`a di Milano Bicocca, Milano, Italy l Universit`a di Roma Tor Vergata, Roma, Italy m Universit`a di Roma La Sapienza, Roma, Italy m Universit`a di Roma La Sapienza, Roma, Italy n Universit`a della Basilicata, Potenza, Italy o AGH - University of Science and Technology, Faculty of Computer Science, Electronics and
Telecommunications, Krak´ow, Poland p LIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain q Hanoi University of Science, Hanoi, Viet Nam r Universit`a di Padova, Padova, Italy s Universit`a di Pisa, Pisa, Italy t Scuola Normale Superiore, Pisa, Italy u Universit`a degli Studi di Milano, Milano, Italy u Universit`a degli Studi di Milano, Milano, Italy – 22 –
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https://openalex.org/W3084069372
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https://www.frontiersin.org/articles/10.3389/fpubh.2020.00446/pdf
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English
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Does Dialogue Improve the Sustainable Employability of Low-Educated Employees? A Study Protocol for an Effect and Process Evaluation of “Healthy HR”
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Frontiers in public health
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cc-by
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STUDY PROTOCOL
published: 08 September 2020
doi: 10.3389/fpubh.2020.00446 STUDY PROTOCOL
published: 08 September 2020
doi: 10.3389/fpubh.2020.00446 Keywords: low-educated employees, employer, dialogue, job control, sustainable employability, effect evaluation,
process evaluation, protocol Does Dialogue Improve the
Sustainable Employability of
Low-Educated Employees? A Study
Protocol for an Effect and Process
Evaluation of “Healthy HR”
Emmelie Hazelzet*, Hans Bosma, Angelique de Rijk and Inge Houkes
Department of Social Medicine, Faculty of Health, Medicine and Life Sciences, CAPHRI Care and Public Health Research
Institute, Maastricht University, Maastricht, Netherlands Department of Social Medicine, Faculty of Health, Medicine and Life Sciences, CAPHRI Care and Public Health Research
Institute, Maastricht University, Maastricht, Netherlands Background: There is a need to develop sustainable employability (SE) interventions that
are better aligned to the needs of low-educated employees. This group needs to get a
voice in intervention development and implementation. In this study, a dialogue-based
approach is proposed consisting of an online step-by-step support toolkit for employers,
“Healthy Human Resources” (HHR). When intervening, this toolkit enables and stimulates
employers to have a continuous dialogue with their low-educated employees. By
improving the employees’ job control, HHR is aimed at cost-beneficially improving SE. This paper describes the protocol of the evaluation study to evaluate the effectiveness
and implementation process of HHR on the SE of low-educated employees. Edited by:
Leah Okenwa Emegwa,
Swedish Red Cross University
College, Sweden Reviewed by:
Victor C. W. Hoe,
University of Malaya, Malaysia
Kaisa Törnroos,
Finnish Institute of Occupational
Health, Finland Methods:
The protocol of the evaluation study consists of: (1) an effect evaluation
with a pretest-posttest design with a 1-year follow-up in five work organizations in the
Netherlands deploying low-educated employees and with SE as the primary outcome
and job control as the secondary outcome. The effect evaluation is expanded with a
budget impact analysis; (2) a mixed-method process evaluation at 6 and 12 months after
the start of HHR to evaluate the whole implementation process of HHR. This includes
the experiences with HHR of various stakeholders, such as employees, human resource
managers, and line managers. *Correspondence:
Emmelie Hazelzet
e.hazelzet@maastrichtuniversity.nl *Correspondence:
Emmelie Hazelzet
e.hazelzet@maastrichtuniversity.nl Specialty section:
This article was submitted to
Occupational Health and Safety,
a section of the journal
Frontiers in Public Health Received: 17 April 2020
Accepted: 20 July 2020
Published: 08 September 2020 Discussion: The effect evaluation will give insight into the effects of HHR on the SE of
low-educated employees. The process evaluation will provide insight into the underlying
mechanisms of the (in) effectiveness of HHR. By improving dialogue, we hypothesize that
HHR, through enhancing job control, will strengthen the SE of low-educated employees. Also for helping with tackling the socioeconomic health gap, if proven effective, the
implementation of HHR on a wider scale can be recommended. INTRODUCTION feeling heard and valued has been found to increase the self-
esteem and self-efficacy of employees (19). Despite
many
attempts
to
reduce
socioeconomic
health
differences, such differences remain large and persistent (1, 2). As, in the work domain, low-educated employees much more
often prematurely leave the labor force due to health-related
problems than their higher-educated counterparts (2–4), it is
worrying that lower-educated employees are often difficult to
reach in research and intervention efforts aimed at improving
their situation (5, 6). Through absenteeism, presenteeism, and
high staffturnover, this has substantial financial implications for
employers too (7). Low-educated employees constitute a group
that needs extra effort in this regard. Employees’ sustainable
employability (SE) has become top priority for employers,
as they aim to foster employees’ health and productivity in a
sustainable way (8). The concept of SE is not one individual
aspect, but rather an interaction between the employee and
the organizational context. The workplace therefore is a good
starting point to reach low-educated employees and improve
their SE (8–10). This group, however, hardly participates in
workplace health interventions (9, 11), and when they do
participate, they tend to benefit to a lesser extent (12). In practice,
many SE interventions are being developed without taking
the perspective of the target group into account. Employers
tend to buy ready-made health programs from (commercial)
third parties, in which implementation takes place via a non-
participatory top-down approach (13). Employees are often
passive receivers in these programs (14, 15). Consequently,
a mismatch occurs between these health programs and the
needs and the world of daily experience of most low-educated
employees. Therefore, low-educated employees need a different
and more intensive approach than their higher-educated
counterparts (16). We propose a dialogue-based approach to stimulate active
employee participation in the development and implementation
of tailored SE interventions. We assume that this will contribute
to a higher job control and SE of low-educated employees. Due to the participatory approach, including the dialogue
component, employees get the opportunity to obtain more self-
direction, experience more job control, which eventually will
improve their health and SE. By lowering sickness absence,
our approach will also be cost-beneficial for employers (7). We
have therefore developed a free online support toolkit named
- Healthy Human Resources’ (HHR) aimed at improving SE
of the low-educated employees. Citation: Hazelzet E, Bosma H, de Rijk A and
Houkes I (2020) Does Dialogue
Improve the Sustainable Employability
of Low-Educated Employees? A
Study Protocol for an Effect and
Process Evaluation of “Healthy HR”. Front. Public Health 8:446. doi: 10.3389/fpubh.2020.00446 September 2020 | Volume 8 | Article 446 Frontiers in Public Health | www.frontiersin.org 1 Study Protocol to Evaluate a Web-Based Toolkit Hazelzet et al. INTRODUCTION With the toolkit, employers
(e.g., HR managers; supervisors), in dialogue with the low-
educated employees, can develop and implement tailored SE
interventions. As long as these are the outcome of a shared
dialogue, the tailored SE interventions can vary widely regarding
size and content and may, e.g., include compliments cards, job
crafting, lifestyle interventions, or leadership training. The online
toolkit HHR has already been developed, also in dialogue with
several stakeholders, such as HR-managers, supervisors, and low-
educated employees. This paper presents the study protocol of the evaluation
study, evaluating the effect and the process of HHR. Particularly
through increasing the low-educated employees’ control at work,
we hypothesize that the use of HHR in organizations, by
integrating a dialogue-based approach, improves the SE of low-
educated employees. We therefore also expect that employees
who are more exposed to the dialogue integrated within HHR
will experience more improvement in SE than employees who are
less or not at all exposed to HHR (dose-response). The conceptual
model of HHR is illustrated in Figure 1. p
There is thus an urgent need to better align SE interventions
to the needs of low-educated employees. To increase the
effectiveness of these interventions, this group needs to have
a say and needs to be actively involved in intervention
development and implementation (6, 17, 18). Active involvement
and participation in decision-making processes is expected to
empower employees by increasing job control and autonomy;
these in turn are expected to improve the employees’ (mental)
health and SE (19–21). Job control is an important determinant
of employee well-being, particularly for low-educated employees
who generally work in low control situations (20, 22, 23). When intervening, we expect that a profound dialogue between
employees and the employer is crucial in increasing job control
and SE among low-educated employees (24–26). Dialogue stands
for an explanatory way of having a conversation in which all
involved stakeholders experience a shared responsibility for the
outcome of the dialogue (27). Instead of one-sided monologs
or directives from the top, during dialogue, employees and
representatives of the employer can think together and share
experiences from different perspectives (25). When employers
engage employees in dialogue, employees feel that their opinions
count and that they are given a voice (28, 29). Previous studies
found positive effects of improved work conditions through
dialogue groups among high-educated physicians (28) and Frontiers in Public Health | www.frontiersin.org METHODS The evaluation framework consists of a quantitative effect
evaluation and an extensive mixed-method process evaluation. The aim of the effect evaluation is to investigate the effect
of HHR on the SE of low-educated employees. The aim of
the process evaluation is to assess the implementation process,
the underlying mechanisms of the HHR’s effectiveness or lack
thereof (the how, what, why), and the HHR experiences of
key stakeholders, such as the employees, HR manager and
supervisors. The effect and process evaluation supplement
each other. Intervention: Healthy HR HHR is a web-based step-by-step support toolkit for HR
managers and/or supervisors aimed at improving SE of low-
educated employees. It supports HR managers and supervisors
by developing and implementing their own tailored SE
interventions by – from the start – involving their low-educated
employees via dialogue. This online toolkit is presented on
the “Healthy Human Resources” website (www.gezondhr.nl) (in
Dutch). It consists of different steps, tasks, and dialogue-based September 2020 | Volume 8 | Article 446 2 Hazelzet et al. Study Protocol to Evaluate a Web-Based Toolkit FIGURE 1 | Conceptual model of HHR and expected outcomes. FIGURE 1 | Conceptual model of HHR and expected outcomes. frequent and more intense, employees will be more exposed and
will experience more improvement on SE as mentioned before. A
detailed description about the development and content of HHR
will be published elsewhere (34). tools for use within a team or department of the participating
organizations. Within HHR eight steps are presented: step
(1) Prepare together; step (2) Measuring is knowing; step (3)
Our problems; step (4) Our solutions; step (5) Action plan;
step (6) Let’s start; step (7) Evaluation, and step (8) Along
the way: obstacles in the process. Each step, is represented
by several underlying tasks (e.g., brainstorming; prioritizing;
communicating) and every task contains one or more supportive
tools. Tools can be questionnaires, working forms, checklists,
communication tips and information, external links, or a library
with simple solutions and evidence-based interventions. Every
task and tool facilitates a certain degree of employee participation
and dialogue. The main outline of the steps, tasks and tools
are presented in Appendix A. Organizations can select the tools
which best fit to their context and their employees’ situation,
thereby developing a tailor-made toolkit for the needs assessment
(HHR step 1–4) and developing and implementing their own
tailored SE interventions (HHR step 5–7). The development of
HHR is based on the Intervention Mapping approach (IM) (30). As IM is a rather detailed and time-consuming approach (30, 31),
we decided to use an adapted version of the IM within HHR
as well; this will make HHR more feasible for employers to put
into practice (32, 33). The HR manager and/or supervisor will
facilitate HHR themselves, without any external consultancy. We
developed HHR in such a way, that it is a self-led intervention. It will be delivered in the participating organization, likely
during working hours. Intervention: Healthy HR HR manager and supervisors are able
to decide by themselves how much time they spend on HHR
and how they are going to integrate HHR in the daily business. However, a rule of thumb is provided within the toolkit by
the researchers. Nevertheless, we expect when using HHR more Effect Evaluation The effect evaluation will be a quantitative study with a pretest-
posttest design with a 1-year follow-up within each participating
organization (T2). The employees’ SE will be compared between
prior to and after the HHR intervention. We will also examine
whether the SE improves more, if employees are more exposed
to HHR. Additionally, a budget impact analysis (BIA) will be
performed to gain more insight into whether HHR is financially
affordable and beneficial for employers deploying low-educated
employees. The primary aim of the effect evaluation is to
investigate the effectiveness of HHR on the SE of low-educated
employees. The main research question is: - What
is
the
effect
of
HHR
on
the
SE
of
low-educated employees? Frontiers in Public Health | www.frontiersin.org Frontiers in Public Health | www.frontiersin.org Secondary outcome y
Job control will be the secondary outcome of the study and will
be measured by means of a self-developed scale consisting of 5
items. The items are inspired by existing lists, such as the Dutch
Questionnaire on the Experience and Evaluation of Work and the
Maastricht Autonomy questionnaire (39, 40). The formulation
of the items was aligned to the linguistic usage and preferences
of the low-educated employees. The response scale ranged from
1 (never) to 5 (always). An example item is: “I have a say in
what happens on my job.” Validity and reliability of this scale will
be analyzed. Primary outcomes
bl
l Sustainable employability (SE) will be the primary outcome of
the effect evaluation and can be considered as a distal outcome
measure. The level of SE is measured by means of two scales,
productivity and health, from the Maastricht Instrument of
Sustainable Employability (MAISE) (35). SE measurement will be
complemented by several proxies of SE: Vitality will be measured by means of the subscale vitality
of the Dutch version of the Utrecht Work Engagement Scale
(UWES) (5 items) (37). The response scale ranged from 1 (never)
to 7 (always/every day). A global measure of work engagement
will be used as well, measured by means of the shortened
Dutch version of the Utrecht Work Engagement Scale (UWES-
3). This short version of UWES-9 is proven to be reliable and
valid (38). Self-perceived health will be measured using a single Data Collection Data for the effect evaluation of HHR will rely upon quantitative
data from similar questionnaires at two time points: baseline (T0)
and follow up (T2, 12 months after the start of step 1) (Figure 2). The baseline questionnaire (T0) will also be used as the needs
assessment instrument in step 2 of HHR. The questionnaire
for the needs assessment and effect evaluation is adapted and
based on the existing Maastricht Instrument of Sustainable
Employability (MAISE) (35). The MAISE has been developed for
measuring SE from an employees’ perspective. The MAISE has
been validated among employees with (on average) intermediate
and higher educational levels. For use among a sample of low-
educated employees and the purpose of serving as a needs
assessment, the MAISE and other (self-developed) subscales,
such as job control, self-efficacy and lifestyle have been adjusted,
to better fit with the language and way of thinking of low-
educated employees. It is our hope that this adaptation improves
the reach and the validity and reliability of our questionnaires. For instance, the use of existing job control scales from existing
questionnaires were still too difficult to understand by the
employees when discussing these items together with them. For
the effect evaluation, additional, well-validated measures were
also used (e.g., vitality). Study Sample and Sample Size Five Dutch work organizations (a manufacturing company, a
meat processing company, a cleaning company, a warehouse
and a governmental institution) participated in the development
of HHR. These organizations will also implement HHR and
participate in the effect evaluation. Employees with lower
educational levels varying from no education to secondary
vocational education [coded according to the 2011 International
Standard Classification of Education (ISCED-11)] will be
included in HHR and the effect evaluation. In this study, we will
focus on employees with lower educational levels, particularly September 2020 | Volume 8 | Article 446 Frontiers in Public Health | www.frontiersin.org 3 Study Protocol to Evaluate a Web-Based Toolkit Hazelzet et al. those employees who perform low-skilled jobs within certain
departments of an organization. item: “In general, what would you say about your health?”
with five response options: excellent; very good; good; fair; and
poor. For sickness absence, self-reported sickness absence will be
measured by using a single item: “In the past 12 months, how
many days were you sick-listed?” and registered sickness absence
data will be drawn from the registers of the organizations. The
sickness absence percentages will be obtained per participating
department of each organization before the start at T0 and after
12 months (T2). A power calculation was performed to determine the sample
size. Based on the mean difference in SE of 0.25 (theoretical range
1 to 5) that was found between high and low-educated employees
in a previous study (35), we expect SE differences between high
and low-educated employees to decrease with 0.25. As the uptake
and output of HHR is organization-specific, we aim to study
the SE improvement in each organization separately, but we will
also pool the data to examine the overall effect. With a power of
80% and a significance level of 5%, the required sample size is a
minimum of 126 employees per organization (36), which implies
an overall sample size of 630 employees. We expect a varied non-
response and dropout rate per organization. The gross number
of employees varies between 40 and 1,200 per organization. For participating organizations with insufficient power, data will
be pooled. Other outcomes
l d d We included several additional proximal outcomes which can be
used to measure potential effects of the tailored SE interventions
per organization: self-efficacy, lifestyle, social climate, social
support, organization of work, adapted work possibilities, and
communication and collaboration. Self-efficacy will be measured
by means of the general self-efficacy scale (GSES-12) using the
subscale effort (5 items) (41). Lifestyle will be measured according
to the five behaviors: physical activity, smoking, alcohol use,
consumption of fruit or vegetables and quality of sleep (42–
44). These five lifestyle behaviors provided a so-called “optimal
lifestyle index.” Each behavior scored “1” when the norm is met
(and “0” when not met). A sum score will be computed of all five
behaviors to create an optimal lifestyle index (43). The variables
social climate (4 items), social support (3 items), organization
of work (9 items), adapted work possibilities (4 items), and
communication and collaboration (5 items) will be measured by
means of self-developed scales. Validity and reliability of these
scales will be analyzed. Information on covariates (gender, age, type of contract (e.g.,
permanent or flex), level of education, ethnicity, shift work) will
be also collected. Finally, to examine whether the SE improves
more when employees are more intensely exposed to HHR
(dose-response), the process indicator dose-received will be
included in the follow-up questionnaire (T2). Dose-received will
be measured by means of a self-developed continuous scale at
employee and organizational level (see also process evaluation). Employees will be asked to what extent they actively aware and
participated in HHR. Process Evaluation The aim of the process evaluation is to evaluate, in each
participating organization, the implementation process and
the underlying mechanisms of the HHR’s effectiveness or
lack thereof (the how, what, why), and the experiences of
key stakeholders with HHR. These key stakeholders might
influence the implementation throughout the process in various
ways and therefore the outcomes. The process evaluation will
have a mixed-method design (45) and will be utilized to
interpret and understand the outcomes of the effect evaluation
(46, 47). The study population of the quantitative process
evaluation (follow-up questionnaire T2) equals that of the
effect evaluation (the employees). The study population of
the qualitative process evaluation includes various stakeholders
(i.e., employees, supervisors, and HR managers) at different
levels of the organizations. We will examine the key process
indicators suggested by Linnan and Steckler presented in
Table 1 (48). Because the organizational context can hinder
or facilitate the implementation process and outcomes, we
will examine both omnibus context (e.g., general context) and
discrete context (e.g., specific events during HHR) in this process
evaluation (46, 47, 49). In the qualitative parts of the process
evaluation, we will generally follow the principles of responsive
evaluation, which is well in line with the participative and
dialogue-based approach of this study (50). This participative
evaluation method explicitly includes the intervention and Data Analysis Descriptive statistics will be used to analyze background
characteristics. Differences over time (T0-T2) on the primary
and secondary outcomes will be analyzed by means of paired
t-tests of mean differences, chi square tests and regression September 2020 | Volume 8 | Article 446 4 Study Protocol to Evaluate a Web-Based Toolkit Hazelzet et al. FIGURE 2 | Overview evaluation moments and data collection. FIGURE 2 | Overview evaluation moments and data collection. involved in HHR. We ensure that the report on both costs
and benefits will be simultaneously available for employers and
HR managers. analyses. The dose-received variable will be used to test the
correlation between the dose and change in the primary
outcome SE. Subgroup analyses (e.g., gender; education; type of
contract) will be performed to examine specifically heightened or
lowered improvements in SE in subgroups. Multilevel analyses
are used to examine the association between the level of
HHR implementation on the company level (level 2) and the
improvement in SE (level 1). If multilevel analyses appear not
to be feasible, other ways of taking account of the nested design
will be considered. Finally, when there is a need for pooling
(one organization has only 40 employees in total), multilevel is
similarly considered (when pooling). Analyses will be performed
using SPSS version 26. Frontiers in Public Health | www.frontiersin.org Budget Impact Analysis We will perform a budget impact analysis (BIA) from the
employer perspective. The main aim of the BIA is to assess
whether the implementation of HHR is financially affordable
for the employer (e.g., time; implementation costs of HHR;
additional cost for HHR) and show the budget impact of HHR. Generally, employers have interest in maintaining a healthy
and productive workforce and, thus, they may be able to offset
decreased sickness absence gains against the costs. Data will
be collected on the direct costs of specific resources needed
to implement HHR (e.g., staff, expertise, supplies, equipment,
working time) by means of interviews. The estimation of the
time spent gathered in interviews will be supplemented with
data from the logbooks of the employers and researchers. The
time spent will be translated to costs by multiplying number of
hours with the average hour salary of for the group of employees September 2020 | Volume 8 | Article 446 5 Study Protocol to Evaluate a Web-Based Toolkit Hazelzet et al. TABLE 1 | Process indicators, stakeholders’ level, operationalization and data collection method. Budget Impact Analysis Process indicators and definition
Stakeholder
level
Operationalization
Data collection method
Context
The contextual factors (omnibus; discrete) and history (i.e.,
barriers, facilitators) that affect HHR implementation or outcomes
Employer
Employees
Description of barriers
Description of facilitators
Documentary evidence (T0–T2)
Logbook (T0–T2)
Focus groups (T1; T2)
Semi-structured Interviews (T1;T2)
Recruitment
Procedures used to approach and attract employees
Employer
Employees
Description of approaches
Logbook (T0–T2)
Focus group (T1; T2)
Reach
Percentage of departments and employees participating in HHR
Employees
Characteristics of departments
Characteristics of employees
Percentage of employees, participated
Drop-out and reasons
Baseline questionnaire and follow-up
questionnaire (T0; T2)
Logbook (T0–T2)
Focus groups (T1; T2)
Semi structured Interviews (T1; T2)
Checklist (T1; T2)
Dose delivered
The extent to which HHR or components actually was delivered
according to the intervention plan
Employer
Employees
Dose delivered items (yes/no)
Logbook (T0–T2)
Questionnaire at follow-up (T2)
Focus groups (T1; T2)
Semi structured Interviews (T1; T2)
Checklist (T1; T2)
Dose received
The extent to which employees actively aware and participated
in HHR
Employees
Dose-response
Participation rate HHR
Questionnaire at follow-up (T2)
Focus groups (T1;T2)
Semi structured Interviews (T1;T2)
Fidelity
The extent to which HHR was delivered as intended
Employer
Employees
Statements (yes/no)
Reasons
Logbook (T0–T2)
Questionnaire at follow-up (T2)
Focus groups (T1; T2)
Semi structured Interviews (T1; T2)
Satisfaction
Employees and employer satisfaction about HHR
Employer
Employees
Satisfaction rate (scale 0–10)
Experiences of employees and employers
Logbook (T0–T2)
Questionnaire at follow-up (T2)
Focus groups (T1; T2)
Semi structured Interviews (T1; T2) Data collection method connects the different perspectives of stakeholders in order to
obtain a more complete picture. participants, special remarks and events by means of a logbook
and will be called monthly by the researchers. The researchers
will also keep a logbook to document events and to keep
documentary evidence for each participating organization. At
T1, we will collect qualitative data about the experiences of
employees and employers with steps 1–4 of HHR. At T2, we
will collect qualitative data about the experiences of employees
and employers with steps 5–7 of HHR (Figure 2). For both T1
and T2, focus groups and individual semi-structured interviews
with the key stakeholders and other third parties (e.g., policy
makers; communication staff) involved in the process will be
conducted. These individual interviews and focus groups are
complementary to each other (52). Budget Impact Analysis The topic lists for the
focus groups and individual semi-structured interviews will be
based on the process indicators and will include open-ended
questions about HHR, the dialogue-based approach, experiences
of stakeholders with HHR, and experienced changes. All focus
groups and individual interviews will be digitally recorded and
qualitative data will be analyzed thematically via a qualitative data
analysis software program (e.g., NVivo). The research questions for the process evaluation are: - How and to what extent has HHR been implemented in
the participating organizations, taking into account the key
process indicators? - What are the experienced changes and the perspectives of the
k
t k h ld
ith HHR? - What are the experienced changes and the perspectives of the
key stakeholders with HHR? key stakeholders with HHR? Data Collection and Analysis Data will be collected throughout the entire process (T0-
T2), at 6 months (T1), and at 12 months (T2) after the
start of step 1 of HHR (Figure 2). In order to gain multiple
perspectives and assure data validity, data source triangulation
will be applied (51). At T2, the follow-up questionnaire of
the effect evaluation will be extended with quantitative process
evaluations questions covering the key process indicators: Reach,
dose delivered, dose received, fidelity and satisfaction. These
quantitative data will be analyzed by means of descriptive
statistics. Data on the process indicators will be collected by
means of different methods and at different stakeholder’s levels
within the organization (Table 1). Throughout the process (T0-
T2), employers have the opportunity to give feedback by means
of a feedback function built within HHR. Employers will keep
track of the progress, number of meetings, time investment, Frontiers in Public Health | www.frontiersin.org FUNDING This research was funded by ZonMw project: The MAISE-toolkit:
a dialogue-based approach to improve the health of workers with
a low socio-economic status (531001405), the Netherlands. DISCUSSION This paper presents the protocol for the effect and process
evaluation of the intervention HHR. HHR is a web-based support
toolkit for employers based on dialogue and aimed at improving September 2020 | Volume 8 | Article 446 Frontiers in Public Health | www.frontiersin.org 6 Study Protocol to Evaluate a Web-Based Toolkit Hazelzet et al. the SE of low-educated employees. We hypothesize that - through
increasing job control - employees who are more exposed to HHR
will experience better SE than employees who are less or not
exposed to HHR. relationship. Third, the setting and context within participating
organizations will be a challenge, due to constant changes (e.g.,
dismissing/attracting flex workers; changing role/attitude of key
stakeholders). Fourth, it may vary per organization how much
time the HR managers and the wider management will allow
to spend by their employees, e.g., for filling in questionnaires
(including the needs assessment) and to work with HHR. This
is also related to the level of commitment and support of the
higher management. These changes might affect the results and
will therefore be well-documented throughout the process and
assessed during the process evaluation moments (e.g., being
dismissed clearly is a low control experience for the employee). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fpubh. 2020.00446/full#supplementary-material ETHICS STATEMENT The extensive process evaluation, including different time
points and data collection methods, will be a strength to better
understand the underlying mechanisms of HHR, experienced
changes and how dialogue and job control is experienced
by different key stakeholders over time. Furthermore, the
process evaluation at T2 allows to gain insight into the specific
tailored SE interventions in each organization and their related
perceived effectiveness next to the experience of HHR as a
whole. Finally, we conduct the evaluation study in five different
sectors and settings, which will increase the generalizability of
our results. The Medical Ethical Committee (METC) of the academic
hospital (MUMC) in Maastricht confirmed that the Medical
Research Involving Human Subjects Act (WMO) does not apply
to this study and that an official approval of this study by the
committee is not required (METC 2017-0311). All participants in
the effect and process evaluation will be asked to sign an informed
consent form when they start their participation in the study. Methodological Challenges EH wrote the original draft. EH, AR, HB, and IH reviewed,
revised, and edited several earlier versions of this paper. All
authors read and approved the final version of the manuscript
and contributed to finalizing the design and protocol for
the study. Despite this extensive study design, several methodological
challenges can be pointed out. First, HHR is a generic toolkit
and organizations will work with the same steps, tasks and
tools. However, the way HHR will be implemented, including
the use of the tools will differ per organization. Employers
are free to choose those tools which best fits their situation
and their specific SE problems. This might lead to differences
in effects and processes across the organizations. Therefore,
it is important to perform subgroup analyses. Second, the
participating organizations appeared to be unable to allocate
a control group, because of time limits and other concerns
within organizations. The lack of a control group is a well-
known issue within research of organizations; this unfortunately
leads to less robust evidence about what is effective in terms
of SE interventions in the workplace (53). Hence, due to the
lack of the control group it is important to study the uptake of
HHR and profoundly assess whether there is a dose-response Strengths of the Protocol This evaluation study provides insight into the effect and
implementation process of HHR, including the underlying
mechanisms that shapes the outcomes. Data triangulation using
different quantitative and qualitative methods and data sources
will be applied to assure the validity of this research. We expect
that HHR as a whole will show positive effects on the SE of low-
educated employees, regardless which organization or the effects
of the tailored SE interventions developed per organization
and the way we organized the process evaluation supports
finding explanations for possible lack of effects. Furthermore,
an economic perspective from the employer is also taken into
account in a form of a BIA. The BIA will address the affordability
of HHR and, together with the report on the benefits and gains
of the intervention, will help employers to decide whether they
want to invest in HHR. Despite these methodological challenges, it is important to
conduct evaluation studies in natural settings of organizations
and among low-educated employees in particular. Their voices
need to be heard, also in research. If HHR is proven to be
effective, HHR for and with this vulnerable group will be a
valuable support toolkit, which can be applied on a wider
scale. HHR is thereby expected to contribute to tackling the
socioeconomic health gap. 2. The
Lancet
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article distributed under the terms of the Creative Commons Attribution License (CC
BY). Frontiers in Public Health | www.frontiersin.org September 2020 | Volume 8 | Article 446 REFERENCES 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
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https://openalex.org/W4386541923
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https://proceedings.unisba.ac.id/index.php/BCSIES/article/download/9588/3940
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Indonesian
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Perbaikan Pelayanan Jasa dengan Menggunakan Model Kano
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Bandung Conference Series. Industrial Engineering Science
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cc-by
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*meidianavindati14@gmail.com, mdzikron@gmail.com Abstract. Barbershop not only provides haircut services, but also provides services
that support the appearance of men. Hairnerds Studio Bandung is a company engaged
in haircut services and is a branch of Hairnerds Studio Jakarta. However, after the
opening of Hairnerds Studio Bandung branch, based on data obtained from April
2019 – November 2020, several times the number of customer arrivals did not meet
the target set by the management. In April, May and October in 2019 but in April
Hairnerds Studio Bandung operated on April 15. Meanwhile, in 2020, the decrease
in subscribers occurred in March, April, May, July, August and September. From
March 18 to the end of May Hairnerds Studio Bandung is not operating due to the
pandemic and will reopen in June. One of the factors that affect the quality of service
is customer satisfaction. Therefore an analysis is needed to determine the level of
customer satisfaction so that the target number of customer arrivals can exceed the
target agreed upon by the management. In this study, using a problem identification
tool using the Kano model and making a repair design using a fishbone diagram. Based on the results of data processing, there are 3 categories that meet 21 service
attributes of Heirnerds Studio, namely 6 one dimensional attributes, 8 attractive
attributes, and 7 must be attributes. The service attribute that the solution will provide
is the overall attractive attribute because the attributes in that category can increase
customer satisfaction very high. Keywords: Quality Control, Kano Model, Fishbone. Abstrak. Barbershop tidak hanya memberikan pelayanan potong rambut saja, tapi
juga memberikan pelayanan yang menujang penampilan pria. Hairnerds Studio
Bandung merupakan perusahaan yang bergerak di bidang jasa potong rambut dan
merupakan cabang dari Hairnerds Studio Jakarta. Namun setelah dibukanya
Hairnerds Studio cabang Bandung, berdasarkan data yang didapat dari bulan April
2019 – November 2020 beberapa kali jumlah kedatangan pelanggan tidak memenuhi
target yang ditetapkan oleh pihak manajemen. Pada bulan April, Mei dan Oktober
ditahun 2019 namun pada bulan April Hairnerds Studio Bandung beroparasi pada
tanggal 15 April. Sedangkan pada tahun 2020 penurunan pelanggan terdapat pada
bulan Maret, April, Mei, Juli, Agustus dan September. Dari tanggal 18 Maret hingga
akhir bulan Mei Hairnerds Studio Bandung tidak beroprasi dikarenakan adanya
pandemi dan buka kembali pada bulan Juni. Salah satu faktor yang mempengaruhi
terhadap kualitas layanan yaitu kepuasan pelanggan. Bandung Conference Series: Industrial Engineering Science Bandung Conference Series: Industrial Engineering Science https://doi.org/10.29313/bcsies.v3i2.9588 Kata Kunci: Pengendalian Kualitas, Model Kano, Fishbone. Perbaikan Pelayanan Jasa dengan Menggunakan Model Kano Meidiana Vinda*, M, Dzikron A
Prodi Teknik Industri, Fakultas Teknik, Universitas Islam Bandung,
Indonesia. *meidianavindati14@gmail.com, mdzikron@gmail.com Corresponding Author
Email: mdzikron@gmail.com A.
Pendahuluan Hairnerds Studio Bandung merupakan perusahaan yang bergerak di bidang jasa potong rambut
dan merupakan cabang dari Hairnerds Studio Jakarta. Perusahaan memilih tempat usaha
disekitar Tamansari dikarenakan perusahaan merasa bahwa daerah Tamansari memiliki tempat
yang ideal berdasarkan pihak manajemen. Namun setelah dibukanya Hairnerds Studio cabang
Bandung, berdasarkan data yang didapat dari bulan April 2019 – November 2020 beberapa kali
jumlah kedatangan pelanggan tidak memenuhi target yang ditetapkan oleh pihak manajemen. Menurut Gaspersz (1), manajemen kualitas dapat dikatakan sebagai “semua aktivitas
dari fungsi manajemen secara keseluruhan yang menentukan kebijaksanaan kualitas, tujuan dan
tanggung jawab, serta mengimplementasikannya melalui alat-alat manajemen kualitas”. Dalam
penerapannya, aktivitas dan fungsi manajemen tersebut harus diawasi untu mempertahankan
tingkat keunggulan yang diinginkan seperti penetapan kebijakan mutu, membuat dan
merencanakan mutu, jaminan dan kontrol kualitas serta peningkatan kualitas. Menurut Lovelock (2), pengertian dari jasa adalah suatu aktivitas ekonomi yang
ditawarkan oleh satu pihak kepada pihak yang lain. sering kali kegiatan yang dilakukan dalam
jangka waktu tertentu, dalam bentuk suatu kegiatan yang akan membawa hasil yang diinginkan
kepada penerima, objek, maupun aset-aset lainnya yang menjadi tanggung jawab dari pembeli. Jasa adalah suatu kegiatan ekonomi yang outputnya bukan suatu produk fisik yang umumnya
dikonsumsi bersama dengan waktu produksi dan memberi nilai tambah seperti kenikmatan,
hiburan santai, kesehatan, bersifat tidak berwujud. Model kano adalah model yang dikembangkan oleh Norioki Kano pada tahun 1984,
Menurut Wijaya (3) yang mengutip penelitian dari Kano, Kano merupakan sebuah model yang
bertujuan mengkategorikan atribut-atribut produk maupun jasa berdasarkan seberapa baik
produk atau jasa tersebut mampu memuaskan kebutuhan pelanggan. Menurut Besterfield (4) Diagram Sebab-Akibat (cause-effect diagram) adalah metode
grafis sederhana untuk membuat hipotesis mengenai rantai penyebab dan akibat serta untuk
menyaring potensi penyebab dan mengorganisasikan hubungan antar variabel. Kegunaan utama
diagram ini adalah untuk menganalisis timbulnya akibat, yaitu dengan mencari atau menemukan
dan menggambarkan faktor-faktor yang menjadi penyebab dari suatu masalah. Berdasarkan latar belakang yang telah diuraikan, maka perumusan masalah dalam
penelitian ini sebagai berikut: Berdasarkan latar belakang yang telah diuraikan, maka perumusan masalah dalam
penelitian ini sebagai berikut: 1. Bagaimana kebutuhan pelayanan jasa untuk meningkatkan kualitas dengan dimens
Kano di Hairnerds Studio Bandung? 2. Bagaimana kepuasan dan harapan pelanggan terhadap atribut kualitas pelayanan jas
Hairnerds Studio Bandung berdasarkan model layanan kualitas jasa kano? 3. Bagaimana rancangan usulan perbaikan jasa layanan pada Hairnerds Studio Bandung? *meidianavindati14@gmail.com, mdzikron@gmail.com Maka dari itu diperlukan analisis
untuk mengetahui tingkat kepuasan pelanggan agar target jumlah kedatangan
pelanggan dapat melebihi target yang telah disepakati oleh pihak manajemen. Pada
penelitian ini menggunakan alat identifikasi masalah dengan menggunakan model
Kano dan membuat rancangan perbaikan dengan menggunakan diagram fishbone. Berdasarkan hasil dari pengolahan data terdapat 3 kategori yang memenuhi 21 atribut
pelayanan Heirnerds Studio yaitu terdapat 6 atribut one dimensional, 8 atribut
attractive, dan 7 atribut must be. Atribut pelayanan yang akan diberikan solusi yaitu
keseluruhan atribut attractive karena atribut pada kategori tersebut dapat
meningkatkan kepuasan pelanggan dengan sangat tinggi. Corresponding Author
Email: mdzikron@gmail.com 746 Perbaikan Pelayanan Jasa dengan Menggunakan Model Kano | 747 A.
Pendahuluan Berdasarkan rumusan masalah yang dikembangkan, penelitian ini dilakukan dengan
tujuan sebagai berikut: Berdasarkan rumusan masalah yang dikembangkan, penelitian ini dilakukan dengan
tujuan sebagai berikut: 1. Mengetahui atribut kebutuhan pelanggan dengan menggunakan dimensi Kano di
Hairners Studio Bandung. g
2. Mengetahui pelayanan yang harus diperbaiki dan dipertahankan oleh Hairnerds Studio g
2. Mengetahui pelayanan yang harus diperbaiki dan dipertahankan oleh Hairnerds Studio
Bandung berdasarkan model layanan jasa kano 2. Mengetahui pelayanan yang harus diperbaiki dan dipertahankan oleh Hairnerds Studio
Bandung berdasarkan model layanan jasa kano. Bandung berdasarkan model layanan jasa kano. 3. Mengetahui usulan perbaikan jasa pelayanan pada Hairnerds Studio Bandung. B.
Metodologi Penelitian Pengumpulan data yang dilakukan dalam penelitian ini yaitu dengan menggunakan kuesioner,
dimana pertanyaannya diperoleh dari hasil wawancara dengan pelanggan dan karyawan
Hairnerds Studio Bandung. Kuesioner disebarkan kepada responden dalam obyek penelitian
yaitu pelanggan Hairnerds Studio Bandung. Untuk mendapatkan informasi sesuai yang
diinginkan, maka daftar pertanyaan digunakan sebagai alat bantu atau sarana pengumpulan data
yang akan diolah. Untuk mendapatkan hasil data yang akan diperoleh maka dilakukan dengan
menyebarkan kuesioner kepada responden dalam bentuk pertanyaan – pertanyaan mengenai
penilaian pada penilaian terhadap tingkat kepentingan dari aspek pelayanan yang sesuai dengan
penilaian pelanggan pada pelayanan yang diberikan Hairnerds Studio Bandung. Kuesioner Industrial Engineering Science 748 | Meidiana Vinda, et al. functional dan dysfunctional Kegunaan tabel evaluasi Kano dibawah ini, untuk
mengelompokkan suatu atribut – atribut dari tiap responden. Tabel 1. Kuesioner Functional dan Dysfunctional Tabel 1. Kuesioner Functional dan Dysfunctional Customer need
Dysfunctional
1 = Suka
2 = Harap
3 = Netral
4 = Toleransi
5 = Tidak Suka
Functional
1 = Suka
Q
A
A
A
O
2 = Harap
R
I
I
I
M
3 = Netral
R
I
I
I
M
4 = Toleransi
R
I
I
I
M
5 = Tidak Suka
R
R
R
R
Q
Notes: A = Attractive, O = One dimensional, M = Mustbe,
Q = Questionable, R = Reverse, I = Indifferent Customer need
Dysfunctional
1 = Suka
2 = Harap
3 = Netral
4 = Toleransi
5 = Tidak Suka
Functional
1 = Suka
Q
A
A
A
O
2 = Harap
R
I
I
I
M
3 = Netral
R
I
I
I
M
4 = Toleransi
R
I
I
I
M
5 = Tidak Suka
R
R
R
R
Q
Notes: A = Attractive, O = One dimensional, M = Mustbe,
Q = Questionable, R = Reverse, I = Indifferent Notes: A = Attractive, O = One dimensional, M = Mustbe,
Q = Questionable, R = Reverse, I = Indifferent C. Hasil Penelitian dan Pembahasan Vol. 3 No. 2 (2023), Hal: 746-754
ISSN: 2828-21 Vol. 3 No. 2 (2023), Hal: 746-754 ISSN: 2828-2132 C.
Hasil Penelitian dan Pembahasan Penentuan kategori kano dari jawaban tiap – tiap atribut berdasarkan jenis functional dan
dysfunctional, dilakukan dengan cara menentukan titik nilai responden ditiap nilai atribut
functional dengan dysfunctional. Maka akan diperoleh titik temu untuk menentukan atribut
tersebut kedalam model kano. Dibawah ini hasil penentuan dalam klasifikasi masing – masing
atribut ke kategori Kano Setelah menentukan klasifikasi pada functional dan disfunctional, selanjutnya dilakukan
pengelompokkan dan perhitungan dari hasil klasifikasi yang sudah didapatkan sehingga untuk
langkah ini hanya menentukan jumlah kategori kano tiap – tiap atribut terhadap hasil semua
responden. Tabel 2. Pengelompokkan dan Perhitungan Hasil Klasifikasi Tabel 2. Pengelompokkan dan Perhitungan Hasil Klasifikasi
Atribut Pelayanan
Kategori Kano
Total
Q
R
I
O
A
M
1
0
0
24
25
13
20
82
2
0
0
19
28
13
22
82
3
0
0
23
12
32
15
82
4
0
0
19
13
18
32
82
5
0
0
26
16
17
23
82
6
0
0
20
16
20
26
82
7
0
0
16
24
32
10
82
8
0
0
19
21
31
11
82
9
0
0
23
9
35
15
82
10
0
0
28
24
23
7
82
11
0
0
18
10
41
13
82
12
0
0
19
27
17
19
82
13
0
0
24
21
17
20
82
14
0
0
18
19
20
25
82
15
0
0
23
7
45
7
82
16
0
0
23
7
21
31
82
17
0
0
22
11
21
28
82
18
0
0
26
7
37
12
82
19
0
0
21
14
16
31
82
20
0
0
11
26
22
23
82 Perbaikan Pelayanan Jasa dengan Menggunakan Model Kano | 749 749 Atribut Pelayanan
Kategori Kano
Total
Q
R
I
O
A
M
21
0
0
16
25
26
15
82 Penelitian ini tidak hanya menyebarkan kuesioner kepada pelanggan Hairnerds Studio
Bandung saja, tetapi juga menyebarkan kuesioner ke Barbershop lain yang memiliki segmen
pasar sejenis dengan Hairnerds Studio Bandung. Kuesioner ini disebarkan setengah dari
kuesioner tertutup dan digunakan untuk membandingkan atribut pelayanan antara Hairnerds
Studio Bandung dan Barbershop lain yang berada di segmen pasar sejenis. Nilai rata-rata hasil
kuesioner yang semakin mendekati angka satu memiliki arti bahwa atribut pelayanan yang
diberikan Barbershop telah memenuhi kepuasan pelanggan. Sampel yang digunakan berasal dari jumlah populasi pelanggan Hairnerds Studio
Bandung setiap bulannya. Industrial Engineering Science C.
Hasil Penelitian dan Pembahasan Setelah diperoleh banyaknya sampel yang dibutuhkan dengan
menggunakan perhitungan dibawah ini maka kuesioner dapat disebarkan kepada pelanggan
Hairnerds Studio Bandung. Berikut rumus perhitungan penentuan sampel dengan menggunakan
rumus Slovin: =
450
1+450(0,12) = 81,82 ≈ 82 sampel =
450
1+450(0,12) = 81,82 ≈ 82 sampel (
)
Maka berdasarkan perhitungan diatas, jumlah responden yang dibutuhkan sebanya
81,82 ≈ 82 responden. Uji validitas dilakukan dengan menggunakan 30 kuesioner dari 82 kuesioner yang
disebarkan. Pengolahan uji validitas data dengan menggunakan tingkat error 10%, sehingga
nilai 𝑟𝑡𝑎𝑏𝑒𝑙 yang diperoleh yaitu 0,306. Menentukan Kategori Kano untuk Tiap Atribut Layanan penentuan dalam kebutuhan
pelanggan sesuai dengan kategori Kano, selanjutnya menentukan kategori Kano tiap masing –
masing atribut layanan dengan menggunakan Blauth’s Formula sebagai berikut: 1. Jika (one – dimensional + attractive + must-be) > (indifferent + reverse + questionable),
maka grade diperoleh dari ujung yang paling maksimum dari (one – dimensional,
attractive, must-be). 2. Jika (one – dimensional + attractive + must-be) < (indifferent + reverse + questionable),
maka grade diperoleh dari ujung yang paling maksimum dari (indifferent, reverse,
questionable). q
)
3. Jika (one – dimensional + attractive + must-be) = (indifferent + reverse + questionable)
maka grade diperoleh yang paling maksimum diantara semua kategori kano yaitu (one –
dimensional, attractive, must-be, indifferent, reverse, questionable). 3. Jika (one – dimensional + attractive + must-be) = (indifferent + reverse + questionable)
maka grade diperoleh yang paling maksimum diantara semua kategori kano yaitu (one –
dimensional, attractive, must-be, indifferent, reverse, questionable). Tabel 3. Menentukan Kategori Kano tiap Masing-masing Atribut Atribut Pelayanan
Kategori Kano
Total
Q
R
I
O
A
M
1
0
0
24
25
13
20
82
2
0
0
19
28
13
22
82
3
0
0
23
12
32
15
82
4
0
0
19
13
18
32
82
5
0
0
26
16
17
23
82
6
0
0
20
16
20
26
82
7
0
0
16
24
32
10
82
8
0
0
19
21
31
11
82
9
0
0
23
9
35
15
82 750 | Meidiana Vinda, et al. C.
Hasil Penelitian dan Pembahasan Atribut Pelayanan
Kategori Kano
Total
Q
R
I
O
A
M
10
0
0
28
24
23
7
82
11
0
0
18
10
41
13
82
12
0
0
19
27
17
19
82
13
0
0
24
21
17
20
82
14
0
0
18
19
20
25
82
15
0
0
23
7
45
7
82
16
0
0
23
7
21
31
82
17
0
0
22
11
21
28
82
18
0
0
26
7
37
12
82
19
0
0
21
14
16
31
82
20
0
0
11
26
22
23
82
21
0
0
16
25
26
15
82 Setelah dilakukan penjumlahan dengan berdasarkan aturan penggunaan dalam Blauth’s
Formula seperti pada tabel diatas maka hasil yang diperoleh bahwa pada kategori Attractive,
One – dimensional dan Must – be memiliki jumlah yang lebih besar dibandingkan jumlah
kategori Questionable, Reverse dan Indifferent yang lebih kecil. Untuk selanjutnya dalam hasil
akhir pada model kano yaitu menentukan tiap – tiap atribut ke dalam kategori Kano sehingga
hasil yang didapat dari hasil nilai tertinggi dari kesesuaian kategori Kano Berdasarkan hasil pengolahan dengan menggunakan model kano Terdapat tiga kategori
dari hasil pengolahan data dengan menggunakan model kano yaitu kategori must be, one
dimensional, dan attractive. Keseluruhan atribut pelayanan yang berada pada kategori attractive
akan dibuat usulan perbaikan nya. Hal ini dilakukan karena atribut pelayanan attractive
merupakan pelayanan yang paling besar pengaruhnya dalam memberikan kepuasan terhadap
pelanggan. Tabel 4. Hasil Pengolahan Tabel 4. Hasil Pengolahan Tabel 4. Hasil Pengolahan
No
Atribut Pelayanan
Kategori
Kano
Tangible
1 Barbershop memiliki WC dan ruang tunggu
One
Dimensional
2 Lokasi Barbershop berada dipusat keramaian
One
Dimensional
3 Area parkir yang mudah diakses
Attractive
4 Kebersihan dan kerapihan Barbershop yang terjamin
Must Be
Emphaty
5 Karyawan Barbershop dapat memberikan kenyamanan pada saat
proses cukur rambut
Must Be
6 Karyawan Barbershop ramah kepada konsumen
Must Be
7 Barbershop memberikan pelayanan yang sama kepada semua
konsumen
Attractive
8 Karyawan Barbershop memberikan saran potongan rambut kepada
konsumen
Attractive
Assurance
9 Adanya tanggung jawab bila salah potong
Attractive Vol. 3 No. C.
Hasil Penelitian dan Pembahasan 2 (2023), Hal: 746-754 Perbaikan Pelayanan Jasa dengan Menggunakan Model Kano | 751 751 No
Atribut Pelayanan
Kategori
Kano
10 Kerapihan karyawan dalam memotong rambut merupakan hal yang
harus diperhatikan
One
Dimensional
11 Karyawan Barbershop jujur kepada konsumen jika terjadi kesalahan
Attractive
Responsiveness
12 Respon karyawan yang baik dan cepat terhadap keluhan konsumen
One
Dimensional
13 Karyawan Barbershop sangat tanggap kepada konsumen
One
Dimensional
14 Kemudahan dalam mendapatkan informasi mengenai Barbershop
Must Be
15 Proses pangkas rambut terhadap konsumen tidak memakan waktu
lama
Attractive
Reliability
16 Barbershop memiliki alat yang lengkap
Must Be
17 Barbershop memiliki alat yang berkualitas
Must Be
18 Tarif merupakan hal yang harus diperhatikan
Attractive
19 Barbershop memiliki kotak tip untuk karyawan
Must Be
20 Proses pembayaran dilakukan dengan mudah
One
Dimensional
21 Proses antri tidak rumit
Attractive No
Atribut Pelayanan
Kategori
Kano
10 Kerapihan karyawan dalam memotong rambut merupakan hal yang
harus diperhatikan
One
Dimensional
11 Karyawan Barbershop jujur kepada konsumen jika terjadi kesalahan
Attractive
Responsiveness
12 Respon karyawan yang baik dan cepat terhadap keluhan konsumen
One
Dimensional
13 Karyawan Barbershop sangat tanggap kepada konsumen
One
Dimensional
14 Kemudahan dalam mendapatkan informasi mengenai Barbershop
Must Be
15 Proses pangkas rambut terhadap konsumen tidak memakan waktu
lama
Attractive
Reliability
16 Barbershop memiliki alat yang lengkap
Must Be
17 Barbershop memiliki alat yang berkualitas
Must Be
18 Tarif merupakan hal yang harus diperhatikan
Attractive
19 Barbershop memiliki kotak tip untuk karyawan
Must Be
20 Proses pembayaran dilakukan dengan mudah
One
Dimensional
21 Proses antri tidak rumit
Attractive Atribut Pelayanan Model kano merupakan metode identifikasi atribut pelayanan yang digunakan pada
penelitian ini. Model kano mengelompokkan setiap atribut berdasarkan kemampuan atribut
tersebut dalam memenuhi kepuasan pelanggan dari sudut pandang pelanggan itu sendiri. Berdasarkan hasil dari pengolahan data terdapat 3 kategori yang memenuhi 21 atribut pelayanan
Heirnerds Studio yaitu terdapat 6 atribut one dimensional, 8 atribut attractive, dan 7 atribut must
be. Atribut pelayanan yang akan diberikan solusi yaitu keseluruhan atribut attractive karena
atribut pada kategori tersebut dapat meningkatkan kepuasan pelanggan dengan sangat tinggi. C.
Hasil Penelitian dan Pembahasan Terdapat 8 atribut pelayanan yang perlu diperbaiki karena masih belum bisa memenuhi
kepuasan pelanggan yaitu area parkir yang mudah diakses, barbershop memberikan pelayanan
yang sama kepada semua konsumen, karyawan barbershop memberikan saran potongan rambut
kepada konsumen, adanya tanggung jawab bila salah potong, karyawan barbershop jujur kepada
konsumen jika terjadi kesalahan, proses pangkas rambut terhadap konsumen tidak memakan
waktu lama, tarif merupakan hal yang harus diperhatikan, dan proses antri tidak rumit Solusi
akan diberikan dengan menggunakan pendekatan Fishbone. Tabel 5. Atribut Layanan yang perlu dilakukan Perbaikan NO
Atribut Layanan yang perlu dilakukan Perbaikan
1
Area parkir sulit diakses
2
Barber Shop tidak memberikan pelayanan yang sama kepada semua konsumen
3
Karyawan Barbershop tidak memberikan saran potongan rambut kepada konsumen
4
Tidak adanya tanggung jawab bila terjadi kesalahan potong
5
Karyawan barbershop tidak terbuka kepada konsumen jika terjadi kesalahan Atribut Layanan yang perlu dilakukan Perbaikan Industrial Engineering Science 752 | Meidiana Vinda, et al. NO
Atribut Layanan yang perlu dilakukan Perbaikan
6
Proses potong rambut terhadap konsumen memakan waktu lama
7
Tarif potong tidak terjangkau
8
Proses antri rumit Berdasarkan hasil pengolahan dengan menggunakan model kano Terdapat tiga kategori
dari hasil pengolahan data dengan menggunakan model kano yaitu kategori must be, one
dimensional, dan attractive. Keseluruhan atribut pelayanan yang berada pada kategori attractive
akan dibuat usulan perbaikan nya. Hal ini dilakukan karena atribut pelayanan attractive
merupakan pelayanan yang paling besar pengaruhnya dalam memberikan kepuasan terhadap
pelanggan. Tarif Potong tidak
terjangkau
Material
Metode
Kualitas pelayanan tidak
sesuai dengan harga
Manusia
Kebijakan perusahaan dalam
menentukan harga
Mesin
Lingkungan
Kualitas potongan rambut
tidak sesuai dengan harga
Biaya alat potong dan bahan
yang mahal
Tarif mengikuti cabang
Jakarta
Biaya perawatan mahal
Mengikuti Pendapatan Kota
Bandung
Gambar 1. Tarif Potong Tidak Terjangkau Material Manusia Kualitas potongan rambut
tidak sesuai dengan harga Kebijakan perusahaan dalam
menentukan harga Tarif Potong tidak
terjangkau Biaya alat potong dan bahan
yang mahal Tarif mengikuti cabang
Jakarta Kualitas pelayanan tidak
sesuai dengan harga Mengikuti Pendapatan Kota
Bandung Lingkungan Metode Gambar 1. Tarif Potong Tidak Terjangkau g
j
g
solusi yang diberikan untuk setiap penyebab masalah tersebut, yaitu sebagai berikut: 1. Kebijakan perusahaan dalam menentukan harga
Kebijakan perusahaan dalam menentukan harga merupakan salah satu hal yang harus
diperhatikan. Solusi yang diberikan yaitu dengan melakukan atau melihat pendapatan rata – rata
masyarakat Kota Bandung dan agar dapat bersaing dengan perusahaan lainnya. 2. Kualitas potongan rambut yang tidak sesuai dengan harga. C.
Hasil Penelitian dan Pembahasan Tarif yang tinggi membuat pelanggan mengharapkan potongan rambut yang diinginkan. Tarif
yang terlalu mahal membuat pelanggan memiliki harapan lebih kepada barbershop. Solusi yang
dapat dilakukan adalah terus meningkatkan kualitas pelayanan jasa didukung dengan pemberian
pelatihan kepada karyawan dan memberikan penghargaan kepada karyawan teladan sehingga
karyawan memiliki keinginan untuk terus maju dan berkembang. 3. Biaya Alat cukur dan bahan yang digunakan mahal
Peralatan yang digunakan oleh perusahaan memiliki harga yang cukup mahal sehingga
mengakibatkan kepada harga yang mahal. Solusi yang dapat dilakukan dengan penyesuaian alat
dan tarif yang diberikan agar dapat bersaing dengan perusahaan lainnya. 4. Biaya perawatan Mahal Vol. 3 No. 2 (2023), Hal: 746-754 ISSN: 2828-2132 753 Perbaikan Pelayanan Jasa dengan Menggunakan Model Kano | 7 Biaya perawatan alat memiliki harga yang cukup mahal sehingga mengakibatkan kepada harga
yang mahal. Solusi yang dapat dilakukan dengan penyesuaian alat dan tarif yang diberikan agar
dapat bersaing dengan perusahaan lainnya. Biaya perawatan alat memiliki harga yang cukup mahal sehingga mengakibatkan kepada harga
yang mahal. Solusi yang dapat dilakukan dengan penyesuaian alat dan tarif yang diberikan agar
dapat bersaing dengan perusahaan lainnya. Biaya perawatan alat memiliki harga yang cukup mahal sehingga mengakibatkan kepada harga
yang mahal. Solusi yang dapat dilakukan dengan penyesuaian alat dan tarif yang diberikan agar
dapat bersaing dengan perusahaan lainnya. 5. Kualitas pelayanan tidak sesuai dengan harga
Banyak pelanggan yang merasa bahwa kualitas pelayanan yang diberikan kurang sesuai dengan
tarif yang harus mereka bayar. Tarif yang terlalu mahal membuat pelanggan memiliki harapan
lebih kepada barbershop. Solusi yang dapat dilakukan adalah terus meningkatkan kualitas
pelayanan jasa didukung dengan pemberian pelatihan kepada karyawan dan memberikan
penghargaan kepada karyawan teladan sehingga karyawan memiliki keinginan untuk terus maju
dan berkembang. 6. Mengikuti pendapatan Kota Bandung
Mengikuti pendapatan warga kota Bandung bertujuan guna menyesuaikan harga dengan pesaing
yang lain. Solusinya yaitu menyesuaikan dengan pendapatan masyarakat kota Bandung g
p
p
g
Mengikuti pendapatan warga kota Bandung bertujuan guna menyesuaikan harga dengan pesaing
yang lain. Solusinya yaitu menyesuaikan dengan pendapatan masyarakat kota Bandung
7
T if
ik i
b
J k 7. Tarif mengikuti cabang Jakarta
Tarif pelayanan mengikuti cabang pusat di Jakarta sehingga tidak sesuai dengan kemampuan
pelanggan yang ada di Bandung. Solusi yang tepat adalah dengan menentukan tarif sesuai
dengan segmen pasar atau meningkatkan kualias pelayanan menjadi lebih baik sesuai dengan
tarif yang diberikan. Industrial Engineering Science D. Hasil dari penelitian yang telah dilakukan oleh peneliti terhadap pelayanan jasa Hairnerds
Studio Bandung dapat disimpulkan: 1. Terdapat 21 atribut pelayanan pada Hairnerds Studio yaitu Barbershop memiliki WC dan
ruang tunggu, Lokasi Barbershop berada dipusat keramaian, Area parkir yang mudah
diakses, kebersihan dan kerapihan barbershop yang terjamin, karyawan barbershop dapat
memberikan kenyamanan pada saat proses cukur rambut, karyawan barbershop ramah
kepada konsumen, barbershop memberikan pelayanan yang sama kepada semua
konsumen, karyawan barbershop memberikan saran potongan rambut kepada konsumen,
adanya tanggung jawab bila salah potong, kerapihan karyawan dalam memotong rambut
merupakan hal yang harus diperhatikan, karyawan barbershop jujur kepada konsumen
jika terjadi kesalahan, respon karyawan yang baik dan cepat terhadap keluhan konsumen,
karyawan barbershop sangat tanggap kepada konsumen, kemudahan dalam mendapatkan
informasi mengenai barbershop, proses pangkas rambut terhadap konsumen tidak
memakan waktu lama, barbershop memiliki alat yang lengkap, barbershop memiliki alat
yang berkualitas, tarif merupakan hal yang harus diperhatikan, barbershop memiliki
kotak tip untuk karyawan, proses pembayaran dilakukan dengan mudah, dan proses antri
tidak rumit. 2. Terdapat 8 atribut pelayanan yang perlu diperbaiki karena masih belum bisa memenuhi
kepuasan pelanggan yaitu area parkir yang mudah diakses, barbershop memberikan
pelayanan yang sama kepada semua konsumen, karyawan barbershop memberikan saran
potongan rambut kepada konsumen, adanya tanggung jawab bila salah potong, karyawan
barbershop jujur kepada konsumen jika terjadi kesalahan, proses pangkas rambut
terhadap konsumen tidak memakan waktu lama, tarif merupakan hal yang harus
diperhatikan, dan proses antri tidak rumit. 3. Solusi perbaikan yang diberikan antara lain memberikan pengetahuan cara parkir yang
rapih kepada tukang parkir sehingga area parkir dapat digunakan untuk kapasitas yang
besar, memberikan SOP tentang pelayanan yang harus dilakukan oleh karyawan dengan
mempertimbangkan waktu pelayanan yang sama untuk setiap pelanggan, memberikan
pendidikan tentang jenis-jenis potongan rambut pada saat pelatihan, menentukan
besarnya gnti rugi yang harus diberikan perusahaan sehingga karyawan dan pelanggan
merasa adil, memberikan teguran atau sanksi terhadap karyawan yang berlaku tidak jujur
dan tidak adil dalam menghadapati pelanggan, memberikan standar waktu pelayanan
agar karyawan tidak terlalu lama dalam melakukan pekerjaannya, tarif pelayanan
disesuaikan dengan pelayanan dan produk yang digunakan dengan hasil yang Industrial Engineering Science 754 | Meidiana Vinda, et al. memuaskan pelanggan, dan saat mengantri pelanggan diberikan nomor antrian beserta
dengan nama karyawan yang akan melayani mereka. Acknowledge
Terimakasih kepada seluruh Pembimbing, kedua orangtua, Pihak Hairnerds Studio Bandung. Acknowledge
Terimakasih kepada seluruh Pembimbing, kedua orangtua, Pihak Hairnerds Studio Bandung. Vol. 3 No. 2 (2023), Hal: 746-754 Acknowledge Acknowledge Daftar Pustaka Daftar Pustaka
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https://openalex.org/W2513340759
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https://repositum.tuwien.at/bitstream/20.500.12708/159/2/Rupprechter%20Guenther%20-%202016%20-%20Surface%20spectroscopy%20on%20UHV-grown%20and...pdf
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English
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Surface Spectroscopy on UHV-Grown and Technological Ni–ZrO2 Reforming Catalysts: From UHV to Operando Conditions
|
Topics in catalysis
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cc-by
| 10,329
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Surface Spectroscopy on UHV-Grown and Technological Ni–ZrO2
Reforming Catalysts: From UHV to Operando Conditions Kresimir Anic1 • Astrid Wolfbeisser1 • Hao Li1 • Christoph Rameshan1 •
Karin Fo¨ttinger1 • Johannes Bernardi2 • Gu¨nther Rupprechter1 Published online: 12 August 2016 Published online: 12 August 2016 Published online: 12 August 2016
The Author(s) 2016. This article is published with open access at Springerlink.com g
The Author(s) 2016. This article is published with open access at Springerlink.com decreased the H2/CO ratio. Operando studies of methane
steam reforming, simultaneously recording FTIR and MS
data, detected activated CH4 (CH3 and CH2), activated
water (OH), as well as different bidentate (bi)carbonate
species, with the latter being involved in the water gas shift
side reaction. Surface science Ni–ZrO2 model catalysts
were prepared by first growing an ultrathin ‘‘trilayer’’
(O–Zr–O) ZrO2 support on an Pd3Zr alloy substrate, and
subsequently depositing Ni, with the process being moni-
tored by XPS and LEED. Apart from the trilayer oxide,
there is a small fraction of ZrO2 clusters with more bulk-
like properties. When CO was adsorbed on the (fully
metallic) Ni particles at pressures up to 100 mbar, both
PM-IRAS and XPS indicated CO dissociation around room
temperature and blocking of the Ni surface by carbon (note
that on the partially oxidized technological Ni particles,
CO dissociation was absent). The Ni nanoparticles were
stable up to 550 K but annealing to higher temperatures
induced Ni migration through the ultrathin ZrO2 support
into the Pd3Zr alloy. Both approaches have their benefits
and limitations but enable us to address specific questions
on a molecular level. Abstract Ni nanoparticles supported on ZrO2 are a pro-
totypical
system
for
reforming
catalysis
converting
methane to synthesis gas. Herein, we examine this catalyst
on a fundamental level using a 2-fold approach employing
industrial-grade catalysts as well as surface science based
model catalysts. In both cases we examine the atomic
(HRTEM/XRD/LEED) and electronic (XPS) structure, as
well as the adsorption properties (FTIR/PM-IRAS), with
emphasis on in situ/operando studies under atmospheric
pressure conditions. For technological Ni–ZrO2 the rather
large Ni nanoparticles (about 20 nm diameter) were evenly
distributed over the monoclinic zirconia support. In situ
FTIR spectroscopy and ex situ XRD revealed that even
upon H2 exposure at 673 K no full reduction of the nickel
surface was achieved. CO adsorbed reversibly on metallic
and oxidic Ni sites but no CO dissociation was observed at
room temperature, most likely because the Ni particle
edges/steps comprised Ni oxide. Surface Spectroscopy on UHV-Grown and Technological Ni–ZrO2
Reforming Catalysts: From UHV to Operando Conditions CO desorption tempera-
tures were in line with single crystal data, due to the large
size of the nanoparticles. During methane dry reforming at
873 K carbon species were deposited on the Ni surface
within the first 3 h but the CH4 and CO2 conversion hardly
changed even during 24 h. Post reaction TEM and TPO
suggest the formation of graphitic and whisker-type carbon
that do not significantly block the Ni surface but rather
physically block the tube reactor. Reverse water gas shift Keywords Nickel Zirconia Model catalysts
Technological catalysts Carbon monoxide Methane
steam reforming Methane dry reforming In situ
spectroscopy Operando spectroscopy Top Catal (2016) 59:1614–1627
DOI 10.1007/s11244-016-0678-8 Top Catal (2016) 59:1614–1627
DOI 10.1007/s11244-016-0678-8 ORIGINAL PAPER ORIGINAL PAPER & Gu¨nther Rupprechter
guenther.rupprechter@tuwien.ac.at & Gu¨nther Rupprechter
guenther.rupprechter@tuwien.ac.at
1
Institute of Materials Chemistry, Technische Universita¨t
Wien, Getreidemarkt 9/BC/01, 1060 Vienna, Austria
2
University Service Center for Transmission Electron
Microscopy, Technische Universita¨t Wien, Wiedner
Hauptstraße 8-10, 1040 Vienna, Austria 1 Introduction 1
Institute of Materials Chemistry, Technische Universita¨t
Wien, Getreidemarkt 9/BC/01, 1060 Vienna, Austria Ni–ZrO2 catalysts are among the most important industrial
catalysts, due to their widespread application for methane
reforming reactions. Steam reforming [1–3], dry reforming
[4–9] and partial oxidation [10, 11] are routes to produce 2
University Service Center for Transmission Electron
Microscopy, Technische Universita¨t Wien, Wiedner
Hauptstraße 8-10, 1040 Vienna, Austria 123 12 3 1615 Top Catal (2016) 59:1614–1627 synthesis gas (CO and H2) of varying composition but in all
cases deactivation by coke (carbon filament) formation is a
major limitation of catalyst performance and lifetime. Several strategies were applied to reduce coking, including
alloying with a second metal (e.g. Au [12–14], Cu [15–25],
intended to decorate the active Ni steps that are prone to
coking with a less active metal) and/or using mixed-oxide
supports [4, 5, 26–33]. Adding CeO2 to ZrO2 to has been
found to improve the oxygen storage capacity and redox
properties, which affects the ability to deliver oxygen from
the lattice to carbon, increasing thermal stability and cat-
alytic activity [3, 5, 26]. chemicals XZO 880/01; calcined at 973 K) with a total
metal weight content of 5 % w/w. After dissolving the
appropriate amount of Ni-nitrate in water, ZrO2 powder
was suspended in the solution, which was dried at 373 K
overnight. Then the powder was heated to 723 K in air with
a heating rate of 5 K min-1 and calcined at 723 K for 2 h. Prior to characterization and reaction the catalyst was
oxidized at 773 K and reduced at 673 K (at *1 bar in
20 % O2 in Ar and 20 % H2 in Ar, respectively, with a flow
rate 50 ml min-1). The catalyst was characterized by high resolution
transmission electron microscopy (HRTEM) including
energy dispersive X-ray fluorescence (EDX) analysis, H2-
chemisorption, infrared spectroscopy (FTIR) with CO as a
probe molecule, X-ray diffraction (XRD), and X-ray
absorption spectroscopy (XAS). To study the catalytic
performance, the catalyst was tested in a continuous flow
setup for methane dry and steam reforming. Operando
spectroscopy provides information about the catalyst under
reaction conditions, i.e. high temperature and reactive gas
atmosphere. In this respect, operando IR, XAS and near
atmospheric pressure X-ray photoelectron (NAP-XPS)
spectroscopy were applied. 1 Introduction After reaction, the used catalyst
was further studied via temperature programmed oxidation
(TPO), in order to get information about the amount of
carbon deposition and the temperature needed to remove
the carbon species via oxidation, and by HRTEM, imaging
carbon deposition. Apart from these modified variants, the ‘‘simple’’ Ni–
ZrO2 catalysts still holds potential for fundamental studies
of the nanoparticle size, shape and surface structure, of the
interaction of the Ni nanoparticles with the oxide support,
of the specific adsorption and reaction properties of metal-,
oxide- and metal-oxide-interface sites, and of the thermal
and chemical stability of the catalysts. Due to the relatively
harsh reforming conditions, especially high temperature,
the exact reaction mechanisms on Ni (and Pt) are still
debated [34–39]. Herein, we describe an approach to
examine fundamental properties of Ni–ZrO2 by performing
surface spectroscopic studies both on technological and
model (ultrahigh vacuum (UHV) prepared) catalysts. Whereas the first provide a link to industrial catalysis, the
latter enable us to reduce complexity and to apply surface
science techniques [40–51]. It is important to note that in both cases structure,
composition and adsorption properties were examined
which clearly reveals analogies but also differences
between applied and model catalysts. For both types of
catalysts we have performed ‘‘in situ’’ studies, i.e. acquir-
ing surface spectra during ongoing processes, in an effort to
obtain meaningful and technologically relevant structure–
activity correlations. In recent years we have applied this
approach to a variety of technological catalysts [52–58]
and single-crystal based model catalysts [59–61]. For the
technological catalysts, when catalytic activity is recorded
simultaneously, the term ‘‘operando’’ spectroscopy is
rather used [62, 63]. 2.2 Ni Nanoparticles on Ultrathin ZrO2 Films
Grown in UHV The experimental setup used to prepare and characterize
the ultrathin ZrO2 support, as well as the Ni nanoparticles,
has been described in detail in references [49, 50]. In brief,
it consists of an ultrahigh vacuum (UHV) chamber for
sample preparation and characterization (LEED, TPD,
XPS, IRAS), which is connected to a UHV-compatible
atmospheric pressure reaction cell (‘‘Rupprechter-design’’
[64]) that also allows for in situ PM-IRAS spectroscopy. In
order to prepare an ultrathin model zirconia thin film with
Ni nanoparticles on top, either a Pd3Zr(0001) or Pt3-
Zr(0001)
single
crystal
was
cleaned
by
sputtering
(8 9 10-6 mbar of Ar, 2 kV) and annealing in UHV. After
cleaning, the Pd3Zr or Pt3Zr single crystal was exposed to
1 9 10-7 mbar of O2 at 673 K for 30 min, followed by
annealing in UHV at 1073 K for 20 min to grow an uni-
form ultrathin ZrO2 layer (for details see [65, 66]). On top
of this ultrathin layer, an amount of Ni equivalent to a
homogeneous layer of nominally 3 A˚ was deposited at
room temperature, utilizing an Omicron electron-beam
triple evaporator (EFM 3T) for physical vapor deposition. The prepared model catalyst surface was characterized by 2 Experimental In the following, the synthesis/preparation of the Ni–ZrO2
catalysts is briefly described. For more detailed descrip-
tions we refer to refs [24, 25]. 3 Results and Discussion The following section is divided into two parts, the first
describing results obtained for the technological catalyst,
and the second describing those for the model catalyst. For
both we first focus on structural properties, then turn to CO
adsorption properties, and finally discuss studies performed
under
elevated
pressure
and/or
elevated
temperature
conditions. 3.1.1 Structure Characterization The band at 2060 cm-1 appearing at
0.1 mbar pressure is attributed to linearly adsorbed CO on
Ni0 and the broad band(s) below 2000 cm-1 are attributed
to threefold hollow bonded CO on Ni0 [69–71]. The band
at 2120 cm-1, shifting to 2127 cm-1 with increasing CO
pressure, is attributed to Ni?–CO. The unusual oxidation 2.1 Impregnated Ni–ZrO2 The technological Ni–ZrO2 catalyst was prepared by
impregnation of ZrO2 (commercial Zr(OH)4 from MEL 12 3 3 Top Catal (2016) 59:1614–1627 1616 LEED and XPS and, to follow the interaction of the surface
with CO, by polarization modulation infrared reflection
absorption spectroscopy (PM-IRAS) [67]. LEED and XPS and, to follow the interaction of the surface
with CO, by polarization modulation infrared reflection
absorption spectroscopy (PM-IRAS) [67]. Fig. 1 XRD (a) und TEM (b, c) of technological Ni–ZrO2 after ex
situ oxidation at 773 K followed by reduction at 673 K. The sample
was exposed to air prior to XRD and TEM, however. The XRD
pattern shows reflections characteristic of monoclinic ZrO2 and cubic
NiO. TEM reveals ZrO2 (b) and Ni/NiO particles (c) of about 50 and
20 nm diameter, respectively 3.1.1 Structure Characterization Applying H2-chemisorption to Ni–ZrO2 after oxidation at
773 K and reduction at 673 K indicated an accessible
metallic Ni surface area of 31.4 m2 g-1 [68]. This results
in a calculated Ni particle size of 21.5 nm (implying
spherical Ni particles) which is in reasonable agreement
with the particle size found by TEM, as described below. Note however, that for XRD and TEM analysis, the
reduced Ni–ZrO2 had to be exposed to air prior to the
measurements. Due to easy reoxidation, at least the outer
shell of the Ni nanoparticles is thus not expected to remain
in the metallic state. The diffractogram in Fig. 1a) displays mostly reflections
characteristic of monoclinic ZrO2. Small peaks of cubic
NiO (at 37.3 and 43.2) are visible, whereas those of
metallic Ni (at 44.4 and 51.8 and also expected to be
weak) are overlapping with ZrO2 reflections. According to
TEM, the particles of the monoclinic ZrO2 support have a
diameter of about 50–100 nm (Fig. 1b). At low magnifi-
cation the low Z-contrast between zirconia and Ni or NiO
prevents the unambiguous identification of Ni/NiO parti-
cles and EDX was thus applied to locate them. Typically,
the particles consisted of a Ni core and a disordered shell
(with a thickness of up to several nanometers). The fcc Ni
particle core is imaged with lattice resolution in Fig. 1b. According to XRD, the shell must be NiO. The Ni/NiO
particles were evenly distributed on the zirconia support
and their size ranged from about 10–30 nm, but most of
them had a diameter of about 20 nm. Fig. 1 XRD (a) und TEM (b, c) of technological Ni–ZrO2 after ex
situ oxidation at 773 K followed by reduction at 673 K. The sample
was exposed to air prior to XRD and TEM, however. The XRD
pattern shows reflections characteristic of monoclinic ZrO2 and cubic
NiO. TEM reveals ZrO2 (b) and Ni/NiO particles (c) of about 50 and
20 nm diameter, respectively Afterwards 0.1, 0.5, 1.0, and 5.0 mbar CO were dosed at
300 K and for each respective pressure FTIR spectra were
acquired (Fig. 2a). Additionally, one spectrum was recor-
ded after evacuation to less than 2 9 10-6 mbar. The peaks at 2185–2190 and 2164 cm-1 are attributed
to Zr4?–CO and Ni2?–CO, respectively. With increasing
CO pressure, the Zr4?–CO peak grows and obscures the
Ni2?–CO peak. Afterwards 0.1, 0.5, 1.0, and 5.0 mbar CO were dosed at
300 K and for each respective pressure FTIR spectra were
acquired (Fig. 2a). Additionally, one spectrum was recor-
ded after evacuation to less than 2 9 10-6 mbar.
The peaks at 2185–2190 and 2164 cm-1 are attributed
to Zr4?–CO and Ni2?–CO, respectively. With increasing
CO pressure, the Zr4?–CO peak grows and obscures the
Ni2?–CO peak. The band at 2060 cm-1 appearing at
0.1 mbar pressure is attributed to linearly adsorbed CO on
Ni0 and the broad band(s) below 2000 cm-1 are attributed
to threefold hollow bonded CO on Ni0 [69–71]. The band
at 2120 cm-1, shifting to 2127 cm-1 with increasing CO
pressure, is attributed to Ni?–CO. The unusual oxidation CH4 þ CO2 2CO þ 2H2
DH
298K ¼ þ261kJ=mol: CH4 þ CO2 2CO þ 2H2
DH
298K ¼ þ261kJ=mol: CH4 þ CO2 2CO þ 2H2
DH
298K ¼ þ261kJ=mol: After oxidation at 773 K in O2/Ar followed by reduction
at 873 K in H2/Ar the Ni–ZrO2 catalyst was exposed to
CH4:CO2:Ar = 10/10/80
with
a
total
flow
rate
of
25 ml min-1 at 873 K for 24 h (total pressure 1 bar). Figure 3 highlights the analysis obtained by gas chro-
matographic (GC) flame ionization detection (FID) and
thermal conductivity (TCD) detection of reactants and
products during methane dry reforming. It was observed
that the CH4 and CO2 conversion as well as the production
rate of H2 and CO hardly changed over time. The H2:CO
ratio achieved was below one due to reverse water gas
shift (RWGS) as a side reaction (CO2 þ H2 CO þ
H2O;
DH
298K ¼ þ41kJ=mol). The C-balance (indicating
coke formation when being below one) increased to nearly
one during the first 3 h. After evacuation, Ni?–CO as well as linear and hollow
bonded CO on Ni0 partly remain on the surface. Overall,
CO FTIR indicates that, in agreement with XAS mea-
surements [68], both reduced and oxidized Ni species are
present on the catalyst surface and that NiO reduction in H2
is not complete at 673 K. This may explain why CO dissociation (and resulting
carbon poisoning) is not occurring for the technological
catalyst upon room temperature CO adsorption. When the
CO dosing was repeated (after evacuation) basically the
same IR spectra were obtained. As shown below, the
supported (fully metallic) Ni nanoparticles prepared in
UHV dissociated CO already around room temperature. The turn-over-frequency (TOF) for H2 production based
on the metallic Ni surface area after reduction at 873 K,
normalized to the number of surface nickel atoms assuming
1.59 9 1019 nickel atoms/m2 [74], was initially 1.2 s-1,
1.2 s-1 after 3 h time on stream and 1.0 s-1 after 24 h time
on stream. In a consecutive experiment (after evacuation) CO
desorption was monitored via heating to elevated temper-
atures. In Fig. 2b the thermal stability of adsorbed CO was
followed by recording FTIR spectra during heating with a
rate of 10 K min-1 in high vacuum. CO desorbed from
Ni? CH4 þ CO2 2CO þ 2H2
DH
298K ¼ þ261kJ=mol: between 300 and 350 K, adsorbed multiply coordi-
nated CO on Ni0 vanished at 400–450 K (in agreement
with single crystal data [64, 73]) and at 450–500 K all CO
(including linear) had desorbed from Ni–ZrO2. Figure 4a shows a TEM image of Ni–ZrO2 taken after
dry reforming at 873 K (24 h). Filamentous carbon has
formed and some Ni particles are located on top of the
carbon nanofibers, tubes or rods. 3.1.2 CO Adsorption In the next step, the interaction of the technological catalyst
with CO was studied by FTIR. For the adsorption experi-
ments Ni–ZrO2 was pre-reduced in 5 mbar H2 at 673 K. 123 123 1617 Top Catal (2016) 59:1614–1627 Fig. 2 a FTIR spectra of Ni–ZrO2 (reduced in 5 mbar H2 at 673 K)
acquired at 300 K during CO adsorption at different pressures and
after evacuation. b The thermal stability of adsorbed CO was Fig. 2 a FTIR spectra of Ni–ZrO2 (reduced in 5 mbar H2 at 673 K)
acquired at 300 K during CO adsorption at different pressures and
after evacuation. b The thermal stability of adsorbed CO was
followed by recording FTIR spectra upon annealing in vacuum
(heating rate of 10 K min-1) followed by recording FTIR spectra upon annealing in vacuum
(heating rate of 10 K min-1) followed by recording FTIR spectra upon annealing in vacuum
(heating rate of 10 K min-1) Fig. 2 a FTIR spectra of Ni–ZrO2 (reduced in 5 mbar H2 at 673 K)
acquired at 300 K during CO adsorption at different pressures and
after evacuation. b The thermal stability of adsorbed CO was state of Ni? is stabilized by the ligand CO [72]. Indeed, the
decreasing peaks of CO on Ni0 and Ni2? and the increasing
CO–Ni? peak are due to a surface reaction in the presence
of CO (Ni2? ? Ni0 ? 2 Ni?; see Kasal et al. [72]). 3.1.3 Methane Dry Reforming (MDR) After 24 h reaction in methane and carbon dioxide,
temperature programmed oxidation (TPO) was performed
with a heating rate of 5 K min-1 in 20 % O2 in Ar. TPO
provides information about the amount of coke formed
during the reaction and the temperature which is required
to burn off the carbon species. This temperature is The most straightforward and direct measurement of the
thermal stability of the Ni–ZrO2 catalyst is via its catalytic
activity at high temperature, such as during methane dry
reforming: 12 3 Top Catal (2016) 59:1614–1627 1618 Fig. 3 a Conversion of reactants and reaction rate of hydrogen and carbon monoxide production and b H2/CO ratio and carbon balance over Ni–
ZrO2 for methane dry reforming reaction at 873 K; feed composition: CH4:CO2:Ar = 10/10/80; feed flow rate = 25 ml min-1 Fig. 3 a Conversion of reactants and reaction rate of hydrogen and carbon monoxide production and b H2/CO ratio and carbon balance over Ni–
ZrO2 for methane dry reforming reaction at 873 K; feed composition: CH4:CO2:Ar = 10/10/80; feed flow rate = 25 ml min-1 Fig. 4 a TEM image of coked
Ni–ZrO2 and b temperature
programmed oxidation with
20 % O2 in Ar after exposure to
CH4 and CO2 at 873 K Fig. 4 a TEM image of coked
Ni–ZrO2 and b temperature
programmed oxidation with
20 % O2 in Ar after exposure to
CH4 and CO2 at 873 K Fig. 4 a TEM image of coked
Ni–ZrO2 and b temperature
programmed oxidation with
20 % O2 in Ar after exposure to
CH4 and CO2 at 873 K activation barrier, a low sticking coefficient and a high
hydrogen kinetic isotope effect [77]. Wang et al. [35]
investigated the reaction pathways for MDR on Ni(111) by
density functional theory and suggested a simplified reac-
tion mechanism. The rate determining step was found to be
CH4 dissociative adsorption. CH3, which is produced by
CH4 dissociation prefers to dehydrogenate into CH2 which
prefers to dehydrogenate to CH. However, CH prefers to be
oxygenated to CHO by surface oxygen, which is formed by
CO2 dissociation (without surface oxygen, dehydrogena-
tion to C and H occurs). Dehydrogenation of CHO into H
and CO then has a very low energy barrier on Ni(111). The
mechanism proposed in this analysis of unsupported Ni is
depicted in Fig. 5. 3.1.3 Methane Dry Reforming (MDR) characteristic for the bond strength of carbon to the cata-
lyst’s surface and for the nature of the carbon species. Figure 4b shows the CO2 production during TPO after dry
reforming. Temperatures of about 800–880 K are needed
to oxidize most of the coke. According to the literature, the
first CO2 evolution maximum around 800–810 K can be
assigned to graphitic carbon [75]. The CO2 evolution
around 870–880 K can be assigned to the oxidation of
whisker-type carbon which does not deactivate the nickel
surface but rather causes a breakdown of the catalyst by
pore plugging [76]. It is also interesting to note that the
TPO acquired after a reaction time of 3 h is nearly identical
indicating
that
coke
deposition
occurs
rather
rapid. Apparently, this type of coking does not deactivate the Ni
surface but rather grows whiskers. The major problem of
this catalyst is thus not the drop in catalytic activity but
rather the physical blocking of the tube reactor. This was
indeed observed when increasing the amount of catalyst in
other catalytic tests. Fig. 5 Simplified mechanism of CO2 reforming on Ni(111). Adapted
from Wang et al. [35] The activation of CH4 and CO2 as well as the reaction of
these molecules on transition metal surfaces have been
studied in great detail [34–39]. Methane activation on
transition metal surfaces is characterized by a high Fig. 5 Simplified mechanism of CO2 reforming on Ni(111). Adapted
from Wang et al. [35] 123 123 1619 Top Catal (2016) 59:1614–1627 increasing the temperature to 573 K adsorbed water van-
ished from the surface, while the CHx species remained. At
about 623 K, H2, CO and CO2 formation (i.e. MSR and
WGS) set in (detected by MS) and a new type of bidentate
bicarbonate appeared on the surface (*1530 cm-1). The
formed CO2 thus accounts for the (re)appearance of the
(bi)carbonates. When temperature and reactivity were
increasing the vibrations of bidentate bicarbonates shifted
from 1550 to 1520 and from 1330 to 1350 cm-1. During
2 h of reaction at 673 K neither the adsorbates nor the
reactivity towards MSR were changing, i.e. no deactivation
was observed. On supported catalysts, it is expected that the step of
methane decomposition occurs on the metal particles
[38, 39] while CO2 activation likely occurs on the support
[36, 37, 78, 79]. Thus, the reaction between CHx and
‘‘activated’’ CO2 might take place on the metal-support
interface. 3.2.1 Preparation and Characterization As described in the experimental section, the ZrO2 model
support was prepared by oxidation/annealing of an ade-
quate intermetallic compound, either Pd3Zr(0001) or Pt3
Zr(0001), producing an ultrathin ZrO2 (O–Zr–O) trilayer. Using an electron beam evaporator, Ni nanoparticles were
then grown on top of the ultrathin ZrO2 layer. The amount
of deposited Ni (nominal thickness 3 A˚ ) was controlled by
a calibrated quartz microbalance. For further details con-
cerning ZrO2 film preparation and cluster growth we refer
to references [65, 66, 82, 83]. CH4 þ H2O ! CO þ 3H2
DH
298K ¼ þ206kJ=mol; obtained by simultaneous FTIR and MS measurements
(Fig. 6). The pre-reduced catalyst was heated in a mixture
of CH4 and H2O (*1.2 bar with a ratio of 1/3) from 300 to
673 K. As reference state, the FTIR spectrum of the
reduced catalyst prior to the reaction is included. The MSR reaction products are again CO and H2, i.e. both methane and water must be activated, e.g. via CH4
dehydrogenation (dissociative adsorption) and H2O disso-
ciation. Once more, water gas shift (WGS; CO þ H2O
CO2 þ H2
DH
298K ¼ 41kJ=mol) may occur, accounting
for the CO2 byproduct [38, 39]. The growth of the current ZrO2 thin film was monitored
by XPS. Figure 7 shows Zr 3d spectra of the clean Pd3Zr
alloy (left), of the ultrathin (O–Zr–O trilayer) ZrO2 (mid-
dle; after oxidation in 1 9 10-7 mbar of O2 at 673 K for
30 min, followed by annealing in UHV at 1073 K for
20 min), and after deposition of 3 A˚ Ni at 300 K onto the
ZrO2 film (right). On the reduced catalyst a small amount of bridged
bidentate carbonates (at 1630, 1430 and 1230 cm-1) as
well as bridged OH-groups (below 3700 cm-1) were
observed by FTIR at room temperature (all typical of
‘‘residual’’ adsorbates on the ZrO2 support). In the reaction
mixture at 373 K adsorbed water (broad peak around
1630 cm-1) but also adsorbed CH3 and CH2 groups were
detected (i.e. methane decomposition starts already at low
temperature),
in
addition
to
OH-groups. By
further The Pd3Zr alloy (left) is characterized by a peak at a BE
of 179.8 eV, after oxidation there are additional compo-
nents at 181.2 eV and 182.5/183.1 eV, due to the trilayer
ZrO2 film and thicker ZrO2 clusters, respectively. 3.1.4 Operando Spectroscopy During Methane Steam
Reforming (MSR) 3.1.4 Operando Spectroscopy During Methane Steam
Reforming (MSR) 3.2 UHV-Grown Model Catalysts of Ni–ZrO2/
Pd3Zr(0001) The high reaction temperature of MDR (873 K) prevented
to acquire operando FTIR spectra. In the following oper-
ando results are presented for methane steam reforming
(MSR) on Ni–ZrO2, 3.1.3 Methane Dry Reforming (MDR) For MDR over Pt/ZrO2/Al2O3 catalysts, a kinetic
model based on this dual mechanism was successfully
correlated with experimental data [7]. For CO2 activation, support materials with basic OH
species increase the interaction of CO2 with the support
and, therefore, increase the CO2 concentration on the sur-
face, e.g. in form of carbonates, and also increase CO2
affinity for surface carbon which minimizes carbon accu-
mulation
(CO2 þ C 2CO
DH
298K ¼ þ171kJ=mol). [80]. In summary, FTIR detected activated CH4 (CH3 and
CH2), activated water (OH), as well as different bidentate
(bi)carbonate species. The latter certainly originate from
the water gas shift side reaction. Indeed, we have previ-
ously examined the reaction of the product CO with surface
OH groups on an oxide surface, forming surface (bi)car-
bonates [79]. However, it remains unclear whether the
bidentate (bi)carbonates are intermediates in the formation
of CO [36, 37] or rather spectator species originating from
readsorption of CO2. Additional concentration modulation
experiments [81] would be required for a conclusive
answer. Altogether, the reaction network is quite complex, and
reforming, RWGS and Boudouard may occur simultane-
ously. Their relative contribution will strongly depend on
the exact reaction conditions which also affect the state of
the metal (CHx coverage), of the support oxide (adsorbed
carbonates, formates and OH), and of the metal-oxide
interface. 3.2.1 Preparation and Characterization The major difference of the two films is the higher surface
defect density of ZrO2/Pd3Zr and, while for Pd3Zr the
interlayer between the alloy and oxide is roughly stoi-
chiometric, for Pt3Zr there is an interlayer of pure Pt. The fitted Zr 3d region of the Ni-covered ZrO2/Pd3Zr is
shown in Fig. 7c. It is evident that the (relative) intensity of
the ZrO2 trilayer film (and that of the alloy substrate)
strongly decreased while the signal of the ZrO2 clusters
was much less affected (note that the exact geometry
between sample, X-ray source and analyzer may vary
between measurements, so that relative rather than absolute
intensities should be compared). 3
y
p
Figure 8a,b shows the LEED pattern (corresponding to
Fig. 7a,b) measured with an electron energy of 79 eV,
revealing the well-ordered (epitaxial) structure of the
ultrathin oxide on the (0001) Pd3Zr surface. It displays the
hexagonal reciprocal lattice of the oxide film (red hexagon)
and of the alloy substrate (blue hexagon), with a ratio of the
reciprocal lattices of oxide to substrate aox/amet of 1.556. According to scanning tunneling microscopy (STM) this
corresponds to real space lattice parameters of 0.56 and
0.351 nm for oxide and alloy, respectively [66]. Further-
more, there are two oxide hexagonal lattices, rotated by an
angle of 6.7 which correlates perfectly with the Fourier
transform of STM images of the oxide film, indicating two
rotational domains [65]. A structure model of the ZrO2 film
on Pd3Zr, derived from combined STM and by density
functional theory (DFT) studies [66], is shown in Fig. 8c. Based on the structural information, the specific XPS
binding energies of (alloy supported) ZrO2 trilayers and of
thicker ZrO2 layers could be explained by density func-
tional calculations [65]. These XPS binding energies pro-
vide a useful reference for alloy (metal) supported ultrathin
ZrO2 as well as for more bulk-like ZrO2 clusters. The pronounced differences in the oxide trilayer and
oxide cluster signals after Ni deposition can be explained
by the different interplay between Ni and the underlying
ZrO2. When Ni grows on the ultrathin oxide it attenuates
the photoelectrons escaping from the underlying trilayer
of ZrO2 (and from the Pd3Zr alloy beneath), as well as
modifying the electronic and geometric structure of the
trilayer (presumably because of its strong binding both to
Zr and O [83]). 3.2.1 Preparation and Characterization Although
oxide clusters are the minority species (about 10 % surface
coverage), due to their thickness of several nanometers 12 3 Fig. 6 a Operando FTIR during
methane steam reforming
(MSR) reaction on Ni–ZrO2 at
different temperatures. b Reactants/products followed
by MS during temperature
programmed MSR
Fig. 7 Preparation of the Ni–ZrO2/Pd3Zr(0001) model catalyst followed by XPS. Top: schematic illustration. Bottom: (a) XPS Zr3d signal of the
clean alloy, and of the oxide before (b) and after Ni deposition (c). All spectra were fitted to distinguish between the respective components
1620
Top Catal (2016) 59:1614–1627
123 Top Catal (2016) 59:1614–1627 1620 Fig. 6 a Operando FTIR during
methane steam reforming
(MSR) reaction on Ni–ZrO2 at
different temperatures. b Reactants/products followed
by MS during temperature
programmed MSR Fig. 7 Preparation of the Ni–ZrO2/Pd3Zr(0001) model catalyst followed by XPS. Top: schematic illustration. Bottom: (a) XPS Zr3d signal of the
clean alloy, and of the oxide before (b) and after Ni deposition (c). All spectra were fitted to distinguish between the respective components Fig. 7 Preparation of the Ni–ZrO2/Pd3Zr(0001) model catalyst followed by XPS. Top: schematic illustration. Bottom: (a) XPS Zr3d signal of the
clean alloy, and of the oxide before (b) and after Ni deposition (c). All spectra were fitted to distinguish between the respective components 3 1621 Top Catal (2016) 59:1614–1627 ZrO2/Pt3Zr we expect that most ([80 %) of the ZrO2
support is covered by Ni, either in the form of closely
spaced nanoparticles or, in case of coalescence, by large
(*10 nm) connected Ni islands (with a height of about
0.2 nm). Locally, the oxide support is still accessible by the
gas phase, however. On ZrO2/Pd3Zr, the nucleation density
(*4 9 1013 cm-2) is about twice as high, so that a
(nearly) closed and thin (*0.1 nm) Ni film is obtained. their contribution to the spectra is comparably larger
(photoelectrons originate from multiple ZrO2 layers instead
of a single ZrO2 layer). Using a laboratory X-ray source the
inelastic mean free path (IMFP) of the photoelectrons is
about 2 nm (sampling depth *6 nm), thus the spectrum is
still dominated by the alloy substrate. For more surface
sensitive (synchrotron-based) XP spectra we refer to our
study of the related ultrathin zirconia film on Pt3Zr [65]. 3.2.1 Preparation and Characterization 9a) show a gradual increase of the absorption
signal (or coverage of) CO at 2075 cm-1, which can be
attributed to CO chemisorbed on top of individual nickel
atoms. Signals of multiply-bonded CO (*1920 cm-1)
were very broad and weak. CO adsorption on the ZrO2
trilayer (around 2190 cm-1 on powder ZrO2; cf. Fig. 2)
was not observed, due to its desorption temperature of
155 K [65]. In order to examine the exact reason of the disappear-
ance of the PM-IRAS absorption signal upon annealing/
redosing, a fresh Ni/ZrO2/Pd3Zr model catalyst was pre-
pared and the surface was saturated with CO at 90 K
already in the preparation chamber (50 L CO exposure). Afterwards XP spectra of the C1 s and Ni2p regions were
measured which revealed a peak at 284.9 eV characteristic
of molecular CO (Fig. 9c), as well as a peak at 852.7 eV
characteristic of metallic Ni (not shown). Afterwards, the
model catalyst was successively heated in UHV up to
550 K. As shown in Fig. 9c a shift in C1 s binding energy
towards lower binding energies could be observed, from
284.9 eV at 90 K (molecular CO) to 283.9 eV at 550 K
(characteristic of sp2-bound carbon). This clearly shows
that CO dissociates on nickel upon annealing, a well-
known property, thereby forming a carbon over-layer on
the nickel surface, which prevents further CO from
adsorbing. During
this
experiment
the
Ni2p
signal
remained unaffected, i.e. there was no Ni sintering (which
would reduce the intensity) and no formation of Ni carbide
(which would induce a shift to 283.5 eV in C1 s [85]). After evacuation of the reaction chamber and measur-
ing another PM-IRAS spectrum at 200 K, on-top CO was
still detected [Fig. 9b; which is expected since CO des-
orbs from Ni(111) and Ni(100) at *425 K [64, 84]]. However, when the sample was gradually heated to
higher temperatures in UHV, the signal of on-top CO
vanished after heating to 300 K. Clearly, this cannot be
due to CO desorption. Upon recooling and redosing 10-4
mbar (or higher) CO at 200 K the signal at 2075 cm-1
did not reappear, as shown in Fig. 9b. 3.2.1 Preparation and Characterization The ZrO2 clusters exhibit a more bulk-
like electronic structure, and seem less influenced by
deposited Ni. This may be due, apart from their size of
*10 nm, to a different growth mode of Ni on ZrO2
clusters (e.g. with a lower nucleation density leaving more
open space between Ni particles). A clear answer cannot
be given at this point. Photoemission spectra of the Ni 2p region (cf. Fig. 10)
show that nickel is deposited in its metallic form, i.e. with
the 2p region showing the satellite structure at 858.2 eV
characteristic of metallic nickel. Note that on the techno-
logical catalyst, even after reduction at 673 K, part of the
Ni surface remained oxidic. Deposition of 3 A˚ Ni at room temperature induces a
significant change in the Zr 3d region (Fig. 7c, top). Based
on corresponding STM studies [83] of Ni cluster growth on Fig. 8 (a, b) LEED pattern and (c) schematics of the ultrathin ZrO2 (111) film on Pd3Zr (0001) annealed at 1073 K [LEED: oxide lattice (red);
alloy substrate (blue)] and (c) schematics of the ultrathin ZrO2 (111) film on Pd3Zr (0001) annealed at 1073 K [LEED: oxide lattice (red) Fig. 8 (a, b) LEED pattern and (c) schematics of the ultrathin ZrO2 (111) film on Pd3Zr (0001) annealed at 1073 K [LEED: oxide lattice (red);
alloy substrate (blue)] Fig. 8 (a, b) LEED pattern and (c) schematics of the ultrathin ZrO2 (111) film on Pd3Zr (0001) annealed at 1073 K [LEED: oxide lattice (red);
alloy substrate (blue)] 12 3 1622 Top Catal (2016) 59:1614–1627 nickel and that the formed coke covered the metal surface
and hindered CO (re-)adsorption. 3.2.2 CO Adsorption and Dissociation CO is, of course, a product of reforming and it is also
involved in the (reverse) water gas shift reaction. In order
to examine the interaction of the model reforming catalyst
with gaseous CO, the freshly prepared 3 A˚ Ni/ZrO2/Pd3Zr
sample was transferred to the high pressure cell [49] and
cooled with liquid nitrogen to 200 K. Then, CO PM-IRAS
spectra were acquired under isothermal conditions from
10-6 mbar to 100 mbar. As described in more detail in Ref. [50], PM-IRAS only displays the surface adsorbed species,
whereas the gas phase signal is removed. The measured
spectra (Fig. 4 Discussion We have examined Ni–ZrO2 reforming catalysts using both
technological (powder) catalysts as well as model (single
crystal based) catalysts [50, 57]. Both approaches have
their specific benefits, the first providing results that may be
technologically relevant and the latter providing access to
examining specific processes on an atomic/molecular level. In both cases, similar characterization methods have been
employed, e.g. microscopy/diffraction for structure analy-
sis (HRTEM, XRD, LEED), X-ray photoelectron spec-
troscopy or fluorescence for composition analysis (XPS,
EDX), and infrared spectroscopy and temperature-pro-
grammed methods for adsorbate characterization (FTIR,
PM-IRAS, TPO). Indeed, corresponding STM studies of Ni clusters on
ZrO2/Pt3Zr indicated that above 500 K metal atoms
migrated through the oxide support into the alloy substrate
[83]. We have thus modeled the XPS intensities of a 3 A˚ Ni
layer above and below the ZrO2 support oxide. The cal-
culations indicate that Ni migration to below the trilayer
would indeed result in a *40 % intensity decrease. The surface science approach to heterogeneous catalysis
often provides very detailed insight into elementary pro-
cesses. Typically, the catalytic systems are somewhat
simplified, such as well-ordered (ultra)thin planar oxide
films
with
deposited
clean
and
well-shaped
metal
nanoparticles [even though these are complex systems
when compared to (noble) metal single crystals]. Such
model systems are not only accessible by many surface
sensitive methods but can also be modelled straightforward
by theoretical methods, which are typically indispensable
to interpret and thoroughly explain the experimental data
[60, 65, 91, 92]. Whether this approach is a realistic sce-
nario for technological catalysis remains a matter of debate
because often rather the disordered structures exhibit the
highest activity. Once more, to confirm the hypothesis of ‘‘sub-oxide
migration’’, corresponding XPS experiments were per-
formed for ZrO2 supported Pd nanoparticles (not shown). Upon heating the Pd nanoparticles to 500 K, the Pd 3d
signal remained unaffected whereas annealing to 800 K
again led to a *32 % decrease of the XPS Pd 3d signal. Consequently, at high temperature the Pd atoms also
migrated through the ultrathin oxide into the underlying
alloy. Further experiments using low energy ion scattering
(LEIS) or synchrotron XPS (which are both more surface Apart from the materials or complexity gap, in many
cases model studies are performed in UHV and at low
temperature, leading to the ‘‘pressure gap’’. 3.2.3 Ni Nanoparticle Stability sensitive than our laboratory XPS) are required to examine
the ‘‘sub-oxide migration’’ in more detail. The thermal stability of Ni particles on ZrO2 was investi-
gated using XPS (in this case for the Pt3Zr substrate,
however). Figure 10 shows the corresponding Ni2p spec-
tra, acquired after Ni deposition and after annealing in
UHV to 550 K. Apparently, the Ni nanoparticles were
stable up to 550 K, as indicated by the nearly identical
spectra. However, annealing to 800 K induced a *40 %
decrease of the Ni 2p intensity, i.e. there was a strong
‘‘loss’’ of Ni. An apparent explanation would be a strong
sintering of Ni nanoparticles. When the dispersion is
reduced, the Ni2p signal decreases. However, it has been
previously reported for ultrathin oxides that metal atoms
may also diffuse through the ultrathin oxide and merge
with the substrate, for example for Pd on alumina/NiAl
[88], for Pd atoms on SiOx [89] and for Pd on thin
FeO(111) film [90]. 3.2.1 Preparation and Characterization Possible explana-
tions are either (1) that the Ni particles/islands had
strongly sintered (less likely in view of the moderate
temperatures) or (2) (more likely) that CO dissociated on Previous studies of mbar pressure CO adsorption on
supported Pd nanoparticles [52] and smooth and defect-
rich Pd(111) single crystals [86, 87] have indicated the
absence of CO dissociation on Pd. To confirm the
hypothesis of coking on Ni, corresponding XPS experi-
ments were thus performed for ZrO2 supported Pd
nanoparticles. As
expected,
upon
heating
the
Pd
nanoparticles up to 550 K, CO desorption (*420 K) but
no CO dissociation (carbon deposits) were detected by
IRAS and XPS. Fig. 9 CO adsorption on the Ni–ZrO2/Pd3Zr(0001) model catalyst
monitored by PM-IRAS and XPS. a CO adsorption at 200 K at
increasing
pressure;
b
PM-IRAS
signal
after
adsorption
and
evacuation, followed by heating to 300 K. Upon readsorption at
200 K the CO band at 2075 cm-1 did not reappear; c C1 s spectra of
stepwise annealing of the CO saturated surface followed by XPS
1 3 Fig. 9 CO adsorption on the Ni–ZrO2/Pd3Zr(0001) model catalyst
monitored by PM-IRAS and XPS. a CO adsorption at 200 K at
increasing
pressure;
b
PM-IRAS
signal
after
adsorption
and evacuation, followed by heating to 300 K. Upon readsorption at
200 K the CO band at 2075 cm-1 did not reappear; c C1 s spectra of
stepwise annealing of the CO saturated surface followed by XPS 123 123 Top Catal (2016) 59:1614–1627 1623 4 Discussion However, there
are a number of ‘‘(near) atmospheric pressure surface sci-
ence methods’’ available that enable to study functioning
catalysts or, at least, model catalysts at elevated gas pres-
sure and elevated temperature. Among the most frequently
applied are PM-IRAS and SFG (sum frequency genera-
tion), both working up to 1 bar, and NAP-XPS (up to a few
mbar)
[50]. Nevertheless,
operando
spectroscopy
is
demanding in general, even for technological catalysts, and
it is even more so for the low surface area model systems. In the current case of Ni–ZrO2 reforming catalysts, the
Ni particles in the powder catalyst are rather large
(*20 nm) and also the Ni islands on the model ZrO2 cover
most of the surface. Clearly, the ‘‘real’’ ZrO2 is hydroxy-
lated whereas the model oxide is not. Nevertheless, routi-
nes have been developed to create OH groups on UHV
grown (ultra)thin oxides as well. Apart from the materials or complexity gap, in many
cases model studies are performed in UHV and at low
temperature, leading to the ‘‘pressure gap’’. However, there
are a number of ‘‘(near) atmospheric pressure surface sci-
ence methods’’ available that enable to study functioning
catalysts or, at least, model catalysts at elevated gas pres-
sure and elevated temperature. Among the most frequently
applied are PM-IRAS and SFG (sum frequency genera-
tion), both working up to 1 bar, and NAP-XPS (up to a few
mbar)
[50]. Nevertheless,
operando
spectroscopy
is
demanding in general, even for technological catalysts, and
it is even more so for the low surface area model systems. Fig. 10 Ni 2p XP spectra of the pristine Ni–ZrO2/Pt3Zr(0001) model
surface, and after annealing to 550 and 800 K in UHV In the current case of Ni–ZrO2 reforming catalysts, the
Ni particles in the powder catalyst are rather large
(*20 nm) and also the Ni islands on the model ZrO2 cover
most of the surface. Clearly, the ‘‘real’’ ZrO2 is hydroxy-
lated whereas the model oxide is not. Nevertheless, routi-
nes have been developed to create OH groups on UHV
grown (ultra)thin oxides as well. Fig. 10 Ni 2p XP spectra of the pristine Ni–ZrO2/Pt3Zr(0001) model
surface, and after annealing to 550 and 800 K in UHV 12 3 1624 Top Catal (2016) 59:1614–1627 With respect to CO adsorption at mbar pressure, dif-
ferences were observed. CO adsorption on the technolog-
ical Ni–ZrO2 was reversible, i.e. 5 Conclusions We have examined Ni–ZrO2 reforming catalysts, employ-
ing both technological materials (powders) as well as sin-
gle crystal based surface science model catalysts. In both
cases the Ni nanoparticles were rather large (20 nm or
larger) but exhibited different properties with respect to CO
dissociation. Whereas the partially oxidized ‘‘technologi-
cal’’ Ni nanoparticles did not dissociate CO around room
temperature, the fully metallic ‘‘surface science’’ Ni par-
ticles did, with the carbon deposits blocking further CO
adsorption. For the technological samples methane dry and
steam reforming were performed, with both reactions
affected by (reverse) water gas shift, modifying the H2/CO
ratio and producing CO2, respectively. For dry reforming
whisker-type carbon was produced, operando studies of
steam reforming revealed CHx, OH, and different bidentate
(bi)carbonate species. The limited thermal stability of
model Ni–ZrO2 prevented in situ reforming studies. Nev-
ertheless, the XPS binding energies of alloy supported
ultrathin trilayer (O–Zr–O) ZrO2 as well as those of more
bulk-like ZrO2 clusters have been determined which pro-
vide useful reference data. Under methane dry reforming conditions, when hydro-
gen is present at high temperature, the Ni particles (and
their edges) are fully reduced and may also partly disso-
ciate the reforming product CO. Clearly, CH4 is the major
carbon source and graphitic and whisker-type carbon were
observed. Reverse water gas shift occurred in parallel and
decreased the H2/CO ratio. For methane steam reforming
operando (FTIR and MS) studies were performed, indi-
cating activated CH4 (CH3 and CH2), activated water (OH),
as well as different bidentate (bi)carbonate species. The
latter originate from CO2 resulting from the water gas shift
side reaction but it is unclear whether these are interme-
diates or spectator species. Altogether the combined technological and surface sci-
ence approach is rewarding and it is fascinating to observe
marked differences despite nominally the same Ni–ZrO2
composition. Of course, ‘‘Ni–ZrO2’’ is a very crude
description
missing
important
features
like
metal
nanoparticle size and morphology, type of surface facets/
steps, oxidation state, supporting oxide (surface) structure,
hydroxylation, defect density, nature of metal-oxide inter-
face sites, etc. Answering all these questions would provide
the molecular details of how catalytic processes work and
is thus a continuing mission. Ni–ZrO2 model catalysts, prepared by surface science
routes, turned out to be significantly more active for CO
dissociation than their technological counterparts. 4 Discussion CO could be adsorbed,
desorbed at higher temperature, and readsorbed without
significant loss of adsorption capacity. There were also no
indications
of
CO
dissociation. In
contrast,
the
Ni
nanoparticles grown in UHV dissociated adsorbed CO
already around room temperature, which led to coking and
irreversible CO adsorption, thus readsorbing CO on the
poisoned surface was not possible. Taking into account the
well-known fact that CO dissociation preferentially occurs
on Ni edges/steps this difference may be explained by the
partial oxidation of the technological Ni nanoparticles
(even after reduction Ni-oxide species were detected in
addition to metallic Ni). Thus, it is likely that the edges of
technological Ni particles are oxidized which prevents CO
dissociation. Unfortunately, CO-FTIR was not able to
differentiate step and terrace sites and also HRTEM was
unable to demonstrate this (due to sample air exposure
prior to microscopy). technological catalyst and care must therefore be taken for
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CO was adsorbed at pressures up to 100 mbar, both PM-
IRAS and XPS revealed CO dissociation around room
temperature and blocking of the Ni surface by carbon. This
is most likely due to the fully metallic Ni particles whereas
the technological particles were partially oxidized even
after reduction at 673 K. CO adsorbed on the powder ZrO2
at room temperature, but it did not on the trilayer ZrO2 (CO
desorption temperature of 155 K [65]). Acknowledgments This work was supported by the Austrian Science
Fund (FWF) under projects F4502-N16 (SFB FOXSI) and I942-N17
(DryRef). We thank Andreas Jentys for helpful discussions. Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
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Screening of the college students at clinical high risk for psychosis in China: a multicenter epidemiological study
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BMC psychiatry
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cc-by
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Screening of the College S
Risk for Psychosis in China
Epidemiological Study
JiaXin Wu
Tongji Hospital of Tongji University, Tongji University
XiangYun Long
Tongji Hospital of Tongji University, Tongji University
Fei Liu
Tongji Hospital of Tongji University
AnSi Qi
Tongji Hospital of Tongji University
Qi Chen
Tongji Hospital of Tongji University
XiaoFeng Guan
Tongji Hospital of Tongji University
Qiong Zhang
Tongji Hospital of Tongji University
YuHong Yao
Tongji University School of Medicine
JingYu Shi
Tongji University School of Medicine
ShiPing Xie
Nanjing Brain Hospital
Wei Yan
Nanjing Brain Hospital
MaoRong Hu
First Affiliated Hospital of Nanchang University
Xin Yuan
First Affiliated Hospital of Nanchang University
Jun Tang
First Affiliated Hospital of Nanchang University
SiLiang Wu
First Affiliated Hospital of Nanchang University
TianHong Zhang Screening of the College St
Risk for Psychosis in China
Epidemiological Study
JiaXin Wu
Tongji Hospital of Tongji University, Tongji University
XiangYun Long
Tongji Hospital of Tongji University, Tongji University
Fei Liu
Tongji Hospital of Tongji University
AnSi Qi
Tongji Hospital of Tongji University
Qi Chen
Tongji Hospital of Tongji University
XiaoFeng Guan
Tongji Hospital of Tongji University
Qiong Zhang
Tongji Hospital of Tongji University
YuHong Yao
Tongji University School of Medicine
JingYu Shi
Tongji University School of Medicine
ShiPing Xie
Nanjing Brain Hospital
Wei Yan
Nanjing Brain Hospital
MaoRong Hu
First Affiliated Hospital of Nanchang University
Xin Yuan
First Affiliated Hospital of Nanchang University
Jun Tang
First Affiliated Hospital of Nanchang University
SiLiang Wu
First Affiliated Hospital of Nanchang University
TianHong Zhang Screening of the College Students at Clinical High
Risk for Psychosis in China: A Multicenter
Epidemiological Study AnSi Qi
Tongji Hospital of Tongji University Qi Chen
Tongji Hospital of Tongji University XiaoFeng Guan
Tongji Hospital of Tongji University Qiong Zhang
Tongji Hospital of Tongji University YuHong Yao
Tongji University School of Medicine JingYu Shi
Tongji University School of Medicine ShiPing Xie
Nanjing Brain Hospital ShiPing Xie
Nanjing Brain Hospital Wei Yan
Nanjing Brain Hospital Page 1/20 Shanghai Mental Health Center
JiJun Wang
Shanghai Mental Health Center
Zheng Lu
(
luzheng@tongji.edu.cn
) Abstract Background: To investigate a 3-stage screening procedure and explore the clinical features of subjects at
Clinical High Risk (CHR) for psychosis in a representative sample of Chinese college students. Methods: An epidemiological survey of the prevalence of CHR syndrome in Chinese college students that
was selected by stratified random sampling from Shanghai, Nanjing and Nanchang cities was done
followed a 3-stage procedure. Participants was initially screened with the Prodromal Questionnaire-brief
version (PQ-B), and whose distress score of PQ-B exceeded 24 would be invited to a telephone
assessment using the subscale for positive symptoms of the Scale of Prodromal Symptoms
(SOPS)/Structured Interview for Prodromal Syndromes (SIPS). Lastly, participants who scored 3 or higher
in any item of the subscale would be administered with the SIPS interview conducted by trained
researchers to confirm the diagnosis of CHR syndrome. Results: 23 063 college students completed the survey during September 2017 to October 2018. 72
students were diagnosed as CHR subjects, and the detection rate in the total sample was 0.3%. The peak
age range for the first diagnosis of CHR was 17 to 20 years. 13 and 46 were set as the cut-off points of
PQ-B total score and distress score to balance the greatest sensitivity and specificity. Binary logistic
regression revealed that 8 items in PQ-B showed significant distinction for detecting CHR subjects. Conclusions: The 3-stage screening method can be utilized in the detection of CHR subjects for psychosis
in general population, during which delusional ideas, perceptual abnormalities and suspiciousness
deserve great attention. Research article Keywords: Clinical high risk, Ultra-high risk, Early detection, College students, Epidemiology
Posted Date: December 3rd, 2020
DOI: https://doi.org/10.21203/rs.3.rs-62868/v2
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 BMC Psychiatry on May 17th, 2021. See the
published version at https://doi.org/10.1186/s12888-021-03229-8. Page 2/20 1. Background Since being proposed by Sullivan in 1927, the notion of early detection and preventive intervention of
psychosis syndromes has received increased attention in the recent two decades [1-4]. As a series of
studies of first-episode psychosis suggests that longer duration of untreated psychosis is significantly
correlated with worse treatment response and prognosis[5], global researchers and clinicians have
reached a consensus that early detection and prevention is central to contain the development and
burden of psychosis. They started to investigate whether there is an earlier period in the development of
psychosis that can be detected before the break, and then gave birth to the concept of ultra-high risk
(UHR) or clinical high risk (CHR) for psychosis[6, 7]. CHR subjects refer to individuals who are identified
as having nonspecific symptoms (e.g. cognitive symptoms) and attenuated or intermittent psychotic
symptoms, or having asymptomatic genetic risk, but do not meet criteria for a syndromal psychosis, and
thereby they are considered to be at increased risk for developing psychotic disorders[4, 8]. Fusar-Poli et
al.[9] reported that 20-35% CHR subjects developed syndromal psychotic disorder in 2 years, and Zhang
et al.[10] discovered that the transition rate at the first 2 years was 26.4% in a Chinese help-seeking
sample, both of which indicated that the risk of developing psychosis in CHR subjects deserved serious
attention. Page 3/20 To diagnose and assess symptoms of CHR, two categories of instruments based on two complementary
criteria have been globally recognized: the UHR criteria that focus on attenuated or brief limited
intermittent psychotic symptoms and genetic risk, and the basic symptoms criteria that focus on
cognitive and perceptive symptoms. The UHR criteria were designed to detect the risk for developing a
first-episode psychosis in 12 months[11]. The most recognized instruments developed from the UHR
criteria include the Structured Interview for Psychosis-risk Syndromes (SIPS)[12] and the Comprehensive
Assessment of At-Risk Mental States (CAARMS)[13]. While the basic symptoms criteria aimed at detect
the risk for psychosis ideally before the appearance of functional impairments[11]. The most
representative instruments are the Schizophrenia Proneness Instrument, Adult (SPI-A)[14] and Child &
Youth version (SPI-CY)[15]. Since the instruments listed above are based on the clinician’s assessments
through clinical interviews, several self-report scales have been established to simplify the screening of
CHR individuals, such as Prodromal Questionnaire-Brief version (PQ-B)[16], Prodromal Questionnaire-16
items (PQ-16)[17], Prime Screen[18] and Early Psychosis Screener (EPS)[19, 20]. 1. Background Among the instruments listed above, SIPS[21], PQ-B[22], and PQ-16[23] have been translated into Chinese
version, which showed good reliability and validity in screen CHR individuals at clinical settings. Notwithstanding, detection of CHR in China is still limited by its passivity that the clinical interviews and
rating scales can almost only be approached in psychiatric clinics or wards, which indicates that only
individuals with help-seeking behaviors can be tested, and doctors should always wait passively for the
suspected cases. While some of them won’t see a doctor (e.g. because of social discrimination, stigma or
poor knowledge of psychosis) before the onset of psychosis or significant functional impairments[24]. In
fact, most researches concerning CHR detection in China were conducted in help-seeking individuals[10,
25, 26]. Besides, as the risk age range of CHR is 15 to 22 years[27], studies carried out in non-clinical
settings should pay more attention to students in university and high schools. Chen et al. established a
two-stage screening method (PQ-16 followed by SIPS) in 579 Chinese university students who were
enrolled in a mental health education course[23]. There were also two researches using self-report scales
to explore the psychotic-like experiences (PLEs) in high-school students and university students in
China[28, 29]. However, an epidemiological study of CHR detection conducted in a larger sample of
college students with established psychosis-risk instruments still lacks in China. Therefore, we
generalized a 3-stage method (consists of PQ-B, telephone interview and SIPS) for the detection of CHR
individuals in college students from Shanghai, Nanjing and Nanchang in China. The present study aimed
to investigate the screening procedure, and explore the characteristics of prodromal symptoms for
psychosis in Chinese college students. Among the instruments listed above, SIPS[21], PQ-B[22], and PQ-16[23] have been translated into Chinese
version, which showed good reliability and validity in screen CHR individuals at clinical settings. 2.1 Participants The Research Ethics Committee at the Shanghai Mental Health Centre (SMHC), Shanghai, China
approved this study in 2016. The participants were college students recruited by three research teams at
three different cities: Tongji Hospital of Tongji University (TJH) at Shanghai, Nanjing Brain Hospital (NBH) Page 4/20 at Nanjing, and The First Affiliated Hospital of Nanchang University (FAHNU) at Nanchang, from
September 2017 to October 2018. Stratified random sampling was conducted at the level of school’s
level, including the first-tier universities, second-tier universities, third-tier universities and the higher
vocational and technical colleges. Print version and online version of questionnaires were distributed to
the selected sample of 27 145 students. Since the written informed consents were not applicable in the
online survey and telephone assessment, they were only obtained from the participants who received a
face-to-face interview. Nevertheless, a detailed explanation of the study and informed consent were
written in their native language in the beginning of the questionnaire, and participants were asked to
complete the questionnaire only if they were voluntary. Similarly, the informed consents were orally made
in the telephone assessment to protect the basic rights of the participants. Every participant receiving the
phone call was informed of the objective and procedure of the telephone assessment, in the beginning of
dialogue. The telephone assessment was conducted after obtaining the permission of every participant. Moreover, for children and adolescents who participated in the study, since most of them were non-native
residents and lived far away from home, the informed consents from their parents were made through
phone-calls before they signed the informed consents. Nevertheless, their parents’ informed consents
were not obtained in the process of completing questionnaires and telephone assessments. Eventually,
26 005 questionnaires were received, and 2942 questionnaires were removed because of data missing
including demographic information and blank in items of the PQ-B. Thus, a sample of 23 063 college
students were investigated in this study. The age range of the investigated sample was 14 to 31
(mean=18.4 years, S.D.=1.2 years), and 11 028 (47.8%) subjects were female. 2.2.1. The Prodromal Questionnaire-Brief version (PQ-B) The PQ-B[16] is a 21-item self-report scale designed to detect individuals at risk for psychosis. It was
modified from the 92-item Prodromal Questionnaire[30] and focus on positive symptoms. Each item was
marked with “Yes/No” by the participants according to the presence or absence of the corresponding
experience in the past month. If an item was marked as “No”, the item was rated as 0; if an item was
marked as “Yes”, a 5-point Likert scale (strongly disagree, disagree, neutral, agree, strongly agree) was
used to rate the distress level of the symptom. The total score was defined as the number of the items
endorsed, and the distress score was the sum of the distress level of the items endorsed. Zhang et al[10] and Xu et al.[22] translated the original version of PQ-B into Simplified Mandarin Chinese,
and reported good reliability and validity in a sample of help-seeking outpatients in China (Cronbach’s
alpha=0.897). They set the cutoff point for the total score and distress score of the Chinese-version PQ-B
as 7 and 24 respectively. The sensitivity and specificity were reported to be 82.0% and 46.8% respectively
when using the cutoff point for the distress score to screen CHR subjects. In the present study, the internal
consistency of the Chinese-version PQ-B was excellent (Cronbach’s alpha=0.928). 2.2.2. The Structured Interview for Psychosis-risk Syndromes/Scale of Prodromal Syndromes Page 5/20 Page 5/20 The SIPS/SOPS[12] were intended to define whether an individual met the CHR criteria through a
clinician-administered, semi-structured clinical interview. The criteria outlined three types of Psychosis-
Risk Syndromes: Attenuated Positive Symptom Syndrome (APSS), Brief Intermittent Psychotic Syndrome
(BIPS), and Genetic Risk and Deterioration Syndrome (GRDS). There are 19 items assessing 4 major
symptom domains on the SIPS/SOPS, including Positive symptoms, Negative symptoms, Disorganized
symptoms, and General Symptoms. Each item was rated from 1 to 6, according to the severity of the
symptom. Rating of 3 to 5 indicates a prodromal range symptom, and 6 indicates a “severe and
psychotic” symptom. Li et al. established the Chinese version of SIPS/SOPS, and it was demonstrated
with good reliability and validity in assessing subjects with Psychosis-Risk Syndromes[21]. The internal
consistency reliability of SIPS/SOPS in this study was good (Cronbach’s alpha=0.848). 2.2.3. Demographic characteristics Demographic characteristics that include name, sex, age, discipline, family history and contact
information were measured with a questionnaire designed by the authors. 2.3 Procedure A 3-stage screening procedure (see Figure 1) was applied in this study. The screening results of the
Chinese-version PQ-B were obtained from 23 063 participants (the investigated sample). According to Xu
et al.[22], the cutoff point of the distress score of PQ-B was set as 24 in this study. 10 330 (44.8%)
participants reached this cutoff point, and were invited to a telephone assessment. Since 666 participants
were failed to be contacted (e.g. dead number, suspended service or didn’t answer for three times) or
declined to receive the telephone assessment, 9664 (41.9%) participants were assessed by trained
researchers, with the items listed in the subscale for positive symptoms of the SIPS/SOPS (P1-P5,
Unusual Thought Content, Suspiciousness/Persecutory Ideas, Grandiosity, Perceptual
Abnormalities/Hallucinations, and Disorganized Communication) to identify the possible presence of the
prodromal or psychotic symptoms. If any item was rated as 3 or higher, the participant would be
considered as positive in the telephone assessment. Thus, 1176 participants were invited to the face-to-
face interviews, and 991 (4.3%) participants eventually consented to receive the SIPS interviews
administered by trained researchers. The participants that met any criterion of the Psychosis-Risk
Syndromes (APSS, BIPS, GRDS) defined by SIPS were diagnosed as CHR individuals, while everyone else
in the investigated sample were classified into non-CHR individuals. All interviewers had received a
professional training for SIPS prior to the study. The inter-rater reliability was good among the
interviewers (n=20, ICC=0.75). A 3-stage screening procedure (see Figure 1) was applied in this study. The screening results of the
Chinese-version PQ-B were obtained from 23 063 participants (the investigated sample). According to Xu
et al.[22], the cutoff point of the distress score of PQ-B was set as 24 in this study. 10 330 (44.8%)
participants reached this cutoff point, and were invited to a telephone assessment. Since 666 participants
were failed to be contacted (e.g. dead number, suspended service or didn’t answer for three times) or
declined to receive the telephone assessment, 9664 (41.9%) participants were assessed by trained
researchers, with the items listed in the subscale for positive symptoms of the SIPS/SOPS (P1-P5,
Unusual Thought Content, Suspiciousness/Persecutory Ideas, Grandiosity, Perceptual 2.4 Statistical analyses The sample size of this study was estimated by the prevalence (p) of CHR syndrome, which was reported
to be 0.3% in a young adults community[31]. Using the formula[32]: Page 6/20 with the relative error (ε) set as 25%, the sample size was calculated to be roughly 20 500. Considering a
response rate of 80%, we were required to survey 25 625 individuals. Using SPSS software (IBM Corp. Released 2017. IBM SPSS Statistics for Macintosh, Version 25.0. Armonk, NY: IBM Corp.), the group differences in age, the total score and distress score as well as each
item score of the PQ-B were compared using Independent-samples t-tests, and the significance level was
set at 0.05 (two-tailed). Likewise, Chi-square tests were used to compare sex between the two groups. Mann-Whitney U test was used to test whether there was a difference in disciplines between the two
groups. Since 60.8% of the data of the family history were missing, family history was not described in
the demographic characteristics. The internal consistency reliability of PQ-B and SOPS were described
using the Cronbach’s alpha. The receiver operating characteristic (ROC) analyses were performed to reset
the cutoff points and to examine the sensitivity and specificity of PQ-B in detecting CHR subjects. The
accuracy of the tests was described through the areas under the ROC curve (AUCs) with the 95%
confidential interval (CI). A Spearman correlation analysis was used to investigate the association
between each item score of PQ-B and the dichotomized SIPS diagnosis. A logistic regression analysis
was conducted to explore the significant PQ-B items in indicating the CHR states based on the SIPS. For
graphical presentation, bar diagrams were created to show the number of the CHR subjects by the age, as
well as its distribution in female and male groups, and a bar-chart (mean and standard error of the mean)
was created to show the differences in the PQ-B item scores between CHR and non-CHR subjects. 3.1 Demographic characteristics 72 college students met the criteria for CHR states according to SIPS. The detection rate in the total
sample was 0.3% (72/23063). Among them, 1 subject met the criteria of GRDS, the other 71 met the
criteria of APSS, and none of them met the criteria of BIPS. The demographic characteristics of CHR
individuals and non-CHR individuals were presented in Table 1. No significant difference in the mean age,
sex, and disciplines was found between the two groups. 1. Demographic characteristics of the CHR and non-CHR individuals Table 1. Demographic characteristics of the CHR and non-CHR individuals Page 7/20 Variables
CHR
(n=72)
Non-CHR
(n=22991)
t / χ2/ U
p
Age mean (S.D.)
18.3 (1.5)
18.4 (1.2)
0.38
0.707
Female (%)
38 (52.8)
10990 (47.8)
0.71
0.399
Disciplines
Arts (%)
19 (26.4)
2502 (10.9)
751769.50
0.243
Humanities (%)
4 (5.6)
1100 (4.8)
751769.50
0.243
Social sciences (%)
3 (4.2)
2482 (10.8)
751769.50
0.243
Natural sciences (%)
3 (4.2)
3355 (14.6)
751769.50
0.243
Applied sciences (%)
40 (55.6)
12769 (55.5)
751769.50
0.243
Unclassified (%)
2 (2.8)
623 (2.7)
751769.50
0.243
Null (%)
1 (1.4)
160 (0.7)
751769.50
0.243
PQ-B total score mean (S.D.)
18.0 (4.4)
8.5 (7.1)
18.22
<0.001
PQ-B distress score mean (S.D.)
66.0 (22.1)
24.6 (21.1)
16.64
<0.001 3.2 Age of CHR subjects at identification The age distribution of CHR subjects was shown in Fig 2. The frequency of CHR identified with SIPS in
this sample peaked at young adulthood (17 to 20 years) and decreased with subsequent age. The peak
age that met the CHR criteria in both males and females were 18 years old. While the age range of female
subjects (15 to 26 years old) was broader than that of male subjects (15 to 21 years old). Table 2. Accuracy of PQ-B total score and distress score in screening CHR 3.3.1 The total score and distress score of PQ-B in detecting CHR Accuracy of PQ-B total score and distress score in screening CHR Page 8/20 PQ-B cutoff
Sensitivity (%)
Specificity (%)
YIa
LR+a
AUCa
95% CI
p
Total score
≥ 10
94.4
61.9
0.56
2.48
0.842
0.806 - 0.877
<0.001
≥ 11
91.7
65.6
0.57
2.67
0.842
0.806 - 0.877
<0.001
≥ 12
91.7
68.9
0.61
2.95
0.842
0.806 - 0.877
<0.001
≥ 13b
91.7
71.5
0.63
3.22
0.842
0.806 - 0.877
<0.001
≥ 14
87.5
74.1
0.62
3.38
0.842
0.806 - 0.877
<0.001
≥ 15
86.1
76.3
0.62
3.63
0.842
0.806 - 0.877
<0.001
Distress score
≥ 43
90.3
78.2
0.69
4.14
0.900
0.863 - 0.937
<0.001
≥ 44
90.3
79.2
0.70
4.34
0.900
0.863 - 0.937
<0.001
≥ 45
90.3
80.3
0.71
4.58
0.900
0.863 - 0.937
<0.001
≥ 46b
90.3
81.2
0.72
4.80
0.900
0.863 - 0.937
<0.001
≥ 47
83.3
82.2
0.66
4.68
0.900
0.863 - 0.937
<0.001
≥ 48
79.2
83.0
0.62
4.66
0.900
0.863 - 0.937
<0.001
a YI: Youden’s index; LR+: positive likelihood ratio; AUC: area under the curve
b cutoff point with the highest YI a YI: Youden’s index; LR+: positive likelihood ratio; AUC: area under the curve
b cutoff point with the highest YI a YI: Youden’s index; LR+: positive likelihood ratio; AUC: area under the curve
b cutoff point with the highest YI 3.3.2. The significant items of PQ-B in detecting CHR To investigate which items were significant in detecting CHR in Chinese college students, independent-
samples t-tests was initially performed to compare the differences in the item scores of PQ-B between
CHR and non-CHR individuals. The results demonstrated that the score for each individual item in PQ-B
was significantly higher in the CHR group (all p<0.001), which was presented in a bar chart (see Figure 3). Furthermore, a Spearman correlation analysis showed that higher item scores of PQ-B were significantly
associated with the diagnosis of CHR (all p<0.001). Therefore, we did a conditional backward logistic
regression analysis to explore the key items involved in the detection of CHR subjects. All PQ-B item
scores were set as the independent variables and the diagnosis (CHR or non-CHR) was set as the
dependent variable. As presented in Table3, 8 items (PQ1, PQ3, PQ4, PQ12, PQ13, PQ15, PQ17 and PQ18)
were entered the regression model. Table 3. Logistic regression of the CHR/non-CHR classification by PQ-B items
Variables
B
S.E. Wald
Df
p
Exp(B)
PQ1
0.223
0.086
6.764
1
0.009
1.249
PQ4
0.225
0.077
8.489
1
0.004
1.252
PQ12
0.240
0.094
6.504
1
0.011
1.271
PQ13
0.204
0.082
6.138
1
0.013
1.226
PQ15
0.282
0.103
7.527
1
0.006
1.326
PQ17
0.182
0.088
4.317
1
0.038
1.200
PQ18
0.199
0.064
9.666
1
0.002
1.220
PQ21
0.188
0.094
3.995
1
0.046
1.206
Constant
-9.813
0.468
439.389
1
<0.001
0.000
Overall
model
fit
test
χ2 = 252.494, p<0.001; Hosmer–Lemeshow test value = 8.309, p =
0.306. Associated strength
Cox-Snell R2 = 0.011; Nagelkerke R2 = 0.263 3.3.1 The total score and distress score of PQ-B in detecting CHR Compared to non-CHR individuals, both the total score and distress score of PQ-B were significantly
higher in CHR individuals (see Table 1). To examine the accuracy and to reset the cutoff points for
Chinese college students, the diagnosis based on SIPS was set as the state variable, and the ROC
analyses were performed for the PQ-B total score and distress score, whose AUCs were also calculated
(see Figure 2). Both the total score and distress score yielded significant value in assessing CHR
individuals. The total score of PQ-B yielded an AUC of 0.842 (95% CI: 0.806, 0.877) for screening CHR,
and the AUC of the distress score was 0.900 (95% CI: 0.863, 0.937). At different cutoff points for the total
score and distress score of PQ-B, the sensitivity and specificity values were calculated and presented in
Table 2. The cutoff point of 13 for the total score, and 46 for the distress score balanced the greatest
sensitivity and greatest specificity. For the total score, a cutoff point of 13 or higher possessed a
sensitivity of 91.7%, and a specificity of 71.5%. While the sensitivity for the distress score when setting 46
or higher as the cutoff point was 90.3%, the corresponding specificity reached 81.2%. Table 2. 4. Discussion Page 9/20 Page 9/20 Page 9/20 Detection of CHR samples in both the clinical [10, 33-36] and non-clinical settings[31, 37, 38] had caught
great attention across the world, while the detection rate of CHR subjects in those studies varied greatly. Previous studies reported that the prevalence of CHR in the clinician-referred samples was 32-42%[33-35],
which were roughly consistent with each other. While the detection rate in samples of help-seeking
individuals ranged from 4.2% to 80%, which differed from site to site[10, 39-41]. As for the general
population (primarily focused on young adults and adolescences), the annual incidence of new cases of
CHR was estimated to be 1/10 000[33], and the prevalence was reported to range from 0.3% to 2.4%[31,
37, 38]. The diagnostic instruments were considered to be a great factor contributing to the inconsistency
of the detection rate. Schultze et al.[31] screened CHR in a young adult community with SIPS and SPI-A,
and reported that 2.4% of the participants meet at least one criterion of CHR, while only 0.6% met the
criteria of APS identified by SIPS. Salazar et al.[42] reviewed 56 studies and suggested that the 0.3% of
the general non-help-seeking young population met the DSM-5-APS criteria, while 1.3% of the general
population met the SIPS-APSS criteria. In this study, the detection rate of CHR subjects identified with the
SIPS criteria in Chinese college students was 0.3%. To our knowledge, this is the first multicenter
epidemiological study on the detection of CHR subjects in Chinese college students. The participants
were recruited from three different cities, including highly developed cities like Shanghai and Nanjing, and
also a second-tier city like Nanchang, through a random sampling way. Also, the colleges involved
included both general higher educational institutions and vocational colleges. Therefore, the sample in
the present study was representative for Chinese college students. Although the detection rate reported in
our study was lower than 1.1% reported in a single-centered study conducted in Chinese college
students[38], but was generally in line with other studies of this kind mentioned above. Since first-episode psychosis usually occur in late adolescence and early adulthood, CHR or prodromal
psychosis were believed to have an earlier onset[43, 44]. 4. Discussion Our research demonstrated that the peak age
range of the CHR identification by SIPS was between 17 to 20 years in the sample of Chinese college
students, which was largely in line with previous studies but a little bit narrower (e.g. SHARP reported 16
to 21 years in a Chinese sample)[10, 27]. The mean age of CHR was 18.3 (SD=1.5) years in this sample,
which was similar to the sample of NAPLS[33], but younger than that of SHARP[10], and older than that
of RAP[27]. The demographic features of recruited participants in these studies may account for the age
discrepancy. As the participants recruited in this study were college students with an age range of 14 to
31 years, while NAPLS recruited clinician-referred subjects aged 12 to 35 years, SHARP included help-
seeking subjects aged 15 to 45 years, and RAP included help-seeking subjects aged 12 to 22 years. Similar to previous studies using PQ-B to screen CHR in Chinese population[16, 22, 45-47], our research
proved that PQ-B could be a feasible first-step instrument for screening suspected CHR subjects in college
students. Both the sensitivity and specificity of PQ-B were excellent in this study. Also, the results showed
that 8 items of PQ-B including the PQ1 (asking “Do familiar surroundings sometimes seem strange,
confusing, threatening or unreal to you?”), the PQ3 (asking “Do things that you see appear different from
the way they usually do (brighter or duller, larger or smaller, or changed in some other way)?”), the PQ4
(asking “Have you had experiences with telepathy, psychic forces, or fortune telling?”), the PQ12 (asking
“Do you worry at times that something may be wrong with your mind?”), the PQ13 (asking “Have you ever Page 10/20 Page 10/20 felt that you don't exist, the world does not exist, or that you are dead?”), the PQ15 (asking “Do you hold
beliefs that other people would find unusual or bizarre?”), the PQ17 (asking “Are your thoughts
sometimes so strong that you can almost hear them?”) and the PQ18 (asking “Do you find yourself
feeling mistrustful or suspicious of other people?”) had significant predictive value of SIPS diagnosis,
which indicated that delusional ideas, perceptual abnormalities and suspiciousness were key symptoms
in identifying CHR subjects. Similarly, Xu et al.[22] and Schultze et al.[31] also reported that perceptual
abnormalities, delusional ideas and suspiciousness were the most prevalent psychosis-risk symptoms. 4. Discussion Nevertheless, our results recommended higher cut-off points for the total score and distress score of PQ-B
(13 and 46) than those of SHARP did in the Chinese population (7 and 24)[22], which were also much
higher than the cut-off points reported in the initial validation study of PQ-B (3 and 6)[16]. Despite the
cultural background and language may lead to the differences of PQ-B cut-off points[48], there must still
be other reason underlying the differences between the present study and SHARP. First, the participants in
this study were college students who were tested with PQ-B in non-clinical settings (online or in the
colleges), while SHARP recruited help-seeking out-patients from a psychological counselling center[22]. As prodromal symptoms mentioned in PQ-B were often mixed with psychotic-like experiences (PLEs) that
commonly existed in the general population[49-52], subjects who only had PLEs may also been tested
positive with PQ-B, causing false positives. Also, being rated in a clinical setting, rather than a non-clinical
setting, may help the participants better understand the prodromal symptoms mentioned in the scale, and
thus reducing false positive errors. Indeed, if we used the same cut-off points as SHARP did in their
screening procedure, 72.0% (16595/23063) of the participants would be screened positive with PQ-B in
this study, while the positive rate of PQ-B in SHARP was 62.1% (1681/2705)[10], which may indicate that
our false positive rate in PQ-B screening was higher. Second, the screening procedure differed. SHARP
employed a 2-stage method where the subjects tested positive with PQ-B would all be invited to the SIPS
interview, and 18.3% of the subjects tested negative with PQ-B were also interviewed[10]. However, we
used a 3-stage method that applied a telephone interview to subjects screened as positive with PQ-B and
only subjects screened as positive in the telephone interview were invited to the SIPS interview. Moreover,
further investigations were not provided to the negative subjects in the present study. These variance in
the screening procedure may have contributed to the underestimation of CHR subjects, which would
interfere the examination of the psychometric properties of PQ-B. As discussed in the last paragraph, the telephone assessment may cause the missed diagnosis of CHR,
which was a limitation of this study, whereas, it saved a lot of human resources in the process of CHR
screening, indeed, particularly in a large general population. 4. Discussion It is worth mentioning that the telephone
assessment employed in this study was not a semi-structured or structured interview that can be seen in
other studies, although we also use SIPS as the assessing instrument[31, 53, 54]. In the present study, it
only required the researchers to ask questions following the subscale for positive symptoms, and rate the
severity for each domain according to the subjective reports from the participants. Therefore, it only took
5 minutes in average, which was much time-saving compared to a structured/semi-structured interview. In fact, it also enhanced the screening efficiency by having precluded almost 90% of the subjects tested Page 11/20 Page 11/20 positive with PQ-B from receiving a face-to-face SIPS interview, despite which was, of course, based on
the sacrifice of the detection rate. Another limitation is that, due to resource limitations, the information obtained from the participants was
incomplete. On one hand, we missed some important clinical characteristics of the subjects, including
family history and psychiatric comorbidity. On the other hand, the CHR subjects were not followed after
being diagnosed, and thus, the transition to psychosis or other mental disorders was not investigated in
this study. 5. Conclusions In conclusion, the 3-stage method is a useful strategy in screening CHR individuals in general population,
especially when the population is large. Among college students, those with experiences of delusional
ideas, perceptual abnormalities or suspiciousness were more likely to be screened as CHR subjects. For
future researches, a 1- to 2-year follow-up of the CHR subjects is essential to investigate the conversion of
CHR subjects, and to explore the related risk factors. Availability of data and materials The dataset(s) generated during the current study are not publically available due to ethical restrictions
but are available from the corresponding author on reasonable request. 6. List Of Abbreviations NBH: Nanjing Brain Hospital
FAHNU: The First Affiliated Hospital of Nanchang University
APSS: Attenuated Positive Symptom Syndrome
BIPS: Brief Intermittent Psychotic Syndrome
GRDS: Genetic Risk and Deterioration Syndrome
ROC: Receiver operating characteristic
AUCs: Areas under the curve
SHARP: The Shanghai-At-Risk-for-Psychosis Study
NAPLS: The North American Prodrome Longitudinal Study
RAP: The Recognition and Prevention Program Consent to publish Not applicable. Ethics approval and consent to participate This study was approved by the Research Ethics Committee at the Shanghai Mental Health Centre
(SMHC), and carried out in accordance with the Declaration of Helsinki. Written informed consent was
received from all applicable participants prior to inclusion. Competing interests The authors declare no competing interest. We gratefully acknowledged the participation of all individuals involved in the study. We gratefully acknowledged the participation of all individuals involved in the study. Authors’ Information JX.W. and XY.L. are PH.D. candidates studying at the Department of Psychiatry, Tongji Hospital of Tongji
University, Tongji University School of Medicine. F.L., AS.Q., Q.C. and XF.G. are M.D. at the Department of
Psychiatry, Tongji Hospital of Tongji University, Tongji University School of Medicine. Q.Z. is a clinical
psychotherapist at the Department of Psychiatry, Tongji Hospital of Tongji University, Tongji University
School of Medicine. YH.Y. and JY.S. are Ph.D. at Tongji University School of Medicine. SP.X. and W.Y. are
M.D. at the Department of Psychiatry, Affiliated Nanjing Brain Hospital, Nanjing Medical University. MR.H.,
X.Y., J.T. and SL.W. are M.D. at the Department of Psychiatry, The First Affiliated Hospital of Nanchang
University. TH.Z. and JJ.W. are Ph.D. at Shanghai Mental Health Center, Shanghai Jiaotong University
School of Medicine, Shanghai Key Laboratory of Psychotic Disorders. Authors’ contributions Z.L. conceived of the study. XY.L., F.L., AS.Q., SP.X., MR.H., TH.Z., JJ.W. and Z.L. contributed to the design
of the study. JX.W., XY.L., F.L., AS.Q., Q.C., XF.G., Q.Z., YH.Y., JY.S., W.Y., X.Y., J.T. and SL.W. contributed to
the patient recruitment and data acquisition. JX.W. contributed to the data interpretation and statistical
analysis, as well as the drafting of the paper. XY.L. and Z.L. revised the manuscript critically. All the
authors read and approved the final manuscript. Funding This study was supported by grants from Ministry of Science and Technology of China
(2016YFC1306805), Priority of Shanghai Key Discipline of Medicine (2017ZZ02020), National Natural
Science Foundation of China (81974214), Clinical Research Plan of Shanghai Shen Kang Hospital Page 13/20 Development Center (16CR1032B). The funder had no role in the design, collection, management,
analysis, and approval of manuscript or decision to submit the manuscript for publication. Development Center (16CR1032B). The funder had no role in the design, collection, management,
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Figures Figures Page 18/20 Page 18/20 Figure 4 Comparison of PQ-B item scores in CHR and non-CHR individuals: the means of PQ-B items are
significantly higher in CHR individuals (p<0.001) Comparison of PQ-B item scores in CHR and non-CHR individuals: the means of PQ-B items are
significantly higher in CHR individuals (p<0.001) Figure 1 Sample flowchart (TJH: Tongji Hospital of Tongji University; NBH: Nanjing Brain Hospital; FAHNU: The
First Affiliated Hospital of Nanchang University) First Affiliated Hospital of Nanchang University)
Figure 2
Age distribution for the total sample, females and males meeting CHR criteria
Figure 3
ROC curves for the total score and distress score of PQ-B as indicator of CHR diagnosis Figure 2
Age distribution for the total sample, females and males meeting CHR criteria Figure 2 Age distribution for the total sample, females and males meeting CHR criteria Age distribution for the total sample, females and males meeting CHR criteria Figure 3
ROC curves for the total score and distress score of PQ-B as indicator of CHR diagnosis Figure 3 ROC curves for the total score and distress score of PQ-B as indicator of CHR diagnosis ROC curves for the total score and distress score of PQ-B as indicator of CHR diagnosis Page 19/20 Figure 4
Comparison of PQ-B item scores in CHR and non-CHR individuals: the means of PQ-B items are
significantly higher in CHR individuals (p<0.001) supplements.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. supplements.docx Page 20/20 Page 20/20
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The KATP channel in migraine pathophysiology: a novel therapeutic target for migraine
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The Journal of headache and pain
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Citation for published version (APA):
Al-Karagholi, M. A-M., Hansen, J. M., Severinsen, J., Jansen-Olesen, I., & Ashina, M. (2017). The KATP
channel in migraine pathophysiology: a novel therapeutic target for migraine. Journal of Headache and Pain, 18,
[90]. https://doi.org/10.1186/s10194-017-0800-8 The KATP channel in migraine pathophysiology
a novel therapeutic target for migraine Document version
Publisher's PDF, also known as Version of record university of copenhagen university of copenhagen Download date: 24. Oct. 2024 © 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. Abstract Background: To review the distribution and function of KATP channels, describe the use of KATP channels openers
in clinical trials and make the case that these channels may play a role in headache and migraine. Discussion: KATP channels are widely present in the trigeminovascular system and play an important role in the
regulation of tone in cerebral and meningeal arteries. Clinical trials using synthetic KATP channel openers report
headache as a prevalent-side effect in non-migraine sufferers, indicating that KATP channel opening may cause
headache, possibly due to vascular mechanisms. Whether KATP channel openers can provoke migraine in migraine
sufferers is not known. Conclusion: We suggest that KATP channels may play an important role in migraine pathogenesis and could be a
potential novel therapeutic anti-migraine target. Keywords: Migraine, KATP channel, KATP channels, Headache, Levcromakalim, Cromakalim Keywords: Migraine, KATP channel, KATP channels, Headache, Levcromakalim, Cromakalim controlling vascular tone, and protecting cells against
metabolic stress [2, 4, 5]. Introduction
d Adenosine 5′-triphosphate-sensitive K+ (KATP) channel
openers have been used in clinical trials for the treatment
of hypertension and asthma. The most common side ef-
fect mentioned during treatment with KATP channel
openers was headache (62, 64, 66–79) (Tables 2 and 3). However, only little attention has been focused on the role
of KATP channels in migraine pathophysiology. Over the past three decades, some preclinical evi-
dence has emerged indicating that KATP channels may
play an important role in migraine pathophysiology. In particular, the vasodilation effect of KATP channels
is
relevant,
since
it
is
has
been
established
that
endogenous neurotransmitters that trigger migraine
attacks are often associated with dilation of cranial
arteries [6]. KATP channels were originally identified in cardiomyo-
cytes [1], but have also been found in several tissues, in-
cluding
pancreatic
α-
and
ß-cells,
smooth
muscle,
skeletal muscle and central neurons [2, 3]. The channels
belong to the family of inwardly rectifying K+ channels
that are inhibited at physiological intracellular levels
ATP/ADP ratio. When intracellular ATP is reduced
under conditions of metabolic challenges they open. KATP channels are critical in regulating insulin secretion, Here we review preclinical and clinical studies on
KATP channels and discuss the KATP channel as a novel
therapeutic target for migraine treatment. The KATP channel in migraine
pathophysiology: a novel therapeutic target
for migraine Mohammad Al-Mahdi Al-Karagholi1, Jakob Møller Hansen1, Johanne Severinsen1, Inger Jansen-Olesen1,2
and Messoud Ashina1* Document license:
CC BY Citation for published version (APA):
Al-Karagholi, M. A-M., Hansen, J. M., Severinsen, J., Jansen-Olesen, I., & Ashina, M. (2017). The KATP
channel in migraine pathophysiology: a novel therapeutic target for migraine. Journal of Headache and Pain, 18,
[90]. https://doi.org/10.1186/s10194-017-0800-8 Download date: 24. Oct. 2024 The Journal of Headache
and Pain Al-Karagholi et al. The Journal of Headache and Pain (2017) 18:90
DOI 10.1186/s10194-017-0800-8 Open Access * Correspondence: ashina@dadlnet.dk
1Danish Headache Center, Department of Neurology, Rigshospitalet Glostrup,
Faculty of Health and Medical Sciences, University of Copenhagen, Nordre
Ringvej 57, DK-2600 Copenhagen, Denmark
Full list of author information is available at the end of the article Molecular structure and isoforms The KATP channel is a hetero-octameric complex that
consists of four pore-forming K+ inwardly rectifying
(Kir) subunits and four regulatory sulfonylurea receptor
(SUR) subunits [7]. * Correspondence: ashina@dadlnet.dk
1Danish Headache Center, Department of Neurology, Rigshospitalet Glostrup,
Faculty of Health and Medical Sciences, University of Copenhagen, Nordre
Ringvej 57, DK-2600 Copenhagen, Denmark
Full list of author information is available at the end of the article The Kir6.x subunit exists in two isoforms, Kir6.1 and
Kir6.2. The SUR subunit belongs to the ATP-binding
cassette
(ABC)
transporter
family,
regulated
by Page 2 of 9 Page 2 of 9 Page 2 of 9 Al-Karagholi et al. The Journal of Headache and Pain (2017) 18:90 Channel function sulfonylurea, with three isoforms, SUR1, SUR2A, and
SUR2B [7, 8]. KATP channel activity is controlled
by
changes
in
concentrations of intracellular ATP and magnesium ad-
enosine diphosphate (Mg-ADP). KATP channels couple
the metabolic state of the cell to the membrane potential
and thus play a crucial role in many tissues under both KATPchannels have specific tissue expression with
different compositions of Kir6.x and SUR subunits
which lead to distinct functional properties (Figs. 1
and 2 and Table 1). Fig. 1 Molecular structure and isoforms. a Two major Kir6.x isoforms (Kir6.1 and Kir 6.2) and three major SUR isoforms (SUR1, SUR2A and
SUR 2B) have been identified. b Kir.x subunits combine tissue-specifically with different SUR subunits to form various native KATP channels. Pancreatic, cardiac and smooth muscle KATP channels are made up of Kir6.2/SUR1, Kir6.2/SUR2A and Kir6.1 (or Kir6.2)/SUR2B, respectively
[2]. Kir, inwardly rectifying K+ channels; SUR, sulfonylurea receptor Fig. 1 Molecular structure and isoforms. a Two major Kir6.x isoforms
SUR 2B) have been identified. b Kir.x subunits combine tissue-specific
Pancreatic, cardiac and smooth muscle KATP channels are made up o
[2] Ki
i
dl
if i
K+
h
l
SUR
lf
l Fig. 1 Molecular structure and isoforms. a Two major Kir6.x isoforms (Kir6.1 and Kir 6.2) and three major SUR isoforms (SUR1, SUR2A and
SUR 2B) have been identified. b Kir.x subunits combine tissue-specifically with different SUR subunits to form various native KATP channels. Pancreatic, cardiac and smooth muscle KATP channels are made up of Kir6.2/SUR1, Kir6.2/SUR2A and Kir6.1 (or Kir6.2)/SUR2B, respectively
[2]. Kir, inwardly rectifying K+ channels; SUR, sulfonylurea receptor Al-Karagholi et al. The Journal of Headache and Pain (2017) 18:90 Page 3 of 9 Fig. 2 Schematic diagram of the KATP channel. Kir6.x subunits have two transmembrane domains, and a large cytoplasmic domain including an
inhibitory binding site for ATP [8, 84]. SUR subunits have many transmembrane domains and two intracellular nucleotide binding domains (NBD1
and NBD2), which stimulate opening of the channel after binding to MgADP [85] Fig. 2 Schematic diagram of the KATP channel. Kir6.x subunits have two transmembrane domains, and a large cytoplasmic domain including an
inhibitory binding site for ATP [8, 84]. Channel function SUR subunits have many transmembrane domains and two intracellular nucleotide binding domains (NBD1
and NBD2), which stimulate opening of the channel after binding to MgADP [85] blockers (like glibenclamide, second generation of sulfo-
nylurea and PNU37883A) directly activate or inhibit the
vascular KATP channels, respectively [9] (Fig. 3). physiological and pathological conditions [9]. K+ chan-
nels participate in the regulation of vascular tone, in-
cluding cerebral arteries [10]. When intracellular ATP is
reduced, KATP channels become activated; K+ efflux hy-
perpolarize the membrane and close voltage-operated
Ca2+-channels (VOCC). The result is a decrease in cyto-
solic Ca2+ concentration followed by relaxation of vascu-
lar smooth muscle cells and an increase in blood flow
[11]. The same applies if cells are exposed to metabolic
stress such as ischemia or hypoglycemia [12]. Closure of
K+ channels leads to membrane depolarization and con-
striction of the vessels [11]. In addition an increase in
intracellular cAMP and cGMP levels activate KATP chan-
nels to produce vasodilation [11]. Synthetic KATP chan-
nel openers (like levcromakalim and cromakalim) and Trigeminal ganglion and trigeminal nucleus caudalis Trigeminal ganglion and trigeminal nucleus caudalis
Kir6.1, Kir6.2, SUR1 and SUR2 are expressed in the tri-
geminal ganglion and trigeminal nucleus caudalis [22]
(Table 1). In trigeminal neurons Kir 6.1 and Kir 6.2 immu-
noreactivity were expressed in cells with all soma sizes in
all three divisions of the trigeminal ganglion [23]. Intracranial arteries KATP channels are present and functional in intracranial
arteries [13–15]. They are found in vascular smooth
muscle cells and vascular endothelial cells [16, 17]. In
rat cerebral arteries, the distribution of KATP channels
varies with vessel size and brain region [18]. Real time
polymerase chain reaction (RT-PCR) analysis revealed
Kir6.1 and SUR2B subunits in middle meningeal artery
(MMA) and middle cerebral artery (MCA) in rats and
pigs [19, 20]. This profile of KATP channels is also identi-
fied in human MMA [21] (Table 1). Table 1 Distribution of KATP channels
Subtypes of KATP
channels
Tissue
expression
Migraine related structures
Kir6.2/SUR1
Pancreas and
brain
DRG, TG and TNC from rats
(20–24, 26). Kir6.2/SUR2A
Cardiac and
skeletal muscle
Kir6.2/SUR2B
Smooth
muscle
DRG, TG, TNC, BA and MCA from
rats(20–24, 26). Kir6.1/SUR2B
Smooth
muscle
MMA from rats, pigs and human;
MCA from rats and pigs; BA, DRG,
TG and TNC from rats (20–24, 26). DRG Dorsal root ganglia, TG trigeminal ganglion, TNC trigeminal nucleus
caudatus, BA basilar artery, MMA middle meningeal artery, MCA middle
cerebral artery Table 1 Distribution of KATP channels
Subtypes of KATP
channels
Tissue
expression
Migraine related structures
Kir6.2/SUR1
Pancreas and
brain
DRG, TG and TNC from rats
(20–24, 26). Kir6.2/SUR2A
Cardiac and
skeletal muscle
Kir6.2/SUR2B
Smooth
muscle
DRG, TG, TNC, BA and MCA from
rats(20–24, 26). Kir6.1/SUR2B
Smooth
muscle
MMA from rats, pigs and human;
MCA from rats and pigs; BA, DRG,
TG and TNC from rats (20–24, 26). DRG Dorsal root ganglia, TG trigeminal ganglion, TNC trigeminal nucleus
caudatus, BA basilar artery, MMA middle meningeal artery, MCA middle
cerebral artery Table 1 Distribution of KATP channels Nitric oxide (NO) In humans, infusion of the NO donor, glyceryl tri-
nitrate, and inhibition of the breakdown of cGMP by
sildenafil [24] provoke migraine attacks in migraineurs
[25–27]. The NO-cGMP signaling pathway is involved
in the relaxation of vascular smooth muscle [28]. In
vitro studies with cerebral arteries isolated from rat
and piglet and extra-cerebral arteries from rabbit re-
ported that activation (opening) of KATP channels
contributed
to
both
cAMP-
and
cGMP-mediated
vasodilation [29–31]. Yuan et al. [32] reported that
sildenafil-induced vasodilation in porcine retinal arte-
rioles was significantly inhibited by glibenclamide and
suggested that cGMP signaling triggers opening of
KATP channels. In contrast, NO-induced dural and
pial artery dilation in rats was not attenuated by the
KATP channel blocker, glibenclamide [33]. Together,
these data suggest that interspecies differences are
likely to explain the discrepancy in findings of the
role of KATP channels in NO-induced vasodilation. KATP channels openers and migraine signaling
pathways A number of endogenous vasoactive signaling molecules
have been implicated in migraine [6], and KATP channels
may interact with these molecules. Al-Karagholi et al. The Journal of Headache and Pain (2017) 18:90 Page 4 of 9 Fig. 3 Opening of vascular ATP sensitive K channels. Endogenous molecules (ATP, cAMP and cGMP) and exogenous pharmacological agents
(cromakalim and glibenclamide) regulate the activity of KATP channels, which help controlling the vascular tone Fig. 3 Opening of vascular ATP sensitive K channels. Endogenous molecules (ATP, cAMP and cGMP) and exogenous pharmacological agents
(cromakalim and glibenclamide) regulate the activity of KATP channels, which help controlling the vascular tone man and animals are innervated by CGRP-containing
nerve fibers [34–36]. Efficacy of CGRP antagonism is
established in acute [37, 38] and preventive treatment
of migraine [39]. CGRP activates vascular smooth
muscle KATP channels indirectly through adenylate cy-
clase and protein kinase A (PKA) phosphorylation
(Fig. 4) [40–43]. In rats, CGRP-induced dilation of
the dural and pial arteries in vivo was shown to be
inhibited by glibenclamide [33], but KATP channel
openers do not interact with CGRP release in trigemi-
nal ganglion and trigeminal nucleus caudalis [22]. This suggests that KATP channels are involved in
CGRP-induced intracranial vasodilation. Pituitary adenylate cyclase activating polypeptide
(PACAP) Pituitary adenylate cyclase activating polypeptide (PACAP)
is a potent endothelium independent vasodilator of vari-
ous vascular beds, including cerebral arteries [44, 45]. In
vivo and in vitro studies have demonstrated that PACAP
dilates cranial arteries in different species, e.g. human
cerebral arteries [34, 46, 47], pig pia artery, canine basilar
artery, cat cerebral arteries, rabbit posterior cerebral arter-
ies and rat middle cerebral arteries [48–52]. Emerging Headache induced by KATP channels openers In the late 80’s there was a tremendous interest in deve-
loping novel KATP channel openers for hypertension, an-
gina pectoris and asthma. Three pharmacological drugs
were developed, pinacidil, nicorandil and levcromakalim. One of most common adverse events after treatment
reported in these studies was headache [58–63]. Six clinical trials with pinacidil have been published
for treatment of essential hypertension. Between 7% and
21% of the patients reported headache as an adverse
effect (Table 2). Nicorandil was tested for the treatment of angina
pectoris and ischemic heart disease. 23% to 88% of
the patients reported headache as an adverse event
(Table 3). The high incidence of headache is likely
due to the mixed KATP channel opener and NO
donor properties of nicorandil which thus cause vaso-
dilation via two separate mechanisms. Calcitonin gene-related peptide (CGRP) PACAP has three
types of receptors; Pituitary adenylate cyclase PAC1
(pituitary adenylate cyclase receptor 1), VPAC1 (vasoactive
intestinal peptide and pituitary adenylate cyclase receptor
1) and VPAC2 (vasoactive intestinal peptide and pituitary
adenylate cyclase receptor 2) [54] the two latter ones are
also activated by vasoactive intestinal peptide and all three
receptors are found in cerebral artery smooth muscle cells
[55]. Through these receptors, PACAP leads to an in-
crease in intracellular cAMP, which activates PKA
and
produces
vasodilation
by
several
mechanisms
including activation of KATP channels (Fig. 4) [45]. Interestingly,
glibenclamide
could
partially
inhibit
PACAP induced vasodilation in cerebral, coronary
and pulmonary arteries, suggesting that PACAP may
also activate KATP channels [44, 45]. Calcitonin gene-related peptide (CGRP) CGRP is one of the most potent endogenous vasodila-
tors and major arteries in the intracranial circulation of Al-Karagholi et al. The Journal of Headache and Pain (2017) 18:90 Page 5 of 9 Fig. 4 Signaling pathways through vascular smooth muscle KATP channels. Numerous endogenous vasodilators activate vascular smooth muscle
KATP channels through adenylate cyclase and PKA phosphorylation. Conversely, endogenous vasoconstrictors inhibit vascular smooth muscle KATP
channels through DAG and PKC phosphorylation. CGRP, calcitonin gene-related peptide; PGI2, prostaglandin I2; VIP, vasoactive intestinal peptide;
AngII, angiotensin II; NPY, neuropeptide Y; NA, noradrenaline; 5-HT, 5-hydroxytryptamine; Gs, G-protein-coupled receptor alpha stimulation; Gi,
G-protein-coupled receptor alpha i/q; DAG, diacylglycerol; PKA and PKC, protein kinase A and C, respectively Fig. 4 Signaling pathways through vascular smooth muscle KATP channels. Numerous endogenous vasodilators activate vascular smooth muscle
KATP channels through adenylate cyclase and PKA phosphorylation. Conversely, endogenous vasoconstrictors inhibit vascular smooth muscle KATP
channels through DAG and PKC phosphorylation. CGRP, calcitonin gene-related peptide; PGI2, prostaglandin I2; VIP, vasoactive intestinal peptide;
AngII, angiotensin II; NPY, neuropeptide Y; NA, noradrenaline; 5-HT, 5-hydroxytryptamine; Gs, G-protein-coupled receptor alpha stimulation; Gi,
G-protein-coupled receptor alpha i/q; DAG, diacylglycerol; PKA and PKC, protein kinase A and C, respectively KATP channel activity in vascular smooth muscle prepa-
rations by cAMP-dependent PKA activation [57] (Fig. 4). data suggest that PACAP or it receptors are a promising
target for migraine therapeutics [53]. PACAP has three
types of receptors; Pituitary adenylate cyclase PAC1
(pituitary adenylate cyclase receptor 1), VPAC1 (vasoactive
intestinal peptide and pituitary adenylate cyclase receptor
1) and VPAC2 (vasoactive intestinal peptide and pituitary
adenylate cyclase receptor 2) [54] the two latter ones are
also activated by vasoactive intestinal peptide and all three
receptors are found in cerebral artery smooth muscle cells
[55]. Through these receptors, PACAP leads to an in-
crease in intracellular cAMP, which activates PKA
and
produces
vasodilation
by
several
mechanisms
including activation of KATP channels (Fig. 4) [45]. Interestingly,
glibenclamide
could
partially
inhibit
PACAP induced vasodilation in cerebral, coronary
and pulmonary arteries, suggesting that PACAP may
also activate KATP channels [44, 45]. data suggest that PACAP or it receptors are a promising
target for migraine therapeutics [53]. Prostaglandins Prostacyclin (PGI2) activates and sensitizes meningeal
sensory afferents, and provokes immediate migraine-like
attacks in migraine sufferers [56]. PGI2 also increases Levcromakalim was investigated for the treatment of
asthma and essential hypertension. In these studies Table 2 Headache incidences registered during randomized controlled trials (RCT) and open label clinical trials with pinacidil
Paper
Study design
Dose (daily)
Indication
No. of patients
Headache No. Muiesan et al. 1985, Eur. J. Clin. Pharmacol [86]. RCT
30–75 mg
Essential hypertension
30
2 (7%)
Laher & Hickey 1985, J. Int. Med. Res [87]. Open label
12.5 mg
Healthy volunteers
12
1 (8%)
D’Arcy et al. 1985, Eur. J. Clin. Pharmacol [88]. Open label
20–100 mg
Essential hypertension
23
4 (17%)
Zachariah et al. 1986, Eur. J. Clin. Pharmacol [89]. RCT
62 mg (mean)
Essential hypertension
23
——
Sterndorff & Johansen 1988, Acta Med. Scand [90]. RCT
25–100 mg
Essential hypertension
71
7 (10%)
Goldberg 1988, J. Cardiovasc. Pharmacol [91]. RCT
25–100 mg
Essential hypertension
145
31 (21%) Al-Karagholi et al. The Journal of Headache and Pain (2017) 18:90 Page 6 of 9 Table 3 Headache incidences registered during randomized controlled trials (RCT) and open label clinical trials with nicorandil
Paper
Study design
Dose (daily)
Indication
No. of patients
Headache No. Camm & Maltz, 1989, Am. J. Cardiol [92]. RCT
20–60 mg
Angina pectoris
8
20 mg 50%
40 mg 88%
60 mg 67%
Raftery et al. 1993, Eur. Heart Journal [93]. RCT
20 mg and 40 mg
Angina pectoris
18
11 (61%)
Roland 1993, Eur. Heart Journal [94]. Review
10–80 mg
Angina pectoris
1680
36%
Wolf et al. 1993, Eur.J.Clin.Pharmacol [95]. RCT
20–200 μg i.v. Healthy volunteers
48
19 (40%)
Witchitz & Darmaon, 1995, Cardiovasc. Drugs& Therap [96]. Open label
20–40 mg
Angina pectoris
197
45 (23%)
Dunn et al. 1999, Pharmacoepidemiology
and Drug safety [97]. Prescription-event
monitoring (PEM) study
Varying
Angina pectoris &
ischemic heart disease
13,260
477 (4%) between 29% and 76% of the patients reported headache
as an adverse event (Table 4). induced headache and accompanying symptoms are
lacking, these data support a role of KATP channels in
migraine headache. Because KATP channel openers were
tested for other indications, there are no available data
on the potential migraine-inducing effects of pinacidil
and levcromakalim in migraine patients. It is conceivable
that both headache and migraine are underreported as
adverse events, as was found for the phosphodiesterase
inhibitors, cilostazol and sildenafil [73, 74]. Prostaglandins The selective synthetic KATP channel openers levcroma-
kalim and pinacidil have been shown to induce dilation in
rat cranial arteries [13, 15, 19] and in isolated human
cerebral arteries [64]. Moreover, the arterial dilation can
be inhibited by synthetic KATP channel blockers like glib-
enclamide [10, 33] and PNU37883A [21, 65] (Fig. 3). These findings suggest that high incidences of headache
could be due to vasoactive effect of the KATP channel
openers in pain-sensitive extra- and/or intracerebral
arteries. In addition to the vasoactive effects, the KATP channels
might also tap into other parts of the migraine cascade. For a number of patients, migraine attacks are associated
with transient focal neurological symptoms called the
aura [75], possibly caused by cortical spread depression
(CSD) [76]. During CSD K+ conductance is increased,
and CSD may be inhibited by Kir antagonist [77]. The
fact that KATP channels open under cellular stress, as
seen during long lasting depolarizations, could provide a
link between KATP channels, CSD and migraine aura. Discussion and future perspectives KATPchannels are expressed in migraine-related structures
such as the cranial arteries, TG and TNC [18–22, 66]. KATP
channels are also connected to a number of key molecules
in migraine
pathogenesis,
particularly
nitric
oxide,
CGRP, PACAP and PGI2 known to provoke migraine
attacks [56, 67–71]. Therefore, the KATP channels are
interesting in migraine context. With regard to the migraine pain, it is worth noting that
KATP channels are also found in peripheral nociceptive
fibers [78] and activation of these channels play a crucial
role in anti-nociception at both spinal and supra-spinal
levels [23, 79]. The exact role of these findings in the
headache induced by KATP channel openers is unknown. Human experimental models have demonstrated that
the activation of the cAMP and cGMP pathways can
trigger headache in healthy volunteers and migraine at-
tacks in migraine sufferers [6, 71, 72]. The cAMP and
cGMP signaling pathways are crucial in the activation of
KATP channels, which result in the relaxation of smooth
muscle [29–31]. Furthermore, synthetic KATP channel
openers
like
levcromakalim
and
pinacidil
trigger
headache in non-migraine patients [58–63]. Although a
detailed description of levcromakalim- and pinacidil- If KATP channel openers are in fact able to trigger
migraine, the next step to consider is whether KATP
channel antagonists can relieve migraine. KATP blockers
for the treatment of migraine should be selective for the
Kir6.1/SUR2B subtype because of its dominant presence
in vascular tissue (Table 1). The necessity of a subtype Table 4 Headache incidences registered during randomized controlled trials (RCT) and open label clinical trials with levcromakalim
Paper
Study design
Dose (daily)
Indication
No. of patients
Headache No. Singer et al. 1989, J. Hypertens [98]. RCT
1.5 mg
Essential hypertension
8
4 (50%)
Williams et al. 1990, Lancet [60]. RCT
1.5 mg
Asthma
16
10 (62%)
Kidney et al. 1993, Thorax [62]. RCT
0.125–0.5 mg
Asthma
25
19 (76%)
Suzuki et al. 1995, Arzneim.-Forsch./Drug Res [99]. Open label
0.5–1.0 mg
Essential hypertension
14
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1 1Danish Headache Center, Department of Neurology, Rigshospitalet Glostrup,
Faculty of Health and Medical Sciences, University of Copenhagen, Nordre
Ringvej 57, DK-2600 Copenhagen, Denmark. 2Danish Headache Center,
Department of Neurology, Glostrup Research Park, Rigshospitalet Glostrup,
Copenhagen, Denmark. Received: 3 July 2017 Accepted: 15 August 2017 Received: 3 July 2017 Accepted: 15 August 2017 Conclusion Emerging evidence suggests that KATP channels could be
involved in the pathophysiology of migraine. KATP chan-
nels exist in structures which are believed to be linked to
the pathophysiology of migraine, including cerebral and
meningeal arteries and the trigeminal system [19–22]. It is
established that the cAMP signaling pathway and possibly
cGMP signaling pathway are involved in the activation of
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|
https://openalex.org/W1973406332
|
https://zenodo.org/records/2525142/files/article.pdf
|
German
| null |
Überdrucknarkosenapparate
|
Deutsche Zeitschrift für Chirurgie
| 1,913
|
public-domain
| 5,331
|
Aus der K6niglichen chirurgischen UniversitS.tsklinik zu Berlin
(Geheimrat Bi e r). Aus der K6niglichen chirurgischen UniversitS.tsklinik zu Berlin
(Geheimrat Bi e r). Von Dr. W. Gerlach, VolontS.rassistent der Klinik.
(Mit I Abbildung.) Um den operativen Pneumothorax zu beseitigen, mug man
den Luftdruck an der Miindung des menschlichen Atemrohres
um einiges erh6hen gegentiber dem Luftdruck, der fiber der frei-
gelegten Lunge herrscht. Bekanntlich hat man dies auf doppelte
Weise erreieht : e r s t e n s, indem man den Druck fiber der Lunge
unter den no.rmalen Luftdruck herabgesetzt oder zweitens,
indem man den Druck an Mund und Nase erh6ht hat. Beide
Arten, das Unter- wie das {)berdruckverfahren linden heute aus-
gedehnte Anwendung. Es mag darum auffallen, dab wir uns
vornehmen, in dieser Arbeit nur das {3berdruckverfahren in Be-
tracht zu ziehen, und die Methode des Unterdrucks vernach-
1/issigen, obwohl gerade das Unterdruckverfahren die grogen
Erfolge auf dem Gebiet der Thoraxchirurgie veranlal3t hat und
seitdem nicht unbetr/ichtlich vervollkommnet wurde. Dazu kommt,
dab neuere Untersuchungen z. B. yon Cloftta
und Tiegel
keinen Zweifel darfiber gelassen haben, dab das Unterdruck-
verfahren in geringerem Grad als das {)berdruckverfahren die
physiologischen Verhfiltnisse bei der Respiration st6rt. Wenn
wir trotzdem unsere Ausfiihrungen auf die Methode des {~rber-
drucks beschr/inken, so geschieht es aus der {5"berzeugung, dab
diese Methode allein technisch eine solche Vereinfachung und
Verbilligung der Apparate zul/il3t, dab sie auch im Betrieb des
kleineren Krankenhauses und in der Hand des weniger geiibten
Chirurgen sich erm6glichen und bew/ihren. 206 GERLACH Und dies ist das Ziel, das wir zu erreichen streben: dab die
Thoraxchirurgie nicht wie bisher ausschlieBliches Dominium der
grogen Krankenh/iuser bleibe, sondern dab sie ein Bet/itigungsfeld
fiir die allgemeine chirurgische Kunst wird. Freilich e iner Vor-
aussetzung fiir ihre Anwendung rout3 die Methode des Uber-
drucks, wenn sie schon die physiologischen Verhfiltnisse nicht und
in noch geringerem Mal3 als das Unterdruckverfahren aufrecht
erh/ilt, streng gerecht werden, n/imlich, dab die Einatmung der
Druckluft als solcher in keinerWeise sch/idlich auf den Patienten
wirkt. Dies ist bei m/iBiger Druckdifferenz der Fall. S auer-
b ruch hat bekanntlich immer an der Anschauung Iestgehalten,
dab das Unterdruckverfahren physiologisch besser sei, abet auch
er muBte schliel31ich zugeben, dab in praxi bei geniigender Vor-
sicht nichts gegen die Methode des Oberdrucks einzuwenden ist
und nach den zahlreichen Erfahrungen der letzten Jahre pflichten
hierin alle Autoren seinem Urteil bei. Diese Vorbedingung ist
also erfiillt. Aber dariiber hinaus stellt der Chirurg, der einen
Oberdruckapparat bei seinen Operationen benutzen will, eine
Reihe yon Anforderungen, denen ein leistungsfiihiger, brauchbarer
Apparat geniigen muff. Von Dr. W. Gerlach, VolontS.rassistent der Klinik.
(Mit I Abbildung.) Diese sollen in Kiirze zusammengefaBt
und zugleich soil gezeigt werden, inwieweit ihnen die bisher kon-
struierten Apparate Rechnung tragen. Als Hauptgesichtspunkte bei der Beurteilung jedes Apparates
kommen in Betracht I. die Atmung selbst, 2. die Asepsis, 3. die Narkose, 4. die Vermeidung von St6rungen, 5" die Erfiillung praktischer Bediirfnisse. Auf Grund der in der Literatur niedergelegten Erfahrungen
lassen sich die Anforderungen zu I. folgendermaBen prttzisieren: Auf Grund der in der Literatur niedergelegten Erfahrungen
lassen sich die Anforderungen zu I. folgendermaBen prttzisieren: Den Lungen
muB zu jedem
Atemzug
eine ge-
niigend groBe Menge Luft, die sich unter m6glichst
gleichm~iBigem, gegenfiber demAtmosphtirendruck
etwas
erh6htem
Druck
befindet,
zur
Verfi.igung
stehen. Man hat auf die Druckkonstanz der Atemluft nicht immer
Wert gelegt. Im Anfang der Entwicklung des (}berdruckver- {3berdrucknarkosenapparate. 207 fahrens hielt man es gerade ffir einen Vorteil, mit der In-
sufflation der Luft die darauffolgende Aspiration zu verbinden
und diese Anschauung kam in zahlreichen Apparaten, besonders
franz6sischer
und amerikanischer
Chirurgen,
zum
Ausdruck. Druckpumpe und besonders der Blasebalg dienten als {)berdruck-
quelle bei dieser Methode, die ja eigentlich nichts anderes war als
eine kfinstliche Atmung. Als nun Sauerbruch
durch seine
grundlegenden Untersuchungen fiber kiinstliche Atmung zeigte,
dab die gewaltsame Anderung des Bl~ihungsgrades der Lunge
mancherlei Sch/idigungen ffir das Lungengewebe und ffir die Zir-
kulation, daneben noch vermehrten Wfirmeverlust und eine Ver-
gr613erung der Infektionsgefahr zur Folge hat, war auch das
Urteil fiber die Apparate, welche die Folgen des operativen Pneu-
mothorax
durch
kfinstliche
Atmung
beseitigen
wollten, ge-
sprochen. Die Richtigkeit der Sauerbruchschen Ansicht ist heute
nicht mehr bestritten, doch begegnete sie anfangs manchem Wider-
spruch. Es wurden verschiedentlich auch nach ihrer Mitteilung,
die im Jahr 19o 4 erfolgte, noch Apparate, welche inkonstanten
Luftdruck zur Einblasung bzw. Einatmung beniitzten, angegeben,
aber immer folgte rasch ihre Modifikation zur Druckkonstanz. So ging es dem yon Brat 19o 5 angegebenen Apparat, in dessen
Beschreibung die ,,Simderung des Atmungsmodus als erwfinscht"
bezeichnet wurde; drei Jahre sp/iter wurde er yon Brat und
S c h m i e d e n gemeinsam in Anerkennung, dab die Lrberlegenheit
der Druckdifferenz fiber die kfinstliche Atmung feststehe, ab-
ge~indert. Ebenso wurde in dem yon Meltzer
und Auer an-
gegebenen Insufflationsapparat der inkonstanten Druck liefernde
Blasebalg gleichzeitig yon deutscher wie yon amerikanischer Seite
durch Nordmann
und Elsberg
ersetzt durch einen Druck-
konstanz garantierenden elektrischen Antrieb. Der letztgenannte
Autor hat dabei allerdings der Theorie yon der Niitzlichkeit der
Druckschwankungen noch einmal das Wort geredet. Von Dr. W. Gerlach, VolontS.rassistent der Klinik.
(Mit I Abbildung.) Doch ist, wie gesagt, die Erbereinstimmung fiber diesen fahrens hielt man es gerade ffir einen Vorteil, mit der In-
sufflation der Luft die darauffolgende Aspiration zu verbinden
und diese Anschauung kam in zahlreichen Apparaten, besonders
franz6sischer
und amerikanischer
Chirurgen,
zum
Ausdruck. Druckpumpe und besonders der Blasebalg dienten als {)berdruck-
quelle bei dieser Methode, die ja eigentlich nichts anderes war als
eine kfinstliche Atmung. Als nun Sauerbruch
durch seine
grundlegenden Untersuchungen fiber kiinstliche Atmung zeigte,
dab die gewaltsame Anderung des Bl~ihungsgrades der Lunge
mancherlei Sch/idigungen ffir das Lungengewebe und ffir die Zir-
kulation, daneben noch vermehrten Wfirmeverlust und eine Ver-
gr613erung der Infektionsgefahr zur Folge hat, war auch das
Urteil fiber die Apparate, welche die Folgen des operativen Pneu-
mothorax
durch
kfinstliche
Atmung
beseitigen
wollten, ge-
sprochen. Die Richtigkeit der Sauerbruchschen Ansicht ist heute
nicht mehr bestritten, doch begegnete sie anfangs manchem Wider-
spruch. Es wurden verschiedentlich auch nach ihrer Mitteilung,
die im Jahr 19o 4 erfolgte, noch Apparate, welche inkonstanten
Luftdruck zur Einblasung bzw. Einatmung beniitzten, angegeben,
aber immer folgte rasch ihre Modifikation zur Druckkonstanz. So ging es dem yon Brat 19o 5 angegebenen Apparat, in dessen
Beschreibung die ,,Simderung des Atmungsmodus als erwfinscht"
bezeichnet wurde; drei Jahre sp/iter wurde er yon Brat und
S c h m i e d e n gemeinsam in Anerkennung, dab die Lrberlegenheit
der Druckdifferenz fiber die kfinstliche Atmung feststehe, ab-
ge~indert. Ebenso wurde in dem yon Meltzer
und Auer an-
gegebenen Insufflationsapparat der inkonstanten Druck liefernde
Blasebalg gleichzeitig yon deutscher wie yon amerikanischer Seite
durch Nordmann
und Elsberg
ersetzt durch einen Druck-
konstanz garantierenden elektrischen Antrieb. Der letztgenannte
Autor hat dabei allerdings der Theorie yon der Niitzlichkeit der
Druckschwankungen noch einmal das Wort geredet. Doch ist, wie gesagt, die Erbereinstimmung fiber diesen
Punkt eine allgemeine und die Ansicht eines einzelnen kann da-
gegen nicht ins Gewicht fallen. Rein aus praktischen Grfinden
ist nun zwar auch N o r d m a n n von seinem zuerst eingenommenen
Standpunkt in allerletzter Zeit wieder abgekommen und empfiehlt
in einer der letzten Nummern
der Deutschen medizinischen 208 GERLACI-I Wochenschrift den alten Meltzer-Auerschen Apparat mit Blase-
balgantrieb. Von Dr. W. Gerlach, VolontS.rassistent der Klinik.
(Mit I Abbildung.) Aber auch seine Behauptung, auf die er sich we-
sentlich stfitzt, dais die Druckschwankungen praktisch bedeutungs-
los seien, und dab der /dberdruck in den Lungen trotz der
Schwankungen des Manometers ein gentigend konstanter sei,
k6nnen uns nicht hindern, den Versuch der Wiedereinffihrung
kiinstlicher Atmung an Stelle von Druckdifferenz als Riickschritt
anzusehen. Das Bestreben, den LTberdruckapparat zu verein-
fachen, ist natiirlich anzuerkennen, aber es darf nicht soweit gehen,
dab man sich ohne weiteres fiber so wohlbegriindete Meinungen wie
die yon der Sch~idlichkeit der Druckschwankungen hinwegsetzt. y
g
g
Wir lehnen also Apparate zur kfinstlichen Atmung fiir unsern
Zweck in jeder Form ab und verlangen, dab die TJberdruckluft
unter gleichm~il3igem Druck zugeffihrt wird; wir verlangen nun
aber auch welter, dab dieser Druck sich in bestimmten Grenzen
hglt, vor allem mul3 eine dauernde Aufbl~ihung der Lunge ver-
mieden werden. Wir miissen uns hier gleich mit einem Apparat
auseinandersetzen, der auf eine dauernde Aufbl~ihung der Lunge
zwar jetzt verzichtet, sie aber doch anfangs erstrebt hat. Es ist
dies der bereits erw~ihnte, von den amerikanischen Chirurgen
Meltzer
und Auer angegebene und von Elsberg
vielfach
angewendete und erprobte Insufflationsapparat. Eine R6hre, z. B. ein Katheter, wird dabei in die Trachea eingeffihrt; sie miindet
an der Teilungsstelle in die beiden Bronchial~iste. Durch die
R6hre fliel3t die unter /dberdruck stehende Luft zu und, damit
die verbrauchte Luft ungehinderten Abzug habe, darf das Lumen
der R6hre nur die H~ilfte des Durchmessers der Trachea betragen. Letztere wird also in eine Doppelr6hre zerlegt. Die Absicht ist
nun, dutch einen dauernden Luftstrom, der an den Alveolen-
w~inden vorbeistreicht, die n6tige Sauerstoffmenge zuzufiihren. Der l)ruck wird, urn dies zu erreichen, ziemlich h6her genommen
als
bei
deu
anderen
Apparaten,
nach
Elsbergs
Angabe
~-2ommHg und mehr. Es hat sich nun gezeigt, dag einmal die
Gefii[te der Lunge dabei eine nicht unbetr~ichtliche Kompression
erfahren und das rechte Herz dementsprechend eine gr613ere
Arbeit leisten mug, vor allem aber hat sich die Annahme, dag ein
an den Alveolenw~inden vorbeiziehender Luftstrom durch diese
Art der Insufflation entstehe, nicht besttitigt. D u Bois-Rey- fdberdrucknarkosenapparate. 209 mo n d und S c hl e sin g e r konnten nachweisen, dab der Luft-
strom, wenn die Alveolen einmal gefiillt sind, unmittelbar unter-
halb der Miindung der eingefiihrten R6hre sich nach aul3en um-
kehrt. Von Dr. W. Gerlach, VolontS.rassistent der Klinik.
(Mit I Abbildung.) Von sich aus sind die elastischen Fasern nicht imstande,
gegen den stark erh6hten Druck die Luft aus den Alveolen aus-
zudriicken- es kommt also dort zu einer gef~ihrlichen Stagnation
der Luft, zur Anh/iufung von Kohlens~iure und zum Mangel an
Sauerstoff. Auf diese iibeln Folgen dauernder Blfihung hat iibri-
gens auch Nor dmann
in seiner neuesten Publikation wieder
aufmerksam gemacht. Nur durch hSufiges Kollabierenlassen der
Lunge kann ihnen begegnet werden. Beobachtet man diese Mal3-
regel, die auch E ls b e r g durchaus fiir notwendig h/ilt, so liil3t sich
allerdings gegen die Insufflationsmethode nichts weiter sagen,
als dab eben dann die h/iufigeren Druckschwankungen wieder
mit in Kauf genommen werden mfissen. Sicher ist es, damit die
innere Atmung sich ohne St6rung weiter vollzieht, am schonend-
sten
und
zweckm/il3igsten,
der
Lunge
ihren
natiirlichen
Wechsel von Inspirations-
und Exspirationsstellung
zu belassen, ihn jedenfalls nur wenig zu beschr/inken, wenn die
Operation bei beweglicher Lunge auf Schwierigkeiten st613t. Der
fiir gew6hnlich zu wfihlende Mitteldruck betr/igt @ IO mmHg. Nur
beim Verschlu[3 der Pleurah6hle wird man, um die Lunge in (lie
Pleurawunde einzupressen und dadurch alle Luft aus dem Pleura-
raum zu entfernen, yon h6herem Druck Gebrauch machen. Sehr
oft wird es aber auch genfigen, einen Druck yon -~-6 oder
@ 8 mm Hg anzuwenden. Wir haben ausgeffihrt, dab alle gewaltsamen Druckschwan-
kungen sch/idlich und deshalb zu vermeiden sind und wir haben
deshalb alle Apparate, welche nicht einen konstanten Luftstrom
erzeugen, als unzweckmSl3ig zur/ickgewiesen. Es erhebt sich nun
die Frage, wie die Druckschwankungen zu beurteilen sind, welche
dureh die Atmung selbst, wenn sie in geschlossenem Raum er-
folgt, in h6herem oder geringerem Grad veranlal3t werden. Wir
stehen auch hier auf dem Standpunkt, dab es gut ist, diese
Schwankungen m6glichst zu eliminieren, dab man sie aber, da
sie weit geringer und weniger br/isk sind als jene durch Pumpe
oder Blaseba!g hervorgerufenen, wenn sich andere Vorteile dafiir
bieten, auch ohne Gefahr unberiicksichtigt lassen kann. 14 Deutsche Zeitschrift f. Chirurgie. i2 3. Bd. 2 I 0 GERLAClt Damit sind wir zur Besprechung der anderen Haupttypen von
0-berdruckapparaten gekommen, die neben der bereits abgehan-
delten Insufflationsmethode in der Entwicklungsgeschichte des
f)berdruckverfahrens von Wichtigkeit sind, den Kasten-, Masken-
und Intubationsapparaten. Wir halten es nun nicht ftir n6tig,
jeden Apparat im einzelnen zu beschreiben, zumal da manche
wenig voneinander verschieden sind, sondern es scheint dem
Zweck dieser Arbeit zu geniigen, wenn wir uns mit den Haupt-
vertretern jeder Gruppe befassen. Von Dr. W. Gerlach, VolontS.rassistent der Klinik.
(Mit I Abbildung.) Js interessiert uns, nachdem die Notwendigkeit der Druck-
konstanz einmal anerkannt ist, auch weiter nicht, ob dieselbe auf
elektrischem Wege, durch Wasserdruck oder durch eine Bombe,
die komprimierte Luft enth/ilt, erreicht wird, ebensowenig wie die
Beschaffenheit des an der Abflul3stelle der verbrauchten Luft
angebrachten Ventils, durch das in den meisten Apparaten der
Druck von riickw/irts reguliert werden kann; vom Standpunkt der
Erzielung einer m6glichst physiologischen Atmung kommt nur in
Betracht die Art und Weise, wie die Atemluft den Atemorganen
angeboten wird, ob in einem grogen Kasten oder in kleiner Maske
oder durch Zuleitung in einer R6hre. Denn davon h/ingt, wie wir
schon angedeutet haben, ab, in welchem Mal3 durch die Atmung
selbst Druckschwankungen entstehen. g
Der Typus eines sogenannten Kastenapparates ist der von
13rauer
I9O 5 angegebene. Der ftir uns wesentliche Teil des-
selben ist ein I5ol Luft fassender, den ganzen Kopf des Pa-
tienten samt den HS.nden des Narkotiseurs einschliet3ender Kasten. Die Luft str6mt unter einem bestimmten Druck gleichmiil3ig an
einer Stelle ein und verl/il3t den Kasten wieder an anderem Ort
durch ein Ventil, das den Druck im Kasten reguliert und konstant
erh~ilt. Hier, bei Zwischenschaltung eines so grogen Raumes
zwischen Druckquelle und Atemorgan ist es klar, dab auch die
Anwendung eines Blasebalgs z. B. zur Druckerzeugung keine
Schwankungen jenseits des Kastens hervorrufen kSnnte; eben-
sowenig kann durch die Aspiration von Luft der Druck im Kasten
auch nur fiir einen Augenblick merkbar erniedrigt oder durch
Exspiration erh6ht werden. Es gibt keine Atemschwankungen
und dieser Apparat ist, was die Atmung betrifft, einwandsfrei. Dasselbe kann man sagen yon dem ebenso, nur etwas kleiner 13berdrucknarkosenapparate. 2II konstruierten K a r e w s k i schen Apparat und dem eigentlich schon
als 1)berdruckkammer zu bezeichnenden Apparat yon Engel-
ken, in dessen Kasten auger dem Kopf des Patienten noch der
Narkotiseur und eine Sauerstoffbombe Platz haben. Wird der Kasten so klein, dab er nur noch Mund und Nase
einschliel3t, so haben wir das charakteristische Stiick der Mas-
kenapparate. Die Zuflu136ffnung und Abflu136ffnung der Luft
bleibt genau, wie bei den Kastenapparaten eben beschrieben;
ebenso ist ein regulierendes Ventil vorhanden. Die vielen an-
gegebenen Apparate unterscheiden sich meistens nur durch ihre
i)berdruckquelle, durch verschiedene Arten der Ventile, tiber-
haupt durch die gr613ere oder geringere Kompliziertheit ihrer
Apparatur. In Hinsicht des Atemvorgangs verhalten sie sich
ziemlich gleich. g
Bei ihrer Anwendung n/imlich lassen sich die Atemschwan-
kungert nicht mehr verhtiten, sie k6nnen h6chstens durch be. Von Dr. W. Gerlach, VolontS.rassistent der Klinik.
(Mit I Abbildung.) sondere Vorrichtungen gemildert werden. Sie kommen so zu-
stande, dab in derselben Zeit, in der Luft aus der Maske aspiriert
wird, nicht ebensoviel durch das zuleitende Rohr nachstr6mt, der
Druck also sich herabmindert und nach T iegel sogar negativ
werden kann. Umgekehrt vermag bei Exspiration nicht sofort
die vermehrt in die Maske eindringende Luft durch das Ventil
abzufliel3en, es kommt voriibergehend zu weiterer Druckerh6hung. Dasselbe ist der Fall, wo die Luft durch eine R6hre direkt in den
Larynx eingefiihrt wird wie beim K u h n schen Intubationsapparat
oder bei den anderen Apparaten, mit denen Intubation verbunden
werden kann, z.B. dem Brat-Schmiedenschen. Die Schwan-
kungen sind um so gr613er, je enger das Lumen des Zuleitungs-
rohres ist. Sie k6nnen gemildert werden durch eine Erweiterung
des Rohres, wie sie beim Tiegelschen Apparat angebracht ist
in Gestalt eines elastischen Ballons, der seinerseits mit der Maske
kommuniziert. Seine Wirkungsweise bedarf wohl keiner weiteren
Erkl/irung. Wird der Druck in der Maske durch die Aspiration
niedriger, so str6mt sofort vom Ballon Luft nach und seine elasti-
sche Wand hebt den Druck wieder auf das friihere Niveau und
ganz /ihnlich ist es bei der Exspiration. Je gr6ger dieser druck-
kompensierende Luftraum ist und je n/iher er Mund und Nase
liegt, desto besser. Es lassen sich auf diese Weise die Schwan- 14 * 14 * 212 GF.RLACH kungen auf wenige Millimeter Hg reduzieren und sind dann ohne
Belang. Unser Urteil fiber die Maskenapparate kommt zu dem
Ende, dal3 sie, immer nur in Rficksicht auf die Atmung, alle
brauchbar sind, und zwar um so besser, je weniger die Atem-
schwankungen auftreten. Wesentlich rascher werden wir uns orientieren, wenn wir
jetzt das Verh/iltnis der Oberdruckapparate zu den Erfordernissen
der Asepsis prfifen. Man kann sich eigentlich begnfigen, zu
sagen, dab die M6glichkeit aseptischen Operierens im selben Ver-
h/iltnis zunimmt, als die nicht sterilisierbaren und den Operateur
eventuell behindernden Apparatteile, besonders soweit sie an den
Kopf des Patienten zu liegen kommen, an Umfang geringer
werden. So werden den Kastenapparaten die Maskenapparate und
diesen wieder die Intubations- und Insufflationsapparate vorzuziehen
sein. Natfirlich mug auch in Rechnung gezogen werden, ob der
dem Gesicht anliegende Teil einer ihn bedienenden Hand bedarf
oder ob die ganze Konstruktion des Apparats h/iufigere St6rungen
der iJberdrucknarkose herbeifiihrt, welche ihrerseits wieder die
Asepsis gef/ihrdende Manipulationen n6tig machen. Von Dr. W. Gerlach, VolontS.rassistent der Klinik.
(Mit I Abbildung.) Wir sehen
also, dab die gute Asepsis wieder in nicht geringem Mal3 yon einer
guten N a r k o s e abh/ingig ist. Was mtissen die Apparate in dieser
Hinsicht leisten? Sie m/issen m6glich machen, dab zugleich mit
der Z)ruckluft das Narkotikum einstr6mt; es ist gut, wenn dasselbe
genau do sierbar ist; es mug ferner die Zufuhr des Narkotikums
rasch unterbrochen werden k6nnen, ohne dab der Luftstrom eine
Veriinderung erleidet. Es fr/igt sich vielleicht, ob aul3er der M6g-
lichkeit, I)ruckluft mit und ohne Narkose zuzuleiten, noch eine
Einrichtung erforderlich ist, die erlaubt, Narkose ohne Uberdruck
anzuwenden. Es gibt ja wohl Stadien w/ihrend der Operation,
wo ein Kollaps der Lunge erwfinscht ist, ohne dab die Tiefe der
Narkose darunter Not leiden darf. Ffir diesen Fall ist eine ent-
sprechende Vorrichtung sicher von VorteiI. Aber als unumg/ing-
lich m6chten wir sie, da doch die Lunge kaum 1/ingere Zeit im
kollabierten Zustand belassen wird, nicht ansehen. Ebenso-
wenig wie die Armierung des Apparats mit einem Sauerstoff-
bombe, wie es Garr~ verlangt. Uberhaupt Sauerstoff an Stelle
der Luft zu verwenden, liegt kein Grund vor, es k6nnte sich h6ch-
stens um eine Zufuhr von Sauerstoff zum Luftstrom handeln bei l~berdrucknarkosenapparate. 213 irgendwelchen bedrohlichen Atmungsst6rungen. Nun lassen sich
diese aber, wenn man nur die Vorsicht gebraucht, der Lunge
ihren natiirlichen Bl~ihungswechsel zu belassen oder ihr yon
Zeit zu Zeit eine vollkommene Zusammenziehung zu erlauben,
ziemlich sicher vermeiden. Und kommt es doch einmal zu einer
St6rung, so haben wir in der geeignet regulierten Zufuhr von Luft
ein geniigendes Hilfsmittel dagegen. B r ii n i n g sagt am Schlul3
einer ausffihrlichen experimentellen Arbeit fiber die Anwendung
von Sauerstoff und komprimierter Luff zur Narkose, dab bei
Narkosezuf~illen die kfinstliche Atmung a llein stets hinreichend
sei und die Sauerstoffatmung keine Vorteile biete. Derselbe Autor macht noch auf die Wichtigkeit einer anderen
Sache bei der Zufiihrung komprimierter Luft aufmerksam. Das
ist auf ihren Feuchtigkeitsgehalt. Er konnte an M/iusen nach-
weisen, daB, wenn trockene iJberdruckluft angewendet wurde, es
in den Alveolen zu starkem Blutk6rperchenaustritt kam, die A1-
veolen wurden bei l~ingerer Dauer mit Blut ausgeffillt und die
Tiere gingen zugrunde. Bei den Apparaten, welche keine Bomben
benfitzen, also ihre Luft aus dem Operationssaal selbst entnehmen,
hat diese ja wohl immer den n6tigen Feuchtigkeitsgrad; aber
einem Apparat, wie dem T iegelschen gegeniiber, wo Luft oder
Sa'uerstoff aus Bomben direkt eingeatmet wird, ist diese Fest-
stellung von groBer Bedeutung. Von Dr. W. Gerlach, VolontS.rassistent der Klinik.
(Mit I Abbildung.) Wir mfissen hier noch eine Behauptung erw~ihnen, die von
E 1 s b e r g aufgestellt, allerdings nicht unwidersprochen geblieben
ist. Elsb erg glaubte n~imlich, dab der ./~therverbrauch bei der
yon ihm gefibten Insufflationsmethode ziemlich geringer sei als
bei den anderen Apparaten. Er gab an, dab es ihm nicht ge-
lungen sei, auf diese Art einen A_thertod beim Hund zu veranlassen. Wir haben schon auf die von D u B o i s- R e y m o n d und S c h 1 e-
singer gefundene Tatsache der Umkehr des Luftstroms am Ka-
theterende hingewiesen und k6nnten deshalb einen geringeren
A_therverbrauch nicht verstehen. Tats/ichlich hat Nor dInann auch Narkosetod beim Hund
durch die Insufflationsmethode herbeigeffihrt gesehen und S c h 1 e-
sin g e r bestreitet nach seinen zahlreichen Experimenten, dab der
2i.therverbrauch geringer sei als sonst. 2 14 GERLACH Wit kommen jetzt dazu, auf einige besondere StSrungen noch
einzugehen, die durch die Narkose oder durch die fdberdruckluft
hervorgebracht werden k6nnen. Es sind dies : Das Erbrechen, die
Magenaufbl~ihung und der Pneumothorax der anderen Seite. Bei Anwendung von Kastenapparaten hat das Er b r e che n
keine gr6Bere Bedeutung als bei einer gew6hnlichen Narkose. Die H~inde des Narkotiseurs sind bereit, ohne irgendeine StSrung
sonst hervorzurufen, die Mundh6hle des Patienten zu reinigen und
Aspiration zu verh/iten. Dies ist ganz anders bei allen Masken-
apparaten; das Erbrochene wird in die Maske entleert, verlegt die
0ffnungen ftir die Luft und wird yon dort aus aspiriert, veranlaBt
also fast immer eine sofortige Unterbrechung der Uberdruck-
narkose durch Abnahme und Reinigung der Maske. Das kann
unter Umst~inden eine schwere St6rung der Operation bedeuten. Als beste Vorkehr empfehlen deshalb die Autoren, den Patienten
immer erst in tiefe Narkose zu bringen, ehe der Uberdrucknarkosen-
apparat in T~itigkeit tritt. Diese MaBregel mag meistens geniigen. Tritt aber doch einmal Erbrechen ein, so miissen alle Maskem
apparate, aulSer etwa dem Tiegelschen, dessen oben erw~ihnter
Gummiballon als Rezipient fiir das Erbrochene dienen kann, ihre
Arbeit unterbrechcn. Bei den Intubationsapparaten soll es, da die Glottis often ist
und die Tube iiberallhin abgedichtet wird, nicht zum Erbrechen
kommen. Dasselbe g~ilte dann auch fiir die Insufflationsmethode. Letztere soll den weiteren Vorzug haben, dab die Speichel-
driisen weniger sezernieren und dab alles, was in die Trachea ein-
dringen will, dutch den zwischen Tracheal- und Katheterwand
nach auBen ziehenden Luftstrom mitgerissen wird. Von Dr. W. Gerlach, VolontS.rassistent der Klinik.
(Mit I Abbildung.) M a g e n d i 1 a t a t i o n e n wurden bei Kastenapparaten nie be-
obachtet. Dagegen traten sic bei Maskenanwendung 6fret ein;
es wurde auch ein Todesfall infolge davon berichtet. Zur Auf-
bl~ihung des Magens kommt es, nach der gew6hnlichen Meinung,
nicht dutch Verschlucken der Luft, sondern dadurch, dais der
h6here Druck den Sphincter pharyngis ausdehnt und die Luft
so in den 0sophagus eindringt. Gerade die Tatsache, da{3 bei den
Kastenapparaten nie eine Magenbl~ihung vorkam, beweist die
Richtigkeit dieser Annahme. Denn dort liegt zur luftdiehten Be- 0berdrucknarkosenapparate. 2t5 festigung des Kastens immer eine elastische Manschette um den
Hals, die eine Erweiterung des Osophagus verhindert. Da eine Einftihrung der luftzuleitenden R6hre tier in die
Trachea, wie bei der Insufflation, eine Magenaufbls-hung aus-
schliel3t, so ist nur bei den Masken- und Intubationsapparaten
dieser M6glichkeit nicht vorgebeugt. Andererseits diirfen wit an einer Mitteilung S chlesingers
nicht vortibergehen, die sich auf eine sonst nicht erw5-hnte Gefahr
bei der Insufflation bezieht. Er beobachtete mehrmals am Tier
einen Pneumothorax
der anderen Seite, und zwar konnte er
direkt w5-hrend der Operation sehen, wie die Luftblase in der
nicht er6ffneten Pleurah6hle allm/ihlich gr6ger wurde. Unger
beobachtete desgleichen ws-hrend einer Insufflationsnarkose bei
nicht er6ffneter Pleura einen Pneumothorax. Man ist sich fiber
die Ursache dieser Erscheinung nicht einig. Sie interessiert uns
bier auch nicht. Aber wichtig ist, dab von einer solchen Be-
obachtung bei Anwendung einer anderen Methode nicht be-
richtet ist. Wir konnten uns bisher bei Besprechung der (3berdruck-
apparate yon eigentlich medizinischen Gesichtspunkten darauf
beschrs-nken, das Typische und Wesentliche zu nennen, ohne auf
ermiidendes Detail einzugehen; wir m6chten dies auch welter tun,
wenn wit jetzt den praktischen Bediirfnissen unsere Aufmerksam-
keit zuwenden. ~Ein Apparat, der die in der Einleitung aufgestellte Forderung
erfiillen will, mug in verschiedener Hinsicht ,,einfach" sein. Er
mug in erster Linie jede LageverS.nderung
des Patienten
m6glich machen. Dies ist ein absolutes Erfordernis, da ein
Wechsel der Lage bei allen in Betracht kommenden Operationen
hs-ufig notwendig ist. Zweitens mug er leicht transportabel
und iiberhaupt von mOglichst kleinem Umfang sein. Das ist
besonders fiir die Kriegschirurgie wichtig. Die Kastenapparate
diirften also diesen Anforderungen nicht geniigen. Bedeutungs-
voll ftir die Kriegschirurgie wie auch fiir das kleinere Krankenhaus
ist es weiter, dal3 der Apparat von bestimmten anderen Ein-
richtungen unabhs-ngig
ist. Man hat da nicht immer eine
Sauerstoffbombe oder elektrischen Anschlul5 zur Verfiigung. Von Dr. W. Gerlach, VolontS.rassistent der Klinik.
(Mit I Abbildung.) Ein
Mann mug zu seiner Bedienung geniigen, und auch dann sollte die 216 GERLACH Arbeit mit dem Apparat m6glichst wenig durch die Kompliziert-
heit der Konstruktion erschwert werden. Es soll keiner beson-
deren ,,Ein/ibung" auf den Apparat bedfirfen; denn wenn be-
sondere Routine zu seiner Bedienung n6tig ist, taugt er wieder
nichts fiir das kleinere Krankenhaus, das keine so groBe Zahl
derartiger Operationen haben wird, dab man sich einfiben k6ante. g
p
I)iese fdberlegungen verurteilen die Masken- und Intubations-
apparate, die teilweise ~iuBerst kompliziert sind, als in unserem
Sinne lange nicht einfach genug. g
g
g
Es bleibt der Meltzer-Auersche
Apparat, d. h. eigent-
lich f~illt er auch schon unter das eben abgegebene Urteil, denn
obwohl die Apparatur einfach ist, bedarf seine Anwendung doch
ganz besonderer Ubung. Ein Katheter mu6 in die Trachea ein-
gefiihrt werden. Das gelingt ohne besondere Kunstfertigkeit
nicht. Manche, welche die Methode versuchten, haben sie zurfick-
gewiesen, weil ihnen die Einf/ihrung
zuviel Zeit und M/ihe
kostete. E1 sb e r g selbst h~ilt die Anwendung des Laryngoskops
dabei fiir notwendig. Aber das ist nicht die einzige Schwierigkeit. Es bedarf groBer Erfahrung, die richtige Dicke des Katheters zu
treffen. Ist er zu dick genommen, kommt es zur Stauung der
Luft in den Lungen under muB herausgenOmmen und ein anderer
eingesetzt werden, bis die richtige Weite gefunden ist. Da die
Weite der Trachea natfirlich mit der Gr6Be des Individuums
schwankt, :so l~iBt sich denken, wie oft in der Wahl des Katheters
fehlgegriffen 'werden mag. Aber es kommt als Drittes hinzu, dab
das Katheterende an der ,,richtigen" Stelle, der T.eilungsstelle der
Trachea in die Bronchial~iste liegen muB, sonst kann es ebenfalls
zu schweren St6rungen kommen. Bei Anwendung dieser Methode wird man sicher ein langes
und hohes Lehrgeld bezahlen m/issen, besondere ,,Einfachheit"
wird ihr wohl auBer dem in ihr Erfahrenen niemand nachrtihmen. Dies gilt natfirlich ebenso fiir die oben erw~ihnte No r d m a n n sche
Modifikation, die wir bereits aus anderem Grund, wegen Vernach-
l~issigung der Druckkonstanz zur/ickgewiesen haben. l~berblicken wir noch einmal, was unsere Er6rterungen uns
gezeigt haben, so ist yon den Kastenapparaten zu sagen, dab sie
zwar eine cute Atmung garantieren und St6rungen fast aus-
geschlossen sind, dab sie aber, weil zu umfangreich und un- Uberdrucknarkosenapparate. 217 handlich, nicht praktisch sind. Die Maskenapparate sind teilweise
kompliziert, unfibersichtlich und nicht billig genug. Sie lassen die
Atemschwankungen bestehen, sind beim Erbrechen gef/ihrlich
und k6nnen Magendilatation veranlassen. Ansatzsttick
ffir die
MundSffnung Von Dr. W. Gerlach, VolontS.rassistent der Klinik.
(Mit I Abbildung.) SchlieBlich entspricht
der Aufbau des Insufflationsapparates zwar den Ansprfichen der
Einfachheit, aber seine Anwendung erfordert groBe Obung und
ist ziemlich umstfindlich. Daneben ist das bei ihm notwendige
6ftere Kollabierenlassen der Lunge st6rend, wirkt aber der Blase-
balg nach Art der kfinstlichen Atmung, so verst6Bt dies gegen eine
grundlegende, allgemein anerkannte Regel. Man sieht, G a r r ~ hatte schon recht, wenn er sagte, er kenne
keinen Apparat, der nur Vorteile habe. Wir wollen auch nicht
behaupten, dab wir einen solchen pr/isentieren k6nnten. Abet
wir glauben, dab ein von Klapp konstruierter, yon der Firma
Windler in Berlin ausgeffihrter Apparat in mancher Hinsicht einen
Fortschritt bedeutet (Fig. I). In ein Gestell sind eingebaut ein Blasebalg ffir FuBantrieb
und zwei luftdicht abzuschlieBende Glasgef~iBe. Das eine ffir
die Aufnahme warmen Wassers, das andere ffir den Ather zur
Narkose. Die Leitung der vom Blasebalg komprimierten Luft
geschieht mit Hilfe von drei R6hren, die folgendermaBen hinter-
einander geschaltet sind : Die erste setzt sich direkt aus dem Blase-
balg fort, erweitert sich zu einem ca. 2000 ccm fassenden Gummi-
ballon und mfindet unter dem Wasserspiegel des ersten, zur H~ilfte
ungef/ihr mit warmem Wasser angefiillten Gef/il3es. Die zweite
hat ihr eines freies Ende in dem fiber dem Vgasser befindlichen
Luftraum, das andere unter dem Flfissigkeitsspiegel im -~_ther-
geffil3. Die dritte endlich beginnt wieder frei im Luftraum fiber
dem Ather, erweitert sich in einiger Entfernung in einen den
ersten Gummiballon an Gr6Be noch fibertreffenden 1/inglichen
Ballon und endigt direkt dahinter in zwei weichen Ansatzstficken,
welche in die beiden Nasenl6cher zu liegen kommen und bis in
den hinteren Nasenraum vorgeschoben werden. Hinter dem /itherhaltenden Gef/iB ist ein Manometer einge-
schaltet. Die aus dem Blasebalg intermittierend str6mende Druck-
luft wird durch den ersten Gummiballon in einen gleichm/iBigeren
Luftstrom verwandelt. Dieser durchzieht zuerst das angew/irmte 2 [ 8 GERLACH Wasser, reinigt sich dort und befeuchtet sich, darauf die ,~ther-
flasche, yon wo er die Atherd/impfe mitreil3t, und gelangt durch Gummiballon, der die Druckschwanku
er weiter ausgleicht, in die Nase und da
r zweite, der Nase naheliegende Ballon hil den zweiten Gummiballon, der die Druckschwankungen vom
Blasebalg her weiter ausgleicht, in die Nase und dann in die
Lungen. Der zweite, der Nase naheliegende Ballon hilft daneben Uberdrucknarkosenapparate. 219 auch die durch die Atmung hervorgerufenen Druckschwankungen
herabsetzen. Der Mund kann selbstverst/indlich nicht often bleiben, da sonst
ein LTberdruck in der Lunge nicht zustande k/ime. Von Dr. W. Gerlach, VolontS.rassistent der Klinik.
(Mit I Abbildung.) Er wird durch
eine Klappe verschlossen, die folgendermal3en konstrui,ert ist. Eine
an zwei Stellen durchlochte Metallplatte kommt zwischen Zfihne
einerseits und Ober- und Unterlippe andererseits zu lieg~en. Die
Z/ihne des Ober- und Unterkiefers greifen dabei nicht iib ereinander,
sondern lassen einen Zwischenraum bestehen, so dal3 die Kiefer
nicht geschlossen sind. Eine Gummipelotte driickt Ober- und Unter-
lippe lest auf die Metallplatte; die Mundh6hle kommuniziert also
mit der Aul3enluft lediglich durch die beiden 0ffnungen in der
Platte. Die eine bleibt ftir den Durchtritt der Luft often und
ist zu seiner Regulierung mit einem Schraubenventil versehen. Dies wirkt so, daB, wenn es ge6ffnet ist, verh/iltnism/igig viel
yon der in den Nasenraum einstr6menden I)ruckluft sofort, ohne
in 'die Lunge zu gelangen, hier wieder abfliel3t und die Exspira-
tionsluft ebenfalls freien Abzug hat. Die Folge ist Verminderung
des Drucks in der Lunge. Umgekehrt erh6ht sich derselbe, wenn
das Ventil die 0ffnung fiir den Luftdurchtritt verengt. Durch die zweite 0ffnung in der Platte wird ein Schlauch
in den Magen eingefiihrt und damit bei eintretendem Brechreiz
jede Gefahr beseitigt. Ferner hat nun die eventuell in den Magen
eingedrungene Luft Gelegenheit, zu entweichen. Die haupts/ichlichsten Bedenken bei Anwendung der Ober-
drucknarkose sind durch diese einfache Vorrichtung gegenstands-
los geworden. Der einzige am Apparat angebrachte Hebel er-
laubt die Ein- und Ausschaltung der Narkose, d. h. 6ffnet oder
versperrt der Luft den Durchgang durch das A_thergefiig. Der
Apparat ist aul3erordentlich iibersichtlich. Um ihn anzuwenden,
mug man lediglich den Schlauch in den Magen und die Ansatz-
stticke in die Nase einftihren. Aus technischen Griinden ist die Anbringung des Mano-
meters zwischen zweitem Gummiballon und Nase nicht m6glich. Dber die Druckschwankungen in der Lunge wissen wir also nicht
mehr, als dab sie, da jenseits des zweiten Ballons, kleiner sein
mtissen, als das Manometer zeigt. Dies ergibt sich aus unseren
theoretischen Betrachtungen, denen nach M6glichkeit Rechnung GERLACH, 0berdrucknarkosenapparate. 220 zu tragen angestrebt wurde. Wenn es dabei auch nicht gelungen
sein dfirfte, die Druckschwankungen in der Lunge vollsttindig zu
beseitigen, so sind sie doch durch die angegebenen Vorrichtungen
vermindert worden, und deshalb daft der Apparat wohl in An-
spruch nehmen, dal3 er in besserer Weise, als die bisher kon-
struierten, den Anforderungen der Praxis und
der Wissenschaft
gerecht wird. Literaturverzeichnis. i. Briining,
A. Deutsche Zeitschr. f. Chit. 1912 , Bd. II 3. 2. S a u e r b r u c h, Ergebnisse der Chirurgie und Orthop~.die, Bd. I, S. 38 I. 3. Cl o ~ t t a. Arch. f. klin. Chir. 98, II. 4. Elsberg. Arch. f. klin. Chir. 96, I. 5. Schlesinger. Arch. f. klin. Chir. 95, If. 6. Nordmann. Arch. f. klin. Chir. 92, If. 7. Dreyer. Beitr. z. klin. Chir. 65, I. 8. Dreyer-Spannaus. Beitr. z. klin. Chir 77. 9. Friedmann. Deutsche Zeitschr. f. Chir. Bd. IIo. Io. N o r dm a n n. Deutsche med. Wochenschr. I913, Nr. 8. II. B r at. Berlin. klin. Wochenschr. 19o5, Nr. 7. II. B r at. Berlin. klin. Wochenschr. 19o5, Nr. 7. 12. Brat-Schmieden. Mfinchn. med. Wochenschr. 19o8, Nr. 47. 12. Brat-Schmieden. Mfinchn. med. Wochenschr. 19o8, Nr. 13. Garrt
und Quincke. Lungenchirurgie I913. 13. Garrt
und Quincke. Lungenchirurgie I913.
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https://openalex.org/W4304123523
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http://jrk.fmipa.unand.ac.id/index.php/jrk/article/download/501/341
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Indonesian
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Teknik Edible Coating dengan menggunakan Campuran Gel Lidah Buaya dan Ekstrak Daun Psidium guajava L. untuk Mempertahankan Sifat Fisikokimia Buah Jambu Biji
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Jurnal riset kimia
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cc-by
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Abstract Corresponding Author:
Refilda
refilda@sci.unand.ac.id Synthetic chemicals are a common substance used to extend the shelf life of
postharvest fruit. But it can have a health impact. The use of edible coatings
from natural ingredients has begun to be developed. Modification of aloe
vera gel by guava leaf extract as the edible coating to increase guava fruit
shelf life has not been reported yet. Guava fruit coated with several
compositions of aloe vera gel and guava leaf extract has been carried out. Observations on physicochemical in fruit on days 0, 3, 6, 9, 12, and 15 were
evaluated. The best quality was found on guava fruit treated with the
composition of aloe vera gel: ethanol guava leaf extract: CMC: glycerol (90:
5:0.25: 0.5) on day 15. It had a weight loss of 9.88%, spoilage of 3.33%, a
decrease of the water content of 11.61%, titratable acidity of 0.5%, total
antioxidant 1.08 mg AA/g FW, total phenolic 0.51 mg GAE/g FW, and
increase of total dissolved solids 3.01o Brix. This result was significantly better
than the uncoated fruit. It can be concluded that guava leaf extract can be
used to maintain the physicochemical properties during storage. Received: December 2021
Accepted: August 2022
Published: September 2022 Received: December 2021
Accepted: August 2022
Published: September 2022 ©Refilda 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. reproduction in any medium,
provided the original author
and source are credited. Keywords: Edible coating, guava, aloe vera, physicochemical, postharvest dari 122.50 hingga 206.00 mg 100 g-1 dan 167
hingga 210 mg 100 g-1 vitamin C[2]. Teknik Edible Coating dengan menggunakan Campuran Gel Lidah Buaya dan
Ekstrak Daun Psidium guajava L. untuk Mempertahankan Sifat Fisikokimia Buah
Jambu Biji Teknik Edible Coating dengan menggunakan Campuran Gel Lidah Buaya dan
Ekstrak Daun Psidium guajava L. untuk Mempertahankan Sifat Fisikokimia Buah
Jambu Biji Teknik Edible Coating dengan menggunakan Campuran Gel Lidah Buaya dan
Ekstrak Daun Psidium guajava L. untuk Mempertahankan Sifat Fisikokimia Buah
Jambu Biji Refilda*, Riga Habib Ngestu, Emil Salim, Yefrida
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Andalas University, Padang,
West Sumatra, Indonesia Keywords: Edible coating, guava, aloe vera, physicochemical, postharvest ©Refilda 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. Pendahuluan Jambu biji (Psidium guajava L.) adalah salah satu
buah-buahan tropis dan subtropis yang banyak
dikonsumsi
oleh
masyarakat. Buah
ini
dilaporkan mengandung mineral dan senyawa
metabolit sekunder seperti karotenoid, asam
askorbat, dan polifenol[1]. Dibandingkan dengan
apel dan pir, jambu biji mengandung lebih
banyak vitamin C, dengan kandungan nutrisi
lain yaitu asam asetat berkisar antara 55.40-
122.13 mol kg-1, total gula 7.93 hingga 8.90%,
gula reduksi 5.04 hingga 5.49%, total padatan
terlarut berkisar antara 9.18 hingga 11.14%,
keasaman 0.28 hingga 0.35% dan asam askorbat Akibat sifat klimaterknya, buah jambu biji
mengalami banyak perubahan fisiologis setelah
masa panen, sehingga mempercepat proses
pematangan
dan
akibatnya
meningkatkan
kerusakan. Faktor-faktor
yang
dapat
mempercepat kerusakan buah jambu biji setelah
panen yaitu temperatur yang tinggi, tingkat
kelembaban atmosfer yang rendah, dan cedera
fisik[1]. Kualitas buah jambu biji pascapanen
dipengaruhi oleh tingkat kematangan buah
serta
metode
penyimpanan
yang
akan
berpengaruh
terhadap
rasa,
penampakan,
aroma dan nutrisi buah[3]. Buah jambu biji DOI: https://doi.org/10.25077/jrk.v13i2.501 163 Vol. 13, No. 2, September 2022 J. Ris. Kim. J. Ris. Kim. memiliki masa simpan pasca panen antara 2-7
hari, agar diperoleh masa simpan yang lebih
lama maka penanganan pascapanen yang baik
perlu ditingkatkan[4]. penambah tekstur dan agen antimikroba) dan
plasticizer (gliserin)[10]. Ada beberapa metode
yang dapat digunakan untuk pelapisan buah
dan sayur, seperti pencelupan, penuangan dan
penyemprotan[11]. Metode
pencelupan
diaplikasikan pada produk yang memiliki
permukaan tidak rata. Bahan kimia sintetik banyak digunakan untuk
meningkatkan umur simpan buah, namun
bahan ini dapat merusak lingkungan dan
konsumen[5]. Salah satu metode yang banyak
digunakan untuk penyimpanan buah pasca
panen
adalah
edible
coating. Teknik
ini
menggunakan
lapisan
tipis
pada
bahan
makanan
untuk
memperpanjang
umur
simpannya. Dalam teknologi pascapanen bahan
pelapis yang dapat dimakan sudah banyak
dikembangkan
dibandingkan
dengan
film
sintetis[6]. Gel lidah buaya menunjukkan hasil
yang
menjanjikan
dan
berpotensi
untuk
digunakan sebagai bio pengawet buah-buahan
dan sayuran[7]. Proses pencelupan dilakukan sampai larutan
coating menempel pada produk dan sisa
larutannya akan dibuang. Di sisi lain, metode
semprot akan lebih efisien karena tidak
membuang larutan sisa coating yang digunakan. Metode ini digunakan untuk produk yang
memiliki
dua
sisi
permukaan
dan
hasil
coatingnya lebih tipis dibanding teknik celup[12]. Ekstrak daun jambu biji memiliki beberapa
kandungan kimia seperti kumarin, minyak
atsiri,
ellagitannin,
flavonoid
khususnya
triterpen, quercetin, saponin, tanin, alkaloid
antrakuinon, phlobatannins, terpenoid, fenolat
dan alkaloid. Ekstrak daun jambu biji telah
dilaporkan memiliki aktivitas antibakteri yang
signifikan
terhadap
berbagai
bakteri
Staphylococcus,
spesies
Shigella,
spesies
Salmonella, spesies Bacillus, E. DOI: https://doi.org/10.25077/jrk.v13i2.501 Pendahuluan coli, spesies
Clostridium dan bakteri pembusuk makanan
seperti spesies Pseudomonas. Senyawa antijamur
tersebut terutama tanin, phlobatannin, saponin,
terpenoid, alkaloid dan polifenol. Ekstrak juga
menunjukkan sifat antioksidan yang dikaitkan
dengan polifenol yang ditemukan di daun[13]. Bahan pelapis alami yang telah banyak
dikembangkan sebagai edible coating adalah
lidah buaya. Gel lidah buaya merupakan yang
memiliki potensi antimikroba yang baik. Gel
lidah buaya mengandung sekitar 99% gel
berwarna keputihan yang terdiri dari asam
amino, sterol, glukomanan (polisakarida) dan
vitamin. Penggunaan
lidah
buaya
pada
pascapanen buah dan sayuran meningkat secara
signifikan dalam beberapa tahun terakhir. Aplikasi
lapisan
berbasis
lidah
buaya
mengurangi pengeringan dan menjaga kualitas
selama penyimpanan pascapanen[8]. Tujuan penelitian ini adalah untuk mengetahui
efektifitas dari ekstrak etanol daun jambu biji
sebagai campuran edible coating pada buah
jambu biji untuk mempertahankan kualitas
selama penyimpanan. Ekstrak daun jambu biji
ditambahkan pada edible coating gel lidah buaya
karena
mengandung
senyawa
metabolit
sekunder yang berperan sebagai anti bakteri[13]. Pada penelitian ini dilakukan teknik pencelupan
untuk
aplikasi
edible
coating,
teknik
ini
digunakan karena permukaan jambu biji tidak
rata[12]. Sifat fisikokimia buah jambu biji
dievaluasi dengan menentukan kadar air dan
penurunan berat buah menggunakan metoda
gravimetri, pembusukan buah jambu biji
melalui penilaian secara visual, total asam
menggunakan metoda titrasi, total padatan Penggunaan pelapis untuk buah dan sayuran
bukanlah konsep baru dan sebenarnya sudah
ada sejak abad ke-12 dan ke-13. Beberapa hasil
penelitian telah melaporkan bahwa gel lidah
buaya mampu menahan laju respirasi dan sifat
fisikokimia jambu biji[1]–[3], anggur[5] dan buah-
buahan segar[7] selama penyimpanan. Namun
penggunaan bahan pelapis gel lidah buaya yang
ditambahkan ekstrak etanol daun biji sebagai
edible coating pada buah jambu bij belum ada
dilaporkan. Edible coating umumnya terbuat dari satu atau
lebih dari empat jenis bahan utama: lipid, resin,
polisakarida, dan protein[9], ditambah dengan
komponen minor seperti aditif (antioksidan, 164 DOI: https://doi.org/10.25077/jrk.v13i2.501 DOI: https://doi.org/10.25077/jrk.v13i2.501 Vol. 13, No. 2, September 2022 J. Ris. Kim. J. Ris. Kim. Ariza di Korong Panggie-panggie, Nagari
Limpato Sungai Sariak, Kecamatan VII Koto,
Kabupaten Padang Pariaman, Sumatera Barat. Buah jambu biji yang diambil adalah jambu biji
yang baru dipanen dengan ukuran, berat dan
kematangannya hampir sama. terlarut menggunakan metode refraktometri,
kadar antioksidan total menggunakan metode
fenantrolin modifikasi dan kadar fenolik total
menggunakan metode Follin ciocalteu. Data
dianalisis secara statistik. terlarut menggunakan metode refraktometri,
kadar antioksidan total menggunakan metode
fenantrolin modifikasi dan kadar fenolik total
menggunakan metode Follin ciocalteu. Data
dianalisis secara statistik. Persiapan reagen Reagen-reagen
yang
digunakan
dalam
penelitian ini disiapkan seperti NaOH 0,1 N,
Etanol 50%, CMC 1%, Gliserol 20%, Asam Sitrat
10%. Edible coating disiapkan dari gel lidah buaya dan
ekstrak daun jambu biji dengan komposisi
seperti pada Tabel 1. Bahan Kimia Sampel Daun jambu biji segar yang telah
dikumpulkan dilakukan dicuci bersih dengan
air mengalir, dikering anginkan kemudian
digiling sehingga diperoleh bubuk[13]. Bubuk
daun jambu biji ditimbang sebanyak 200 g
dimaserasi dengan pelarut etanol 50% sebanyak
1000 mL selama 3 hari sambil diaduk. Filtrat
yang
diperoleh
diuapkan
pelarutnya
menggunakan rotari evaporator pada suhu 40oC
sehingga diperoleh ekstrak[14]. Bahan-bahan yang digunakan pada penelitian
adalah buah jambu biji (Psidium guajava L.),
daun lidah buaya (Aloe vera. L), daun jambu biji,
asam sitrat, asam oksalat (Merck), NaOH
(Merck), etanol 96% (Merck), carboxymethyl
cellulose (Merck), gliserol (Intraco), aluminium
foil, kertas saring whatman, dan akuades. Peralatan Peralatan yang digunakan berupa alat-alat gelas,
neraca analitik (KERN), oven (Memmert),
vacuum
rotary
evaporator,
blender,
refraktometer, cawan porselen, dan desikator. Asam sitrat 10% digunakan untuk merendam
daun Lidah buaya yang telah dicuci dengan air,
dengan waktu perendaman selama 30 menit,
bahagian kulit lidah buaya dibuang dan
bahagian
gelnya
diblender. Dilakukan
penyaringan sehingga diperoleh ekstrak lidah
buaya yang jernih. Preparasi permukaan buah yang akan dilapisi Daun jambu biji diambil secara acak dengan
kondisi muda dan segar dari kota Pariaman. Lidah buaya diperoleh dari perkebunan di
Kampung Apar, Kabupaten Padang Pariaman. Sampel lidah buaya yang diambil adalah lidah
buaya yang sudah dewasa berwarna hijau tua. Buah jambu biji diperoleh dari perkebunan Buah yang digunakan terlebih dahulu disortir
dan dipilih yang kondisinya baik, yaitu
berwarna
hijau
terang
kemudian,
dicuci
menggunakan air mengalir dan dikeringkan. Buah jambu biji yang digunakan sebanyak 180
buah (untuk 3 pengulangan, 10 perlakuan dan 6
buah untuk tiap perlakuan). 165
DOI: https://doi.org/10.25077/jrk.v13i2.501
Tabel 1. Komposisi Pelapis Buah Jambu Biji
Kode
Pelapis
Lidah Buaya (mL)
Ekstrak Daun Jambu Bij
(mL)
CMC 1%
(mL)
Gliserol 20%
(mL)
Air
(mL)
T1
0
0
0
0
2000
T2
950
0
50
50
950
T3
1900
0
50
50
0
T4
900
100
50
50
900
T5
1800
100
50
50
0 Tabel 1. Komposisi Pelapis Buah Jambu Biji Vol. 13, No. 2, September 2022 J. Ris. Kim. Pelapisan Buah Jambu Biji dengan Berbagai
Komposisi
Bahan
Pelapis
dan
Lama
Penyimpanan Penentuan Total Padatan Terlarut Buah Jambu Biji
Nilai TPT dipengaruhi oleh tingkat kematangan
buah, semakin matang buah nilai TPT nya
semakin tinggi, ini disebabkan oleh hidrolisis
karbohidrat menjadi senyawa glukosa dan
fruktosa. Total padatan terlarut ditentukan
dengan alat refraktometer dan dinyatakan
sebagai persentase (°Brix)[17]. Sebelum dilakukan
pengukuran, alat refraktometer distandarisasi
dengan air suling dan disesuaikan dengan
pembacaan 0°Brix. Daging buah jambu biji
dihaluskan menggunakan blender. Kemudian,
diambil sedikit daging buah yang telah
dihaluskan dan ditempatkan pada prisma kaca
refraktometer
dan
dilakukan
pembacaan
rekaman[18]. Buah jambu biji dicelupkan ke dalam larutan
yang komposisinya seperti Tabel 1. selama 15
menit, kemudian dikering anginkan selama 30
menit. Buah yang sudah dilapisi disimpan
dalam ruangan yang bersuhu lebih kurang 280C. Pengujian
sifat
fisikokimia
buah
setelah
perlakuan dilakukan pada hari ke 0, 3, 6, 9, 12,
dan 15 setelah pelapisan. Penentuan Sifat Fisikokimia Buah Jambu Biji Penentuan Penurunan Berat Buah
Buah jambu biji dari masing-masing perlakuan
(T1, T2, T3, T4, dan T5) ditimbang dengan
menggunakan timbangan digital pada hari
pertama
dan
pada
akhir
setiap
interval
penyimpanan yaitu pada penyimpanan 0, 3, 6, 9,
12 dan 15 hari. Perbedaan antara bobot buah
awal dan akhir (penyimpanan 15 hari) dianggap
sebagai penurunan berat total[15]. Penentuan Total Asam Tertitrasi (TAT) Buah Jambu
Biji Total Asam Tertitrasi
ditentukan dengan
metoda titrasi sesuai SNI[19]. Pembuatan Ekstrak Buah Jambu Biji
Daging buah yang diberi perlakuan ±10 g
diblender dengan menambahkan akuades. Ekstrak dipindahkan ke dalam labu ukur 100
mL, dilakukan penambahan akuades sampai
tanda batas dan disaring menggunakan kertas
Whatman. Ekstrak digunakan untuk penentuan
total asam tertitrasi dan antioksidan total. Penentuan Kadar Air Buah Jambu Biji J
j
Metode yang digunakan untuk penentuan
kadar air buah setelah perlakuan adalah metode
gravimetri[16]. Pemanasan buah yang diuji
dilakukan di dalam oven bersuhu 105°C, berat
buah sebelum dipanaskan ditimbang dan berat
buah setelah dipanaskan ditimbang sampai
diperoleh berat yang konstan. Penentuan kadar
air dilakukan 3 kali ulangan. Penentuan Total Asam Tertitrasi Buah Jambu
Biji Standarisasi NaOH dilakukan terlebih dahulu
sebelum digunakan untuk mentitrasi asam
dalam sampel. Sebanyak 25 mL ekstrak jambu
biji dititrasi dengan NaOH 0.1004 N dengan
menggunakan indikator fenolftalein. Penentuan Pembusukan Buah Jambu Biji Penentuan Pembusukan Buah Jambu Biji j
Tingkat
pembusukan
dievaluasi
dengan
mengamati gejala fisik pada permukaan buah
secara visual dengan menggunakan skala: 0
= tidak ada tanda-tanda pembusukan,
1
= 1–10% pembusukan,
2
= 11–25% pembusukan,
3
= 26–40% pembusukan,
4
= 40–50% pembusukan,
5
= 50% pembusukan. = tidak ada tanda-tanda pembusukan, = 1–10% pembusukan, = 1–10% pembusukan, Penentuan Antioksidan Total Buah Jambu Biji
Kadar antioksidan total buah jambu biji
ditentukan
dengan
metode
fenantrolin
modifikasi menggunakan spektrofotometer[20]. Dipipet masing-masing sebanyak 1 mL ekstrak
buah jambu biji dan dimasukkan ke dalam botol
vial, kemudian ditambahkan 2 mL akuades dan
1 mL FeCl3.6H2O 0,1 % serta 1 mL 1.10 orto
fenantrolin 0.1 %. Selanjutnya larutan di
inkubasi selama 20 menit dan diukur absorban
menggunakan spektrofotometer UV-Vis pada
panjang gelombang 510 nm. 2
= 11–25% pembusukan, = 40–50% pembusukan, = 50% pembusukan. Rumus pembusukan (%) = 100 × AB/CD, Analisis Statistik Analisis Statistik Analisis varian (ANOVA) satu arah digunakan
untuk pengolahan data, aapabila diperoleh nilai
P value <0.05 maka dilanjutkan dengan Uji
Berjarak Duncan (Duncan Multiple Range Test)
menggunakan software static SPSS 23.0 for
Windows. Dari semua perlakuan yang dilakukan, persen
penurunan berat yang nilainya paling besar
adalah T1 dimana pada hari ke-15 yaitu hingga
30.48%, Jambu biji perlakuan T1 sebagai buah
kontrol (jambu biji yang tidak diberi lapisan)
porinya tidak tertutup sehingga proses respirasi
dan transpirasi berlangsung dengan sempurna. Penurunan berat T2 sebesar 26.22%, T3 sebesar
16.23%, T4 sebesar 13.9% dan yang paling
rendah nilai penurunan beratnya adalah T5
yaitu 9.88%. Hasil dan Diskusi Sifat fisikokimia yang dievaluasi untuk melihat
pengaruh pelapisan buah adalah penurunan
berat buah, kadar air, pembusukan buah, total
padatan
terlarut,
total
asam
tertitrasi,
antioksidan dan fenolik total. Penentuan Fenolik Total Buah Jambu Biji karena buah jambu biji tetap melakukan proses
respirasi. Untuk menghasilkan energi dalam
buah oksigen diserap bahan-bahan organik
dibakar menggunakan oksigen diserap dan
diikuti oleh pengeluaran sisa pembakaran
berupa gas karbondioksida dan air melalui pori-
pori permukaan buah. Gambar 1 menunjukkan
bahwa penurunan berat yang dialami oleh buah
jambu biji meningkat selama penyimpanan
mulai dari hari ke-3 hingga hari ke-15. Jambu biji
perlakuan T1 sebagai buah kontrol (jambu biji
yang tidak diberi lapisan) memiliki nilai
penurunan
berat
lebih
besar
dari
pada
perlakuan lainnya dan nilai penurunan berat
untuk
perlakuan
T5
paling
rendah
dibandingkan dengan empat perlakuan lainnya. Hal ini disebabkan karena pori-pori dari
permukaan buah ditutupi oleh pelapis yang
dapat mengurangi terjadinya proses respirasi. Kadar Fenolik Total Buah Jambu Biji ditentukan
dengan metoda Follin-Ciocalteu menggunakan
Spektrofotmeter[21]. Ekstrak sampel dipipet
sebanyak 0.4 mL kemudian ditambahkan 0.4 mL
reagen
Folin-Ciocalteau. Selanjutnya
ditambahkan
4
mL
Na2CO3
7%
lalu
dihomogenkan
dengan
cara
pengocokan. Akuades ditambahkan hingga volume 10 mL
lalu diinkubasi selama 90 menit. Selanjutnya
nilai absorban diukur dengan spektrofotometer
UV-Vis pada panjang gelombang 750 nm. Rumus pembusukan (%) = 100 × AB/CD, Rumus pembusukan (%) = 100 × AB/CD, dimana A adalah total nilai tingkat kerusakan, B
adalah jumlah buah pada tingkat ini, C adalah
jumlah buah pada perlakuan dan D adalah
tingkat kerusakan tertinggi[15]. DOI: https://doi.org/10.25077/jrk.v13i2.501 166 Vol. 13, No. 2, September 2022 J. Ris. Kim. J. Ris. Kim. Penentuan Fenolik Total Buah Jambu Biji Penentuan Fenolik Total Buah Jambu Biji Penurunan Berat Buah Jambu Biji Berat buah jambu biji turun selama masa
penyimpanan, menyusutnya berat buah terjadi Gambar 1. Pengaruh komposisi pelapis dan lama penyimpanan terhadap penurunan berat buah jambu
biji. (T1, T2, T3, T4, dan T5) adalah pelapis buah jambu biji dengan komposisi seperti pada Tabel 1. 0
3
6
9
12
15
0
5
10
15
20
25
30
35
Penurunan Berat Buah (%)
Lama Penyimpanan (Hari)
T1
T2
T3
T4
T5 Gambar 1. Pengaruh komposisi pelapis dan lama penyimpanan terhadap penurunan berat buah jambu
biji. (T1, T2, T3, T4, dan T5) adalah pelapis buah jambu biji dengan komposisi seperti pada Tabel 1. DOI: https://doi.org/10.25077/jrk.v13i2.501 167 Vol. 13, No. 2, September 2022 J. Ris. Kim. Tabel 2. Analisis ANOVA penurunan berat buah jambu biji selama penyimpanan
Sumber Varian
Jumlah Kuadrat
df
Rata-Rata Kuadrat
F
Signifikasi
Antar Grup
392.249
4
98.062
1.554
0.217
Dalam Grup
1577.599
25
63.104
Total
1969.848
29
Gambar 2. Pengaruh suhu reaksi terhadap bilangan oksidan. 0
3
6
9
12
15
30
40
50
60
70
Kadar Air (%)
Lama Penyimpanan (Hari)
T1
T2
T3
T4
T5 Tabel 2. Analisis ANOVA penurunan berat buah jambu biji selama penyimpanan Gambar 2. Pengaruh suhu reaksi terhadap bilangan oksidan. Uji statistik nilai penurunan berat buah jambu
biji dapat dilihat pada Tabel 2. Perbandingan nilai penurunan berat dari setiap
perlakuan
ini
membuktikan
penambahan
ekstrak daun jambu biji pada bahan pelapis
dapat mejaga kualitas jambu biji dari kehilangan
berat akibat proses respirasi dan transpirasi. Semakin besar konsentrasi ekstrak daun jambu
biji yang ditambahkan pada pelapisan maka
akan semakin kecil penurunan berat buah jambu
biji selama penyimpanan. Nilai ini lebih kecil
dibandingkan dengan jambu biji yang dilapisi
dengan campuran 85% ekstrak A. vera, 10%
ekstrak A. indica mengalami penurunan berat
yang paling rendah (11.19%) selama 15 hari
penyimpanan[22]. Berdasarkan hasil analisis ANOVA penurunan
berat buah jambu biji selama penyimpanan
memilliki nilai siginifikansi 0.217, nilai P>0.05
berarti rata-rata nilai penurunan berat buah
jambu biji setiap perlakuan tidak berbeda
nyata[24],[25]. Pelapisan sayur dan buah dengan
gel lidah buaya menunjukkan penurunan berat
buah lebih rendah dibandingkan dengan buah
tanpa dilapisi. Kadar Air Buah Jambu Biji Gambar 2. memperlihatkan penurunan kadar
air dalam buah selama perlakuan. Buah jambu biji merupakan salah satu jenis
buah klimaterik. Buah klimaterik adalah buah
yang akan meningkatkan laju respirasinya
seiring dengan meningkatnya kematangan buah
tersebut. Karena sifat klimateriknya jambu biji
juga akan lebih cepat kehilangan berat buahnya,
terlebih
lagi
ketika
mencapai
puncak
klimateriknya[23]. Penurunan berat buah selama
penyimpanan
sangat
dipengaruhi
oleh
penguapan air, selain itu kehilangan air pada
buah dapat menyebabkan kerusakan dan
menurunkan mutu buah[18]. Kadar air dari buah tanpa dan dengan
perlakuan
cenderung
menurun
selama
penyimpanan. Buah
tanpa
perlakuan
T1
memiliki penurunan kadar air yang paling besar
dibandingkan dengan perlakuan lainnya, hal ini
terjadi karena pada T1 tidak ada lapisan yang
membatasi kontak antara buah dan lingkungan
sehingga proses pematangan (respirasi dan
transpirasi)
terus
berlangsung
dan
mempengaruhi
kadar
air
pada
buah. Selanjutnya untuk T2 dan T3 juga mengalami 168 DOI: https://doi.org/10.25077/jrk.v13i2.501 DOI: https://doi.org/10.25077/jrk.v13i2.501 Vol. 13, No. 2, September 2022 J. Ris. Kim. penurunan
kadar
air,
dimana
T3
dapat
mempertahankan
kadar
air
lebih
besar
dibandingkan T2 karena pada T3 lapisan yang
digunakan
mengandung
konsentrasi
lidah
buaya yang lebih tinggi. T4 (54.77%) memiliki
kadar air yang lebih besar dari kadar air T1
(28.42%), T2 (35.51%), T3 (52.45%) selama
penyimpanan karena pada kedua perlakuan ini
dilakukan penambahan ekstrak dari daun
jambu biji, dan kadar air buah jambu biji pada
T5 (56.45 %) lebih tinggi dibandingkan dengan
perlakuan lainnya. Uji statistik nilai kadar air
buah jambu biji dapat dilihat pada Tabel 3. pelapisan dengan gel lidah buaya dapat
mempertahankan kadar air buah papaya setelah
masa panen. Berkurangnya kadar air dalam
buah disebabkan karena proses pematangan
dalam buah tetap berlangsung, dimana dalam
proses pematangan ini terjadi proses transpirasi
dan respirasi. Dalam proses metabolik ini buah
akan kehilangan atau mengalami penurunan
kadar air dan juga senyawa-senyawa organik
lainnya[26]. Pembusukan Buah Jambu Biji Pembusukan pada buah semakin seiring
dengan lamanya waktu penyimpanan. Bahan
pelapis yang digunakan dapat memperlambat
terjadinya pembusukan pada buah jambu biji
selama periode penyimpanan. Berdasarkan Tabel 3 nilai signifikan kadar air
buah jambu biji dengan semua perlakuan yaitu
signifikan 0.766, yang mana berarti data yang
didapat tidak berbeda nyata. penurunan berat
dan kadar air dapat dipertahankan karena efek
pelapisan dengan gel lidah buaya sebagai
penghalang semi permeable terhadap oksigen,
karbon dioksida dan kelembaban, dengan
demikian, mengurangi respirasi, kehilangan air,
dan reaksi oksidasi. hal ini sesuai dengan
penelitian
sebelumnya
dimana
dengan Gambar 3 menunjukkan bahwa buah jambu biji
T5 (3.33 %) lebih baik dalam mengurangi
pembusukan karena dapat memperlambat kerja
dari proses respirasi akibat pelapisan dengan gel
lidah buaya dan penambahan ekstrak daun
jambu biji dimana buah masih berwarna hijau
kekuningan dan memiliki sedikit noda hitam, Tabel 3. Analisis ANOVA kadar air buah jambu biji selama penyimpanan
Sumber Varian
Jumlah Kuadrat
df
Rata-Rata Kuadrat
F
Signifikasi
Antar Grup
314.498
4
78.625
0.458
0.766
Dalam Grup
4292.730
25
171.709
Total
4607.229
29
Gambar 3. Pengaruh pelapisan dan lama penyimpanan terhadap pembusukan buah jambu biji. 0
3
6
9
12
15
0
3
6
9
12
15
Persen Pembusukan (%)
Lama Penyimpanan (Hari)
T1
T2
T3
T4
T5 Tabel 3. Analisis ANOVA kadar air buah jambu biji selama penyimpanan ambar 3. Pengaruh pelapisan dan lama penyimpanan terhadap pembusukan buah jambu biji. Gambar 3. Pengaruh pelapisan dan lama penyimpanan terhadap pembusukan buah jambu biji. DOI: https://doi.org/10.25077/jrk.v13i2.501 169 Vol. 13, No. 2, September 2022 J. Ris. Kim. Tabel 4. Analisis ANOVA pembusukan buah jambu biji selama penyimpanan
Sumber Varian
Jumlah Kuadrat
df
Rata-Rata Kuadrat
F
Signifikasi
Antar Grup
186.008
4
46.502
3.965
0.013
Dalam Grup
293.210
25
11.728
Total
479.218
29 Tabel 4. Analisis ANOVA pembusukan buah jambu biji selama penyimpanan
Sumber Varian
Jumlah Kuadrat
df
Rata-Rata Kuadrat
F
Signifikasi
Antar Grup
186.008
4
46.502
3.965
0.013
Dalam Grup
293.210
25
11.728
Total
479.218
29
Tabel 5. Uji lanjut duncan pembusukan buah jambu biji pada setiap perlakuan
Perlakuan
Rata-Rata Pembusukan
T1
6.67c
T2
5.56bc
T3
1.85ab
T4
1.29ab
T5
0.37a
Keterangan: Huruf superscript yang berbeda menunjukkan bahwa terdapat perbedaan yang signifikan
antar perlakuan (p<0.05) abel 4. Analisis ANOVA pembusukan buah jambu biji selama penyimpanan Tabel 5. Uji lanjut duncan pembusukan buah jambu biji pada setiap perlakuan Tabel 5. Pembusukan Buah Jambu Biji Uji lanjut duncan pembusukan buah jambu biji pada setiap perlakuan
Perlakuan
Rata-Rata Pembusukan
T1
6.67c
T2
5.56bc
T3
1.85ab
T4
1.29ab
T5
0.37a
Keterangan: Huruf superscript yang berbeda menunjukkan bahwa terdapat perbedaan yang signifikan
antar perlakuan (p<0.05) Keterangan: Huruf superscript yang berbeda menunjukkan bahwa terdapat perbedaan yang signifikan
antar perlakuan (p<0.05) dibandingkan dengan T3 (5.56 %) hanya
menggunakan
gel
lidah
buaya
tanpa
penambahan ekstrak dengan kondisi buah
sudah ada noda hitam, berwarna kuning dan
sedikit lembek dan T1 (14.44%) tanpa pelapisan
dimana buah jambu biji sudah mengalami
pembusukan sepenuhnya, sudah banyak noda
hitam warna kuning kecoklatan lembek dan
perubahan bau buah menjadi menyengat. pelapisan dengan gel lidah buaya dan juga
penambahan ekstrak daun jambu biji dapat
mengurangi pembusukan pada buah papaya,
karena adanya sifat antibakteri pada lidah
buaya dan juga pada esktrak daun jambu biji. Pembusukan terjadi karena adanya proses
respirasi. Oksigen akan membakar bahan
organik yang ada dalam buah. Makin banyak
oksigen yang digunakan maka makin aktif
respirasinya[27]. pelapisan dengan gel lidah buaya dan juga
penambahan ekstrak daun jambu biji dapat
mengurangi pembusukan pada buah papaya,
karena adanya sifat antibakteri pada lidah
buaya dan juga pada esktrak daun jambu biji. Pembusukan terjadi karena adanya proses
respirasi. Oksigen akan membakar bahan
organik yang ada dalam buah. Makin banyak
oksigen yang digunakan maka makin aktif
respirasinya[27]. Setelah dilakukan uji Anova pada Tabel 4 untuk
efek lapisan yang dapat dimakan lidah buaya
dan penambahan ekstrak daun jambu biji
terhadap penundaan pembusukan dari buah
jambu biji didapatkan hasil signifikan 0.013
yang berarti bahwa ada perbedaan nyata dari
setiap
perlakuan
terhadap
penundaan
pembusukan dari buah jambu biji. Karena nilai
signifikan lebih kecil dari 0.05 maka dilakukan
uji
duncan
untuk
mengetahui
letak
perbedaannya seperti pada Tabel 5. Total Padatan Terlarut Buah Jambu Biji Nilai TPT dipengaruhi oleh kematangan buah. Buah yang matang memiliki nilai TPT yang
tinggi. Nilai ini akan meningkat selama proses
penyimpanan,
karena
terjadinya
hidrolisis
karbohidrat menjadi senyawa glukosa dan
fruktosa. Gambar 4 menunjukkan bahwa bahwa TPT
setiap perlakuan meningkat selama masa
penyimpanan
selama
15
hari. Semakin
tingginya total padatan terlarut menandakan
bahwa sedang dalam proses pematangan dan
akan terus meningkat hingga terjadi kebusukan. Tabel 5 menunjukkan bahwa nilai pembusukan
pada perlakuan T5 berbeda secara signifikan
dengan perlakuan T1. Hasil ini sesuai dengan
penelitian sebelumnya, dimana dengan adanya DOI: https://doi.org/10.25077/jrk.v13i2.501 170 Vol. 13, No. 2, September 2022 J. Ris. Kim. Gambar 4. Pengaruh pelapisan dan lama penyimpanan terhadap total padatan terlarut buah jambu biji
(0Brix). 0
3
6
9
12
15
5
6
7
8
9
10
11
12
13
Total Padatan Terlarut (obrix)
Lama Penyimpanan (Hari)
T1
T2
T3
T4
T5 Gambar 4. Pengaruh pelapisan dan lama penyimpanan terhadap total padatan terlarut buah jambu biji
(0Brix). Tabel 6. Analisis ANOVA TPT buah jambu biji selama penyimpanan
Sumber Varian
Jumlah Kuadrat
df
Rata-Rata Kuadrat
F
Signifikasi
Antar Grup
20.533
4
5.133
1.558
0.216
Dalam Grup
82.394
25
3.296
Total
102.927
29 Tabel 6. Analisis ANOVA TPT buah jambu biji selama penyimpanan Buah kontrol T1 (12.33 obrix) memiliki nilai
kadar total padatan terlarut paling tinggi, dan
T5 (8.33 obrix) dimana buah dilapisi gel lidah
buaya dengan penambahan ekstrak daun jambu
biji memiliki kadar total padatan terlarut paling
kecil, dibandingkan dengan T3 (9.67 obrix) yang
dilapisi menggunakan gel lidah buaya saja
tanpa penambahan ekstrak daun jambu biji. Total Asam Tertitrasi Buah Jambu Biji Total Asam Tertitrasi Buah Jambu Biji Total Asam Tertitrasi Buah Jambu Biji Total asam tertitrasi menunjukkan penurunan
pada buah jambu biji selama penyimpanan
seperti yang terlihat pada Gambar 5. Buah
kontrol
T1
memperlihatkan
tanda-tanda
penurunan
kadar
TAT
pada
hari
ke-3
penyimpanan, dan merupakan buah jambu biji
yang kadar TAT-nya lebih kecil dibandingkan
dengan perlakuan lainnya pada hari ke 15 yaitu
0.4 %, diikuti dengan T2 (0.7%), T3 (1.5%), T4
(1.83%) dan buah jambu biji pada T5 (2.07%)
memiliki kadar TAT lebih tinggi dibandingkan
perlakuan lainnya. Disini dapat dilihat bahwa
buah jambu biji yang dilapisi dapat menekan
dan mengurangi oksidasi sehingga kadar TAT
yang lebih tinggi dapat dipertahankan selama
masa penyimpanan. Berdasarkan Tabel 6 nilai signifikan TPT buah
jambu biji dengan semua perlakuan yaitu 0.216
dari sini dapat kita lihat bahwa data TPT yang
didapat dari setiap perlakuan selama 15 hari
tidak berbeda nyata. Kandungan TPT dalam
buah berkaitan dengan adanya senyawa seperti
tanin atau getah, pati, glukosa, fruktosa dan
sukrosa terkandung di dalamnya. Kandungan
gula, fruktosa dan sukrosa akan mempengaruhi
kemanisan dari buah, ini sangat dipengaruhi
oleh kematangan dari buah saat dipanen[28]. Tingkat kematangan buah dapat diidentifikasi
dengan nilai TPT. Nilai TPT yang tinggi
menunjukkan bahwa buah semakin matang[29]. Dari uji statisik yang telah dilakukan didapat
hasil bahwa pelapisan dengan gel lidah buaya
dan penambahan ekstrak ini memiliki nilai
signifikan 0.002 (Tabel 7), berarti data total asam
tertitrasi setiap perlakuan berbeda nyata. DOI: https://doi.org/10.25077/jrk.v13i2.501 171 Vol. 13, No. 2, September 2022 J. Ris. Kim. Gambar 5. Kurva pengaruh pelapisan dan lama penyimpanan terhadap total asam tertitrasi buah
jambu biji. 0
3
6
9
12
15
0.5
1.0
1.5
2.0
2.5
Total Asam Tertitrasi (%)
Lama Penyimpanan (Hari)
T1
T2
T3
T4
T5 Gambar 5. Kurva pengaruh pelapisan dan lama penyimpanan terhadap total asam tertitrasi buah
jambu biji. Tabel 7. Analisis ANOVA TAT buah jambu biji selama penyimpanan
Sumber Varian
Jumlah Kuadrat
df
Rata-Rata Kuadrat
F
Signifikasi
Antar Grup
5.134
4
1.284
5.539
0.002
Dalam Grup
5.793
25
0.232
Total
10.927
29
Tabel 8. Uji lanjut duncan TAT buah jambu biji pada setiap perlakuan
Perlakuan
Rata-Rata TAT
T1
1.43a
T2
1.58a
T3
1.93ab
T4
2.21b
T5
2.34b
Keterangan: Huruf superscript yang berbeda menunjukkan bahwa terdapat perbedaan yang signifikan
antar perlakuan (p<0.05) Tabel 7. Analisis ANOVA TAT buah jambu biji selama penyimpanan
Sumber Varian
Jumlah Kuadrat
df
Rata-Rata Kuadrat
F
Signifikasi
Antar Grup
5.134
4
1.284
5.539
0.002
Dalam Grup
5.793
25
0.232
Total
10.927
29 Tabel 7. Total Asam Tertitrasi Buah Jambu Biji Analisis ANOVA TAT buah jambu biji selama penyimpanan Tabel 8. Uji lanjut duncan TAT buah jambu biji pada setiap perlakuan
Perlakuan
Rata-Rata TAT
T1
1.43a
T2
1.58a
T3
1.93ab
T4
2.21b
T5
2.34b
Keterangan: Huruf superscript yang berbeda menunjukkan bahwa terdapat perbedaan yang signifikan
antar perlakuan (p<0.05) Tabel 8. Uji lanjut duncan TAT buah jambu biji pada setiap perlakuan karena nilai signifikansi lebih kecil dari 0.05
selanjutnya
dilakukan
uji
Duncan
untuk
mengetahui letak perbedaannya seperti pada
Tabel 8. buah oleh proses respirasi dan juga oleh
mikroba. Zat dan gizi dalam bahan pangan
dirombak
oleh
mikroorganisme
untuk
pertumbuhan dan perkembangannya. TAT
mempengaruhi rasa dan aroma yang lebih baik
dibandingkan
dengan
pH[30]. Nilai
TAT
merupakan total asam yang terdisosiasi dan
tidak
terdisosiasi,
sedangkan
pH
hanya
mengukur
total
asam
dalam
kondisi
terdisosiasi[21]. buah oleh proses respirasi dan juga oleh
mikroba. Zat dan gizi dalam bahan pangan
dirombak
oleh
mikroorganisme
untuk
pertumbuhan dan perkembangannya. TAT
mempengaruhi rasa dan aroma yang lebih baik
dibandingkan
dengan
pH[30]. Nilai
TAT
merupakan total asam yang terdisosiasi dan
tidak
terdisosiasi,
sedangkan
pH
hanya
mengukur
total
asam
dalam
kondisi
terdisosiasi[21]. Berdasarkan uji lanjut perlakuan pada Tabel 8
menunjukkan nilai rata-rata TAT perlakuan T5
dan T4 berbeda secara signifikan dengan
perlakuan lainnya. Naiknya nilai TAT karena
terjadinya proses penguraian molekul asam-
asam organik menjadi air dan CO2 pada buah
dan penggunaan asam-asam organik di dalam DOI: https://doi.org/10.25077/jrk.v13i2.501 172 Vol. 13, No. 2, September 2022 J. Ris. Kim. J. Ris. Kim. Kadar Antioksidan Total Buah Jambu Biji pelindung pada buah sehingga buah dapat
melakukan kontak langsung dengan O2 dan
faktor-faktor lainnya dari lingkungan yang
dapat menyebabkan teroksidasinya antioksidan
pada buah. T2 dan T3 lebih baik dalam
mengurangi oksidasi antioksidan sehingga
dapat mempertahankan kadar antioksidan yang
lebih tinggi dibandingkan T1, namun dari kedua
perlakuan ini kadar antioksidan T3 lebih tinggi
dari T2 karena konsentrasi lidah buaya yang
digunakan lebih besar. T4 dan T5 memiliki
kadar
antioksidan
yang
lebih
tinggi
dibandingkan dengan perlakuan sebelumnya,
karena buah dilapisi dengan gel lidah buaya dan
dilakukan penambahan ekstrak daun jambu biji. Kadar antioksidan total buah jambu biji
mengalami
penurunan
selama
masa
penyimpanan. Terjadinya penurunan kadar
antioksidan disebabkan karena selama masa
penyimpanan antioksidan khususnya vitamin C
mengalami oksidasi akibat adanya kontak
langsung dengan O2 dari lingkungan. Gambar 6
menunjukkan bahwa buah kontrol mengalami
penurunan kadar antioksidan total yang besar
dibandingkan dengan perlakuan lainnya setelah
penyimpanan pasca panen selama 15 hari. Kadar
Antioksidan
dari
buah
yang
diperlakukan dengan pelapis T1 yaitu 0.93 mg
AA/g FW, diikuti dengan T2 (1.03 mg AA/g FW),
T3 (1.38 mg AA/g FW), T4 (1.48 mg AA/g FW)
dan T5 (1.73 mg AA/g FW). Buah jambu biji
yang dilapisi dengan T5 memiliki kadar
antioksidan paling tinggi dibandingkan dengan
perlakuan lainnya. Nilai signifikan yang didapatkan dari uji
statistik Analysis of variance (Anova) pada Tabel
9 adalah 0.696, berarti tidak terjadi perbedaan
yang nyata dari kadar antioksidan total pada
buah jambu biji untuk semua perlakuan. Buah kontrol tidak dapat mempertahankan
kadar antioksidan karena tidak adanya lapisan Gambar 6. Kurva Pengaruh Komposisi Bahan dan Lama Penyimpanan terhadap Kadar Antioksidan
Total Buah Jambu Biji. 0
3
6
9
12
15
1
2
3
Kadar Antioksidan Total (mg AA/g FW)
Lama Penyimpanan (Hari)
T1
T2
T3
T4
T5 0
3
6
9
12
15
1
2
3
Kadar Antioksidan Total (mg AA/g FW)
Lama Penyimpanan (Hari)
T
T
T
T
T Gambar 6. Kurva Pengaruh Komposisi Bahan dan Lama Penyimpanan terhadap Kadar Antioksidan
Total Buah Jambu Biji. 173
DOI: https://doi.org/10.25077/jrk.v13i2.501
Tabel 9. Uji statistik Pengaruh Komposisi Bahan Pelapis dan Lama Penyimpanan terhadap kadar
antioksidan total buah jambu biji
Sumber Varian
Jumlah Kuadrat
df
Rata-Rata Kuadrat
F
Signifikasi
Antar Grup
0.770
4
0.192
0.556
0.696
Dalam Grup
8.647
25
0.346
Total
9.417
29 . Uji statistik Pengaruh Komposisi Bahan Pelapis dan Lama Penyimpanan terhadap kadar
dan total buah jambu biji DOI: https://doi.org/10.25077/jrk.v13i2.501 173 Vol. 13, No. 2, September 2022 J. Ris. Kim. Gambar 7. DOI: https://doi.org/10.25077/jrk.v13i2.501 Kadar Antioksidan Total Buah Jambu Biji Kurva Pengaruh Komposisi Bahan Pelapis dan Lama Penyimpanan terhadap Kadar Fenolik
Total Buah Jambu Biji. 0
3
6
9
12
15
0.5
1.0
1.5
2.0
Kadar Fenolik Total (mg GAE/g FW)
Lama Penyimpanan (Hari)
T1
T2
T3
T4
T5 Gambar 7. Kurva Pengaruh Komposisi Bahan Pelapis dan Lama Penyimpanan terhadap Kadar Fenolik
Total Buah Jambu Biji. Tabel 10. Uji statistik Pengaruh Komposisi Bahan Pelapis dan Lama Penyimpanan terhadap kadar
fenolik total buah jambu biji
Sumber Varian
Jumlah Kuadrat
df
Rata-Rata Kuadrat
F
Signifikasi
Antar Grup
0.428
4
0.107
0.867
0.481
Dalam Grup
2.982
25
0.119
Total
3.410
29 l 10. Uji statistik Pengaruh Komposisi Bahan Pelapis dan Lama Penyimpanan terhadap kada
ik total buah jambu biji statistik Pengaruh Komposisi Bahan Pelapis dan Lama Penyimpanan terhadap kadar
ah jambu biji total yang lebih tinggi selama penyimpanan
karena dengan pelapisan dapat mengurangi
kontak dengan oksigen, sehingga oksidasi
senyawa fenolat dalam buah dapat dikurangi,
juga lebih tinggi jika dibandingkan dengan
kadar fenolik total pada T3 (0.85 mg GAE/g FW)
yang hanya menggunakan gel lidah buaya tanpa
penambahan ekstrak daun jambu biji. Kesimpulan 4. Krochta, J. M., Baldwin E.A., and Nisperos-
Carriedo, M. O., Edible Coatings and Films to
Improve Food Quality. CRC Press, 2011,
(2002). Berdasarkan
hasil
yang
diperoleh
dari
pengujian sifat fisikokimia buah jambu biji yang
dilapis
dengan
gel
lidah
buaya
yang
dimodifikasi
dengan
penambahan
ekstrak
etanol daun jambu biji dapat memperpanjang
masa simpan sampai 15 hari. Buah Jambu biji
yang dilapisi dengan bahan pelapis T5 dengan
komposisi gel lidah buaya:ekstrak daun jambu
biji:gliswrol
dan
CMC
(1800:100:50:50)
memberikan hasil yang optimal terhadap
kualitas
seperti
susut
bobot,
kadar
air,
pembusukan, TAT, TPT, antioksidan dan
fenolik total dibandingkan dengan buah yang
dilapisi dengan gel lidah buaya saja dan buah
tanpa dilapisi. Dari hasil uji statistik perlakuan
T5 ini memberikan nilai rata-rata yang berbeda
nyata
dengan
perlakuan
lainnya. Dapat
disimpulkan bahwa ekstrak etanol daun jambu
biji efektif digunakan dalam penanganan pasca
panen buah jambu biji. 5. Valverde, J. M., Valero, D., Martínez-
Romero, D., Guillén, F., Castillo, S. &
Serrano, M., Novel edible coating based on
Aloe vera gel to maintain table grape
quality and safety. J. Agric. Food Chem.,
53(20): 7807–7813 (2005). 6. Misir, J., H. Brishti, F. & M. Hoque, M., Aloe
vera gel as a Novel Edible Coating for Fresh
Fruits: A Review. Am. J. Food Sci. Technol.,
2(3): 93–97 (2014). 7. Shabir, R., Riaz, A., Shah, S. M. & Sohail, A.,
Aloe vera gel coating along with calcium
chloride treatment enhance guava (Psidium
guajava L.) fruit quality during storage. Pure Appl. Biol., 10(3): 549–565 (2021). 8. Anjum, M. A., Akram, H., Zaidi, M. & Ali,
S., Effect of gum arabic and Aloe vera gel
based edible coatings in combination with
plant extracts on postharvest quality and
storability of ‘Gola’ guava fruits. Sci. Hortic. (Amsterdam)., 271(May): 109506 (2020). Kadar Fenolik Total Buah Jambu Biji Kadar Fenolik Total Buah Jambu Biji Kadar fenolik total semua jambu biji pada setiap
perlakuan mengalami penurunan selama masa
penyimpanan. Menurunnya kadar fenolik total
dapat
disebabkan
oleh
terjadinya
proses
oksidasi akibat adanya reaksi dengan O2 dari
lingkungan[15]. T1 (buah kontrol) memiliki kadar
fenolik yang lebih kecil dibandingkan dengan
perlakuan lainnya dan T5 memiliki kadar
fenolik total yang paling besar seperti pada
Gambar 7. Setelah dilakukan uji statistik Analysis of variance
(Anova) didapatkan hasil signifikasi 0.481 yang
berarti perbedaan dari setiap perlakuan tidak
signifikan terhadap kadar fenolik total buah
jambu biji. Hal ini sesuai dengan penelitian
sebelumnya dimana dengan pelapisan gel lidah
buaya dapat mempertahankan kadar fenolik
total yang lebih tinggi pada buah jambu biji
selama penyimpanan[27] dan penelitian lainnya
juga melakukan pelapisan buah lengkeng
dengan lidah buaya yang mana dengan adanya
pelapisan ini dapat mempertahankan kadar
fenolik total buah lengkeng selama masa Gambar 7 menunjukkan bahwa T1 (0.53 mg
GAE/g FW) mengalami penurunan kadar
fenolik
yang
cukup
tinggi
dibandingkan
perlakuan lainnya selama masa penyimpanan
karena tidak adanya lapisan yang membatasi
interaksi antar permukaan buah dengan O2 dari
lingkungan yang konsentrasi sehingga dapat
mempercepat terjadinya oksidasi dan membuat
berkurangnya kadar fenolik total pada buah. T5
(1.19 mg GAE/g FW) memiliki kadar fenolik DOI: https://doi.org/10.25077/jrk.v13i2.501 DOI: https://doi.org/10.25077/jrk.v13i2.501 174 Vol. 13, No. 2, September 2022 J. Ris. Kim. J. Ris. Kim. 2. Adrees, M., Younis, M., Farooq, U. &
Hussain, K., Nutritional Quality Evaluation
of Different Guava Varieties. Pak.J.Agri.Sci,
47(1): 1–4 (2010). penyimpanan. Senyawa-senyawa
yang
berkontribusi pada kandungan total fenolik
diantaranya fenolik, tanin[32]. Gugus pada cincin
aromatik dari senyawa fenolik dapat berperan
sebagai donor hidrogen yaitu gugus hidroksi,
oleh karena itu dapat bereaksi dengan senyawa
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Scrophularia striata Extract Supports Rumen Fermentation and Improves Microbial Diversity in vitro Compared to Monensin
|
Frontiers in microbiology
| 2,018
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cc-by
| 10,807
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Scrophularia striata Extract Supports
Rumen Fermentation and Improves
Microbial Diversity in vitro Compared
to Monensin 1 Department of Agriculture, Iranian Research Organization for Science and Technology, Tehran, Iran, 2 Institute of Animal
Nutrition and Functional Plant Compounds, Department for Farm Animals and Veterinary Public Health, University
of Veterinary Medicine Vienna, Vienna, Austria In the search for natural alternatives to antibiotic feed additives, we compared the
efficacy of two doses of Scrophularia striata extract [S. striata-Low at 40 and S. striata-
High at 80 mg g−1 dry matter (DM)] with monensin (monensin) and a negative control
in the modulation of rumen fermentation, methane production and microbial abundance
in vitro. Microbes were investigated using qPCR and 16S rRNA targeted sequencing. Data showed that the addition of S. striata increased production of total short chain fatty
acids (SCFA) in comparison to both monensin and control (P = 0.04). The addition of
S. striata increased acetate production, and increased propionate at the higher dosage
(P < 0.001). Supplementation of S. striata lowered methane production (P < 0.001)
compared to control but with no effect compared to monensin. Ammonia concentration
decreased by 52% (P < 0.001) with S. striata-High supplementation (4.14 mmol L−1)
compared to control, which was greater than that of monensin (36%). The diversity of
rumen bacteria was reduced (P < 0.001) for monensin and S. striata for both the number
of observed OTUs and the Chao1 index. Quantitative analysis of Protozoa showed a
decrease in the monensin treatment (P = 0.05) compared to control. Archaea copy
numbers decreased equally in both S. striata-High and monensin treatments compared
to the control group. Supplementation with S. striata increased relative abundances
of Fibrobacteres (P < 0.001) and Planctomycetes (P = 0.001) in comparison to both
the control and monensin treatments. Significant negative correlations were observed
between the abundances of Bacteroides, Fusobacterium, and Succinivibrio genera and
methane (r > −0.71; P ≤0.001). The abundance of Fibrobacter genera and total
SCFA (r = 0.86), acetate (r = 0.75), and valerate (r = −0.51; P < 0.001) correlated
positively. These results suggest that S. striata supplementation at 80 mg g−1 DM
inclusion, similar to monensin, supports rumen fermentation, lowers methane and
ammonia production. However, S. striata supported rumen fermentation toward higher
total SCFA and propionate production, while unlike monensin still supported a diverse
rumen microbiome and an increase in cellulolytic bacteria such as Fibrobacter. ORIGINAL RESEARCH
published: 19 September 2018
doi: 10.3389/fmicb.2018.02164 Keywords: greenhouse gasses, fermentation profile, phytogenic compounds, ionophores, rumen microbiome,
Rusitec Edited by:
Sharon Ann Huws,
Aberystwyth University,
United Kingdom Reviewed by:
Alex V. Chaves,
The University of Sydney, Australia
Leticia Abecia,
Center for Cooperative Research
in Biosciences, Spain *Correspondence:
Renee Petri
renee.petri@vetmeduni.ac.at Specialty section:
This article was submitted to
Microbial Symbioses,
a section of the journal
Frontiers in Microbiology Received: 17 April 2018
Accepted: 23 August 2018
Published: 19 September 2018 Experimental Design, Rusitec Procedure,
and Sample Collection The experiment was designed as a completely randomized design
with 2 experimental runs, 4 treatments, and 3 replicates per run,
resulting in six independent measurements per treatment. The
treatments consisted of either no additives (control), 0.83 mg g−1
DM of substrate monensin sodium salt (monensin; Sigma
Chemical Co. Ltd.), 40 mg g−1 DM (S. striata-Low), or 80 mg g−1
DM (S. striata-High) of dried of S. striata extract. Monensin and
S. striata were dissolved in 1 ml ethanol [90% (v/v), TechniSolv
R⃝,
pure] and an equal volume of ethanol was added to the control
treatment group (Cardozo et al., 2005). In this regard, monensin, an ionophore widely used as a
growth promoter (Shen et al., 2017), has been previously shown
to inhibit ruminal methanogenesis and promote the production
of propionate (Green et al., 1999; Duffield et al., 2008). However,
because of the increasing concerns about antibiotic resistance and
since the ban of antibiotics in the European Union (Broudiscou
et al., 2000; Shen et al., 2017), considerable efforts have been
devoted to investigating the potential use of plant phytochemicals
such as essential oils, saponins and tannins as alternatives to
antibiotics in ruminant nutrition (´Sliwi´nski et al., 2002; Bodas
et al., 2012). Scrophularia striata is a herbaceous flowering plant,
commonly known as figwort, which is often used in traditional
folk remedies against infections (Monsef-Esfahani et al., 2010;
Mahboubi et al., 2013). Previous research has shown that the
ethanolic extract of S. striata has antimicrobial activity against
Staphylococcus aureus, Eschericia coli, Enterococcus faecalis, and
Newcastle disease (Bahrami and Valadi, 2010; Bahrami, 2011). However, the effects of S. striata extract on the anaerobic
microbiota of the rumen and the associated fermentation have
not been previously reported. g
p
All runs were comprised of 12 fermenters and lasted for
10 days, with sampling on the last 5 days of each run based
on a 4 day adaptation period for the rumen microbes (Soliva
and Hess, 2007). Initial inocula for each experimental run was
collected from three non-lactating rumen-fistulated Holstein
cows, housed at the Dairy Research Station of the University of
Veterinary Medicine Vienna (Pottenstein, Austria). The use of
donor cows was approved by the institutional ethics committee
of the University of Veterinary Medicnie (Vetmeduni). Donor
cattle were fed hay and grass silage ad libitum and free
access to drinking water. INTRODUCTION Iran. Whole plants containing stem and seed were ground in a
centrifugal mill with 0.5 mm screen. Ground plant material was
mixed with 600 ml L−1 aqueous ethanol solution at the ratio
1:25 (w:v) for 1 h in sonicator bath with cooling system (Sonorex
RK 156H, Bandelin, Berlin, Germany). The mixture was filtered
through filter paper and concentrated in a vacuum at 40◦C by a
rotary evaporator (Buchi, Flawil, consisting of water bath B-480,
Rotovapor R-124, and Vacuum Controller B-72, Switzerland). After ethanol evaporation, the particulate was transferred to
beaker flask, frozen at −20◦C and then freeze-dried according to
previously described methods (Goel et al., 2008). Rumen
microbiota
are
essential
for
the
host,
providing
short-chain fatty acids (SCFA), which make up 75% of the
total metabolizable energy for ruminants (Siciliano-Jones and
Murphy, 1989; Bergman, 1990). In dairy cattle, of the major
SCFA, propionate is critical for gluconeogenesis (den Besten et al.,
2013) and therefore has particular importance for meeting the
high glucose demands of lactation. In addition, the production
of propionate is an electron-accepting pathway and serves as a
H+ sink, lowering the substrate availability for methanogenesis
(den Besten et al., 2013). The production of methane not
only has high global warming potential, but also reduces feed
energy efficiency in ruminants (Johnson and Johnson, 1995). Therefore, ruminant nutritionists have been working for decades
to develop dietary strategies capable of modulating both the
rumen fermentation profile to increase propionate production
for high producing cattle and reducing methane production
(McAllister and Newbold, 2008; Hristov et al., 2013). Experimental Design, Rusitec Procedure,
and Sample Collection Animals were cared for according to
the Austrian guidelines for animal welfare (Federal Ministry
of Health, 2004). Rumen contents of the three cows were
mixed in equal parts, the liquor was first filtered through four
layers of medical gauze (1-mm pore size) before adding to
the fermenters and the solid digesta was filled (∼60 g) into
nylon bags and placed into each fermenter on the first day of
each experimental run according to the previously published
methodology (Klevenhusen et al., 2015). In brief, each fermenter
was filled with 100 ml artificial saliva and 600 ml rumen fluid on
the first day of each experimental run (McDougall, 1948). New
substrate was provided to each fermenter daily, based on a 50:50
forage-to-concentrate dairy ration (Supplementary Table S1)
using nylon bags (140 mm × 70 mm, 150-µm pore size, Linker
Industrie–Technik, Kassel, Germany). After 24 h of incubation,
the bags containing solid digesta were replaced with fresh bags
containing the experimental diet. Accordingly, every feed bag was
incubated for 48 h, and then replaced. Artificial saliva buffer was
continuously infused into each vessel (357 ± 19.2 ml/d) through
a 12 channel peristaltic pump (Model ISM 932D, Ismatec, Index
Health and science GmbH, Wertheim, Germany) in accordance
with Soliva and Hess (2007). Feed additives were pipetted into
the fermenter fluid every day at the time of feeding (0900 h) Therefore, this study was designed to evaluate effects of
S. striata on SCFA production, methane production as well as the
diversity and abundance of the rumen microbiome compared to
monensin in a semi continuous in vitro system. We hypothesized
that feeding of S. striata, similar to monensin, could alter the
rumen microbiome leading to beneficial modifications to rumen
fermentation including increased propionate production and an
inhibition of ruminal methane production. Citation: Bagheri Varzaneh M, Klevenhusen F,
Zebeli Q and Petri R (2018)
Scrophularia striata Extract Supports
Rumen Fermentation and Improves
Microbial Diversity in vitro Compared
to Monensin. Front. Microbiol. 9:2164. doi: 10.3389/fmicb.2018.02164 September 2018 | Volume 9 | Article 2164 1 Frontiers in Microbiology | www.frontiersin.org Scrophularia striata and the Rumen Microbiome in vitro Bagheri Varzaneh et al. September 2018 | Volume 9 | Article 2164 Plant Material and Extraction Plant Material and Extraction Scrophularia striata was purchased from a local herbalist’s shop
in Ilam, a province in the west of Iran and was authenticated
as S. striata Bioss family (Scrophulariaceae) with a voucher
specimen being preserved in the pharmacy herbarium (TEH
No: 6916) at Tehran University of Medical Science, Tehran, September 2018 | Volume 9 | Article 2164 Frontiers in Microbiology | www.frontiersin.org 2 Scrophularia striata and the Rumen Microbiome in vitro Bagheri Varzaneh et al. starting on day 0. After each feeding procedure the system
was flushed with N2 gas for 3 min to maintain anaerobic
condition inside the vessels. Daily effluent and fermentation
gasses were collected in a 1 L volumetric flask located in an ice-
filled box and reusable gas-tight aluminum bags (TECOBAG 8L,
Tesseraux ContainerGmbH, Burstadt, Germany), respectively. On sampling days, directly before feed bag exchange, fermenter
fluid samples were collected using a syringe equipped with
a plastic tube. Part of the fluid samples were immediately
analyzed for rumen fermentation parameters and aliquots were
stored in separate tubes at −20◦C for SCFA and ammonia
measurements. Additional fluid samples were snap frozen in
liquid nitrogen and stored at −80◦C for DNA extraction. The
feed residue bags were hand-washed with running water until
the water was clear and kept at −20◦C for chemical composition
analysis. standard calibration curve. Ammonia and phenol were oxidized
by sodium hydroxide in the presence of sodium nitroprusside
and dichloroisocyanuric acid. The absorbance was measured
at 655 nm using U3000 Spectrophotometer (INULA GmbH,
Vienna, Austria) after 90 min of reaction. Analysis of the residual feeds after 48 h of incubation and
the original feed samples were conducted in duplicate. Prior
to analysis, samples were oven-dried at 65◦C for 48 h and
ground using through a 0.75 mm sieve (VDLUFA, 2007). The
DM of feeds and feed residues was measured after oven drying
at 104◦C for 24 h and ash was analyzed by combustion of
samples over night at 580◦C. Crude protein was measured
by Kjeldahl method (VDLUFA, 2007). Neutral detergent fiber
(aNDFom; Udén et al., 2005) and acid detergent fiber (ADFom)
contents were analyzed using Fibretherm FT12 (C. Gerhardt
GMbH and Co. KG, Konigswinter, Germany) based on VDLUFA
(2007). Heat stable α-amylase used in the NDF procedure
and all fiber fractions were expressed exclusive residual ash. DNA Extraction and qPCR q
Frozen rumen fluid samples were thawed on ice, vortexed
shortly to mix the sample, and then samples from d 5
to 10 were pooled per fermenter per run (n = 6 per
treatment). From the pooled sample, 1 mL subsample was
used for DNA isolation. DNA isolation was performed using
the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden,
Germany) and samples were mixed with 1 mL of InhibitEX
buffer provided in the kit and heated at 95◦C for 5 min
to ensure proper lysis of bacteria. After heating, additional
enzymatic (mutanolysin, 2.5 U µL−1; lysozym, 100 mg mL−1;
proteinase K, 20 mg mL−1) and mechanical lysis (0.4 g sterile
ceramic beads, diameter 1.4 mm) using a bead-beater (FastPrep-
24 5G, MP Biomedicals, LLC, Santa Ana, CA, United States)
was performed to disrupt bacterial cells according to previously
published procedures (Kong et al., 2010). This was followed
by chemical removal of cell debris and PCR inhibitors by
centrifugation and supernatants were transferred to fresh tubes
for column based isolation of total genomic DNA using the
QIAcube robotic workstation. Total genomic DNA was eluted
in 200 µL of low-salt buffer. The isolated DNA concentration
was determined by a Qubit 2.0 Fluorometer (Life Technologies,
Carlsbad, CA, United States) using the Qubit dsDNA HS Assay
Kit (Life Technologies) and stored at −20◦C until further
analysis. p
Concentration and composition of SCFA (acetate, propionate,
n-butyrate, isobutyrate, n-valerate, isovalerate, and caproate)
were analyzed by GC, as described previously (Qumar et al.,
2016). Briefly, incubation fluid samples were thawed and then
centrifuged at 20,000 × g for 25 min at 4◦C. The supernatant
(0.6 mL) was transferred into a fresh tube and 0.2 mL of HCl
(1.8 mol L−1) was added, followed by 0.2 mL of the internal
standard (4-methylvaleric acid, Sigma-Aldrich Co. LLC., St. Louis, MI, United States). After centrifugation of the mixture
at 20,000 × g for 25 min at 4◦C, the clear supernatant was
transferred into the GC vial. Analysis of SCFA concentrations
was conducted via a gas chromatography apparatus (GC Model
8060 MS 172 DPFC, No.: 950713, Fisons, Rodano, Italy)
which was equipped with a flame-ionization detector and a
30 m × 0.53 mm ID × 0.53 µm df capillary column (Trace TR
Wax, Thermo Fisher Scientific, Waltham, MA, United States). Injector and detector were at temperatures of 170◦C and
190◦C, respectively. Plant Material and Extraction Disappearance of nutrients as a measure of degradability was
calculated from the difference between content of nutrients
in the diet before and after 48 h incubation in the Rusitec
system. Sample Analysis Immediately after sampling pH and redox potential (seven
MultiTM, Mettler-Toledo GmbH, Schwerzenbach, Switzerland)
of the rumen fermenter liquid were measured. Fermentation
gasses were also measured every 24 h during the sampling
period. Gas volume was determined using the water displacement
technique (Soliva and Hess, 2007), and the concentration of CH4
was measured using a portable infrared detector (ATEX biogas
Monitor Check BM 2000, Ansyco, Karlsruhe, Germany). In brief,
the biogas monitor is attached to the gas collection bag after a
24 h collection period, the sample is measured through pumping
of gas into the monitor for 30 s to provide a consistent reading as
per the manufacturer’s instructions. Frontiers in Microbiology | www.frontiersin.org DNA Extraction and qPCR Helium was used as carrier gas with a
flow rate of 6 mL min−1. Stratos Software (Stratos Version
4.5.0.0, Polymer Laboratories, Church Stretton, Shropshire,
United Kingdom) was utilized for generation and evaluation of
chromatograms. Quantitative PCR Analysis Sequencing
data are available in BioProject SRA database under the accession
number SRP140677. and alignment were performed using UCLUST (Edgar, 2010)
and OTUs were defined using PyNAST (Caporaso et al., 2010)
and the SILVA database (v123; accessed July 19, 2017; Quast
et al., 2012; Yilmaz et al., 2013). The degree of similarity
between sequences was defined as 97% to obtain OTU. Any
OTUs which clustered with less than 10 reads were manually
removed. A total of 969,155 sequences (mean sequences per
sample 38,504) clustered into 2,727 OTUs for further analysis. All OTUs with a relative abundance greater than 0.1% (135
OTUs) were taken in consideration for statistical analysis and
statistically derived means for relative abundance were graphed
in excel. For calculation of the non-parametric species richness
estimators Chao1 and the observed OTUs per sample were used. Beta diversity analysis was performed using weighted Unifrac
dissimilarity metrics and the principal coordinate analysis
(PCoA) plotting in QIIME with rarefaction at 20,133 sequences,
based on the lowest number of sequences in a sample. Sequencing
data are available in BioProject SRA database under the accession
number SRP140677. CA, United States), 1 µL of each primer (Protozoa 400 nM;
Bacteria 100 nM; Archaea and Fungi 200 nM), 7 µL of nuclease-
free water and 1 µL DNA template (4 ng). The amplification
program included an initial denaturation step at 95◦C for 10 min,
followed by 40 cycles of 95◦C for 10 s, primer annealing at 60◦C
for 30 s, and elongation at 72◦C for 30 s. Fluorescence was
measured at the last step of each cycle. Melting curve analysis
was performed to determine the specificity of the amplification. The dissociation of PCR products was monitored by slow
heating with an increment of 0.1◦C s−1 from 55 to 95◦C, with
fluorescence measurement at 0.1◦C intervals. Standard curves
for all primers were generated using 10-fold serial dilutions
(108–103 molecules µL−1) of the purified and quantified PCR
products generated by standard PCR using DNA from the pooled
rumen fluid from both runs of the present experiment. The PCR
efficiency was calculated: E = 10(−1/slope)−1 (Table 1). Results
were analyzed using the associated software (Stratagene MxPro,
QPCR Software, version 2.00). Statistical Analyses y
Analysis of variance was conducted using the PROC MIXED
procedure of SAS (Version 9.2, SAS Institute, Cary, NC,
United States). Treatments were considered as fixed effects,
while experimental run and fermenter within run were defined
as random effects. Degrees of freedom estimation was done
using Kenward-Rogers. All values were reported as least
squares means. Comparison between the control and treatments
was carried with Dunnett–Hsu. Differences were declared as
significant at P ≤0.05. Correlation analysis using Pearsons
correlation coefficient was performed using the PROC CORR
procedure of SAS. Following Hinkle et al. (2003), the r was
interpreted as follows: 0.00–0.30 as negligible; 0.30–0.50 as low;
0.50–0.70 as moderate; 0.70–0.90 as high, and 0.90–1.00 as
substantial. y
One 40 µL aliquot of each DNA sample was sent for
amplicon sequencing using a MiSeq Illumina sequencing
platform and paired-end technology (Microsynth AG, Balach,
Switzerland). Sequencing targeted the V3–V5 hypervariable
region of the 16S rRNA gene using the primer set 357F-
HMP (5′-CCTACGGGAGGCAGCAG-3′) and 926R-HMP (5′-
CCGTCAATTCMTTTRAGT-3′) to produce an amplicon size
of ∼523 bp (Peterson et al., 2009). In brief, libraries were
constructed by ligating sequencing adapters and indices onto
purified PCR products (Nextera XT Sample Preparation Kit,
Illumina, San Diego, CA, United States) according to the
recommendations of the manufacturer. Equimolar amounts of
each of the libraries were pooled together and sequenced on
an Illumina MiSeq Personal Sequencer and the resulting paired
ends were stitched together by Microsynth (Balach, Switzerland)
resulting in a total of 3.8 million unfiltered reads with an average
of 503 nt in length. Sequence quality control was performed
using the QIIME pipeline (Caporaso et al., 2010). Sequences
were first quality filtered following (Q = 20; 27) and then
screened and filtered for chimeras using USEARCH and its
database (USEARCH v8.1; Bokulich et al., 2013). Clustering Quantitative PCR Analysis y
Using qPCR, the abundance of Protozoa, Bacteria, Archaea, and
Fungi present in Rusitec samples were analyzed on a Stratagene
Mx3000P real-time PCR System (Agilent Technologies, Santa
Clara, CA, United States) using established primer pairs
(Table 1). DNA samples and negative controls were assayed in
duplicate in a 20 µL reaction mixture containing 10 µL the Fast-
Plus EvaGreen Master Mix with Low ROX (Biotium, Hayward, The concentration of ammonia was determined using the
indophenol reaction (Weatherburn, 1967). The fermenter fluid
samples were thawed at room temperature and subsequently
centrifuged at 15,115 × g for 10 min. The clear supernatant was
diluted with distilled water to obtain a concentration within the September 2018 | Volume 9 | Article 2164 Frontiers in Microbiology | www.frontiersin.org 3 Scrophularia striata and the Rumen Microbiome in vitro Bagheri Varzaneh et al. TABLE 1 | Primers for kingdom level analysis using quantitative polymerase chain reaction analysis. Primer
Primer sequence 5′-3′1
Product size (bp)2
Tm3 (◦C)
Primer efficiency
Reference
Archaea 16S rRNA
f:CCGGAGATGGAACCTGAGAC
160
60
0.97
Zhou and
Hernandez-Sanabria, 2009
r:CGGTCTTGCCCAGCTCTTATTC
Universal bacteria
16S rRNA
f:CCTACGGGAGGCAGCAG
189
55
1.02
Muyzer et al., 1993
r:ATTACCGCGGCTGCTGG
Total protozoa 18S
rRNA
f:GCTTTCGWTGGTAGTGTATT
233
60
1.04
Sylvester et al., 2004
r:CTTGCCCTCYAATCGTWCT
Anaerobic fungi ITS1
f:GAGGAAGTAAAAGTCGTAACAAGGTTTC
110-115
60
0.99
Denman and McSweeney,
2006
r:CAAATTCACAAAGGGTAGGATGATT
1f: forward primer, r: reverse primer. 2Product size in basepairs (bp). 3Tm: melting temperature in Celcius. TABLE 1 | Primers for kingdom level analysis using quantitative polymerase chain reaction analysis. and alignment were performed using UCLUST (Edgar, 2010)
and OTUs were defined using PyNAST (Caporaso et al., 2010)
and the SILVA database (v123; accessed July 19, 2017; Quast
et al., 2012; Yilmaz et al., 2013). The degree of similarity
between sequences was defined as 97% to obtain OTU. Any
OTUs which clustered with less than 10 reads were manually
removed. A total of 969,155 sequences (mean sequences per
sample 38,504) clustered into 2,727 OTUs for further analysis. All OTUs with a relative abundance greater than 0.1% (135
OTUs) were taken in consideration for statistical analysis and
statistically derived means for relative abundance were graphed
in excel. For calculation of the non-parametric species richness
estimators Chao1 and the observed OTUs per sample were used. Beta diversity analysis was performed using weighted Unifrac
dissimilarity metrics and the principal coordinate analysis
(PCoA) plotting in QIIME with rarefaction at 20,133 sequences,
based on the lowest number of sequences in a sample. RESULTS Rumen Fermentation Profile, Methane
Production, and Nutrient Degradability Rumen Fermentation Profile, Methane
Production, and Nutrient Degradability
The pH in the fermenters remained constant throughout both
experimental runs (Supplementary Figure S1 and Table 2). The pH in the fermenters remained constant throughout both
experimental runs (Supplementary Figure S1 and Table 2). September 2018 | Volume 9 | Article 2164 Frontiers in Microbiology | www.frontiersin.org 4 Scrophularia striata and the Rumen Microbiome in vitro Bagheri Varzaneh et al. However, redox potential in the fermenters was lower for
all treatments when compared to the control (P = 0.007). Analysis showed that the ammonia concentration decreased
(P < 0.001) with monensin and S. striata supplementation. The extent of ammonia reduction was 35, 26, and 52%,
respectively, by monensin, S. striata-Low and S. striata-High
treatments. Total SCFA production was decreased by monensin,
while S. striata supplementation increased it compared with
control (P = 0.001). All individual SCFA were affected
by supplementation (Table 2). Monensin decreased acetate
production by 9%, whereas S. striata had no effect in comparison
with control. Propionate was increased by 186, 83, and 35%
by feeding monensin, S. striata-High and S. striata-Low,
respectively. As a result, monensin and S. striata-High had
a lower acetate to propionate ratio (A:P) in comparison to
control (P < 0.001). Supplementation decreased iso-butyrate
concentrations in comparison to control (P < 0.001). Butyrate
was decreased by monensin and S. striata-High (P < 0.001) in
comparison to control. In contrast, the addition of S. striata-
Low increased the concentration of iso-valerate above that of
the control group (Table 2). Monensin decreased the proportion
of iso-valerate to 8.1% of the control concentration and
increased of n-valerate by 97% (P < 0.001). Supplementation
with monensin, S. striata-Low and S. striata-High caused a
drop in methane production by 67, 29, and 36%, respectively
(P < 0.003). The DM, OM, and ADF degradation were not
affected by supplementation (Table 2). However, S. striata-High decreased CP degradation (P = 0.03) and all additives decreased
NDF degradability (P = 0.02) compared to the control
group. Quantitative Analysis at the Kingdom
Level of Taxonomy Results
of
qPCR
analysis
are
presented
in
Figure
1. Supplementation
did
not
affect
the
abundance
of
total
Bacteria
and
Fungi
populations
compared
to
control. Monensin remarkably lowered the gene copy number of
Protozoa (1.00 × 102), though it was significant only when
compared to S. striata-High group (3.15 × 105; P = 0.05). Copy numbers of total Archaea were lower with S. striata-
High and monensin compared to S. striata-Low (P = 0.001),
and tended to be lower with monensin compared to control
(P = 0.08). Alpha and Beta Microbial Diversity striata-High), and monensin on copy number of Bacteria
(16S rRNA), Fungi (ITS Gene 1), Protozoa (18S rRNA), and Archaea (16S rRNA) analyzed by qPCR. Data is presented in total copy numbers of the target gene. ∗In
tendency lower than control (P = 0.08). a,bTreatments with different letters differ significantly to the Control P < 0.05. FIGURE 1 | Effect of supplementation of Scrophularia striata extract, in two doses (S. striata-Low and S. striata-High), and monensin on copy number of Bacteria
(16S rRNA), Fungi (ITS Gene 1), Protozoa (18S rRNA), and Archaea (16S rRNA) analyzed by qPCR. Data is presented in total copy numbers of the target gene. ∗In
tendency lower than control (P = 0.08). a,bTreatments with different letters differ significantly to the Control P < 0.05. TABLE 3 | Effect of supplementation of Scrophularia striata extract and monensin on number of sequences, observed operational taxonomic units (OTU) and the Chao1
index. Treatments1
Parameter
Control
Monensin
S. striata-Low
S. striata-High
SEM2
P-value
Number of sequences
38560
39202
36323
36470
5141.0
0.91
Observed OTUs
1884a
726b
1319b
1003b
103.1
<0.001
Chao1 index
3536a
1655b
2727b
2068b
137.9
<0.001
1Control: no additive; monensin: monensin, contained 0.83 mg g−1 of DM of substrate monensin sodium salt; S. striata-Low and S. striata-High contained 40 and
80 mg g−1 of DM of substrate dried plant extract, respectively. 2SEM: standard error of the mean. a,bMeans within the same row with different superscripts differ to the
Control treatment at P < 0.05. Bacteroidetes and reduced the Firmicutes taxa, whereas monensin
increased Fusobacteria, Synergistetes, and Proteobacteria phyla
(Figure 3A). The addition of all additives decreased the relative
abundance of Elusimicrobia and TM7 in comparison to control. Fusobacterium and Firmicutes were, respectively, 4 and 13%
more abundant in the monensin in comparison to the S. striata-
High supplementation (Figure 3). At the genera level, Prevotella
was the most abundant genus ranging from 38.5% in control
to 46.1% in S. striata-High and was significantly increased
with S. striata supplementation. Bacteroidetes-associated genera
represented the largest proportion of the in vitro microbiota
despite the fact that samples were taken from the rumen
liquid (Figure 3B). Firmicutes had the largest diversity in
identified genera with a significant effect of supplementation
(Figure 3C). A drastic increase in Fusobacterium spp. was
found in monensin supplementation compared to the other showed a clear clustering of microbial communities for each
treatment group. Alpha and Beta Microbial Diversity p
y
Statistical analysis of both the intra- (alpha diversity; Table 3) and
inter- (beta diversity; Figure 2) sample diversity was performed. Despite no differences in the total numbers of sequences found
within a sample (P = 0.91), there were differences between
treatments in the number of OTUs (P < 0.001) and the Chao1
index (P < 0.001). For both indices, the diversity within a sample
was highest in the control, followed by S. striata-Low, S. striata-
High, and monensin showed the least microbial diversity. PCoA TABLE 2 | Effect of supplementation of Scrophularia striata extract and monensin on rumen fermentation profile and nutrient degradation. Parameter
Treatments1
SEM2
P-value
Control
Monensin
S. striata-Low
S. striata-High
pH
6.58
6.57
6.56
6.56
0.033
0.95
Redox (mv)
−263.0a
−224.7b
−214.6a
−233.2b
7.89
0.01
NH3 (mmol L−1)
8.65a
5.56b
6.39b
4.14b
0.472
<0.001
CH4 (ml day−1)
119a
38.7b
84.5b
75.9b
4.46
<0.001
CH4 reduction (% of Control)
–
67.2
28.8
35.7
3.50
0.003
Total SCFA (mmol d−1)
35.3a
32.7b
39.4b
38.5b
0.69
<0.001
Individual SCFA (mmol d−1)
Acetate (A)
17.4a
14.7b
19.5b
19.5b
0.675
<0.001
Propionate (P)
4.2a
11.2b
6.4b
8.4b
0.50
<0.001
Isobutyrate
0.20a
0.10b
0.18b
0.10b
0.001
<0.001
n-Butyrate
4.8a
1.9b
4.2a
3.7b
0.72
<0.001
Isovalerate
3.30a
0.27b
4.85b
2.88b
0.338
<0.001
n-Valerate
1.87a
3.70b
2.23a
2.47b
0.264
<0.001
Caproate
3.55a
0.92b
2.07b
1.37b
0.129
<0.001
A:P
4.34a
1.33b
3.18b
2.36b
0.418
<0.001
Nutrient degradation (%)3
Dry matter
62.5
61.4
61.0
60.1
1.00
0.16
Organic matter
59.2
58.0
57.7
56.7
1.04
0.29
Crude protein
69.5a
68.1a
66.9a
65.2b
1.03
0.03
Neutral detergent fiber
24.0a
19.1b
20.4a
18.1b
1.12
0.02
Acid detergent fiber
17.0
14.1
14.8
14.3
1.86
0.11
1Control: no additive; Monensin, contained 0.83 mg g−1 of DM of substrate monensin sodium salt; S. striata-Low and S. striata-High contained 40 and 80 mg g−1 of
DM of substrate dried plant extract, respectively. 2SEM: standard error of the mean. 3Methods of analysis are based on the VDLUFA (2007) methods of feed composition
analysis. a,bMeans within the same row with different superscripts differ to the Control treatment at P < 0.05. September 2018 | Volume 9 | Article 2164 5 Frontiers in Microbiology | www.frontiersin.org Scrophularia striata and the Rumen Microbiome in vitro Bagheri Varzaneh et al. FIGURE 1 | Effect of supplementation of Scrophularia striata extract, in two doses (S. striata-Low and S. Alpha and Beta Microbial Diversity Principle component 1 described 40.8% of
the diversity between samples between original rumen fluid
and experimental treatments, with the monensin treatment
clustering independently (Figure 2). Principle component 2
represented 30.2% of the diversity with a separation of the
S. striata samples from the control and monensin groups. Principle component 3, representing 10.3% of the variation
between samples, was related to the original pooled rumen fluid
sample. 16S rRNA Taxonomy striata-High treatments at the phyla level for significant
differences in the least square means showed of the 15
phyla, eight were significantly different between the two
treatment groups (Figure 4). Of the 30 most abundant
OTUs, 29 were significantly affected by the supplementation
treatment. Bacteroidetes and Firmicutes accounted for 16 and
8 of the OTUs, respectively (Figure 5). Three OTUs were
identified to the species level, Prevotella copri-like OTU2,
Bacteroides uniformis-like OTU10 and Fibrobacter succinogenes-
like OTU24. In comparison to the control, OTU2 was 4.2-
fold and four-fold increased in monensin and S. striata-High,
respectively (P < 0.001). OTU10 accounted for 5.13% of the
relative microbial abundance in the monensin supplemented
group but was less than 0.6% of the abundance in the
other treatments. F. succinogenes-like OTU24 was increased
in
both
the
S. striata-Low
(1.05%)
and
S. striata-High
(0.87%) supplemented groups compared to both control and
monensin. resembles the effects of monensin. However, changes to the
rumen microbiome as a result of S. striata supplementation
were
less
dramatic
than
those
seen
under
monensin
supplementation. In the current study, the degradation of aNDFom and
CP decreased with S. striata-High supplementation in vitro. This agrees with previously reported data showing lower
degradation of DM by addition of some flavonoid-rich plant
extracts and pure flavonid compounds (Oskoueian et al.,
2013; Kim et al., 2015). Previous studies have also reported
reductions in DM and NDF digestibility with monensin
(Poos et al., 1979; Anassori et al., 2012). However, in this
study, S. striata-High supplementation had a similar effect
on degradation as monensin with the exception of CP. This discrepancy among the different studies may be related
to several factors such as diet composition, the period of
adaptation to the product, the time when the samples were
collected, the method of analysis used and the type and
concentrations of the feed additives (Bodas et al., 2012). Ionophores are also generally microbial inhibitors and reduce
both cellulose digestion and microbial yield (Demeyer and
Van Nevel, 1979). Therefore, the effect of supplementation
was assumed to be associated with a decrease in bacterial
abundance. However, bacterial abundance as measured by copy
number and total number of sequences was not affected by
supplementation with either monensin or S. striata. There is
evidence showing that improved feed efficiency is correlated
with lower richness in the rumen microbiota (Shabat et al.,
2016). 16S rRNA Taxonomy Therefore, a possible reason for improved feed efficiency
in previous in vivo studies with monensin might have been
due to lower diversity of the microbiome. As a similar
reduction in microbial diversity was observed with S. striata
supplementation, it can be inferred that S. striata would also
improve feed efficiency. In line with this, we found that S. striata
improved efficiency of fermentation (mM SCFA per unit DM
degraded). 16S rRNA Taxonomy In total, 15 phyla, predominated by Bacteroidetes, Firmicutes, and
Proteobacteria, could be identified into 42 genera. All phyla and
all genera were significantly affected by treatments (Figure 3). Supplementation with S. striata-High increased the total September 2018 | Volume 9 | Article 2164 Frontiers in Microbiology | www.frontiersin.org 6 Scrophularia striata and the Rumen Microbiome in vitro Bagheri Varzaneh et al. FIGURE 2 | Principal coordinate analysis (PCoA) of 16S rRNA using the weighted Unifrac distance metric (PC1 = 40.8%, PC2 = 30.2%, and PC3 = 10.3%). Orange:
riginal rumen fluid sample taken from cows, Red: control, Blue: monensin, Purple: S. striata-Low, and Green: S. striata-High. S. striata: Scrophularia striata extract. FIGURE 2 | Principal coordinate analysis (PCoA) of 16S rRNA using the weighted Unifrac distance metric (PC1 = 40.8%, PC2 = 30.2%, and PC3 = 10.3%). Orange:
original rumen fluid sample taken from cows, Red: control, Blue: monensin, Purple: S. striata-Low, and Green: S. striata-High. S. striata: Scrophularia striata extract. FIGURE 2 | Principal coordinate analysis (PCoA) of 16S rRNA using the weighted Unifrac distance metric (PC1 = 40.8%, PC2 = 30.2%, and PC3 = 10.3%). Orange:
original rumen fluid sample taken from cows, Red: control, Blue: monensin, Purple: S. striata-Low, and Green: S. striata-High. S. striata: Scrophularia striata extract. FIGURE 3 | Mean relative abundance of 16S rRNA sequences identified to 97% with the SILVA128 database at the phyla level (A) and genera level for Bacteroidetes
(B), Firmicutes (C), and other groups (D). All phyla and genera have a significant effect of supplementation (P ≤0.05). FIGURE 3 | Mean relative abundance of 16S rRNA sequences identified to 97% with the SILVA128 database at the phyla level (A) and genera level for Bactero FIGURE 3 | Mean relative abundance of 16S rRNA sequences identified to 97% with the SILVA128 database at the phyla level (A) and genera level for Bacteroidetes
(B), Firmicutes (C), and other groups (D). All phyla and genera have a significant effect of supplementation (P ≤0.05). September 2018 | Volume 9 | Article 2164 September 2018 | Volume 9 | Article 2164 Frontiers in Microbiology | www.frontiersin.org 7 Scrophularia striata and the Rumen Microbiome in vitro Bagheri Varzaneh et al. treatments (Figure 3D). Direct comparison of the monensin
and S. Correlations Between Genera and
Rumen Fermentation Several correlations between rumen fermentation parameters
and
microbial
communities
significantly
altered
by
the
supplementations
were
found
(Table
4). Fusobacterium
spp. showed the greatest number of highly (0.70 < r < 0.90)
negative correlations, including methane, acetate, butyrate,
isovalerate and total SCFA production (P < 0.001) and
positive correlations to propionate (P < 0.001) and valerate
(P < 0.001). Similarly, Bacteroides (phyla Bacteroidetes) and
Succinivibro (phyla Proteobacteria) genera also showed an
inverse relationship to methane production (r = −0.76 and
r = −0.71, respectively) and a positive correlation to valerate
concentrations in vitro (P < 0.001). Furthermore, Bacteroides
spp. was also highly correlated with decreases in butyrate
and isovalerate, similar to Fusobacterium spp. Succinivibrio
showed similar correlations as Bacteroides (Table 4). The
strongest correlation was that of Fibrobacter spp. with total
SCFA concentrations (r = 0.86; P < 0.001), which was supported
by additional positive correlations with acetate and isovalerate
concentrations. Lachnospira spp. showed a highly negative
correlation to ammonia concentrations (P < 0.001). g
In the present study, both monensin and S. striata additions
caused an increase in propionate concentration, resulting in a
decrease in the ratio of acetate to propionate. Effect of S. striata
on decreasing acetate to propionate ratio confirmed results of
a previous study reporting that feeding flavonoids compounds
increased propionate and decreased the acetate to propionate
ratio in dairy cows fed a high concentrate diet (Balcells et al.,
2012). These results are also consistent with those of previous
in vitro studies (Callaway et al., 1997; Shen et al., 2016, 2017). Ionophores work by inhibiting hydrogen producers such as
Ruminococcus and Butyrivibrio and thereby favor propionate
and succinate producers (Schären et al., 2017; Shen et al.,
2017). The suppression of hydrogen transport diverts carbon
away from acetate production toward propionate and increases
irreversible acetate loss in the synthesis of butyrate. This same
mode of action is hypothesized for S. striata based on its
antimicrobial properties. In this study, monensin decreased both
Butyrivibrio and Ruminococcus species (butyrate and acetate
producers, respectively) and increased the total populations
of Bacteroides, Fusobacterium, and Succinivibrio, which are
recognized as major propionate producers (Russell, 2002; Frontiers in Microbiology | www.frontiersin.org DISCUSSION In contrast,
S. striata supplementation increased the relative abundance
of Fibrobacter spp., of which the largest family member is
F. succinogenes (Jeyanathan et al., 2014). F. succinogenes similar
to Succinivibrio (Pope et al., 2011) produces succinate as its
principal fermentation end product, while other rumen microbes
(e.g., Selenomonas ruminantium; Scheifinger and Wolin, 1973)
produce propionate via the succinate pathway (Shen et al.,
2017). While in this study Fibrobacter spp. was significantly
increased in the S. striata treatments, lactate-producing microbes
such as Selenomonas were not found to be significantly affected
by treatments, indicating that other microbes are potentially
responsible for the production of propionate in the S. striata
supplemented groups. One possibility for the differences between
groups is the changes in the Bacteroides group, which was
significantly increased in the S. striata supplementation and
is predominated by the genus Prevotella spp. Prevotella spp. is a well known group of bacteria with extremely diverse
metabolic capabilities (Bekele et al., 2010). It is possible that
a member of this group is able to convert succinate produced
by the Fibrobacter spp. to propionate, explaining the variation
between the monensin and S. striata groups. Interestingly, the
total number of Butyrivibrio also increased with increasing
dosages of S. striata, which is in direct contrast to the effects
of monensin supplemented groups found in this experiment. Similar to a previous study (Shen et al., 2017), we observed that
the relative abundance of Butyrivibrio and Pseudobutyrivibrio Puniya et al., 2015). However, supplementation with S. striata,
despite decreasing methane production did not show the
same increases in Bacteroides, Fusobacterium, and Succinivibrio
and only minor decreases in Ruminococcus. In contrast,
S. striata supplementation increased the relative abundance
of Fibrobacter spp., of which the largest family member is
F. succinogenes (Jeyanathan et al., 2014). F. succinogenes similar
to Succinivibrio (Pope et al., 2011) produces succinate as its
principal fermentation end product, while other rumen microbes
(e.g., Selenomonas ruminantium; Scheifinger and Wolin, 1973)
produce propionate via the succinate pathway (Shen et al.,
2017). While in this study Fibrobacter spp. was significantly
increased in the S. striata treatments, lactate-producing microbes
such as Selenomonas were not found to be significantly affected
by treatments, indicating that other microbes are potentially
responsible for the production of propionate in the S. striata
supplemented groups. One possibility for the differences between
groups is the changes in the Bacteroides group, which was
significantly increased in the S. DISCUSSION We
hypothesized
that
the
supplementation
of
S. striata,
a
plant
with
known
antimicrobial
properties
(Monsef-Esfahani et al., 2010; Mahboubi et al., 2013), would
elicit similar propionigenic and methano-static effects to
the ionophore monensin as a result of changes in the
rumen microbiome. The most important findings of this
study were a decrease in methane production, an enhanced
propionate
proportion
and
a
higher
total
concentration
of SCFA as a result of S. striata supplementation. The
increasing effect of S. striata on propionate concentration,
while decreasing effect on methane and ammonia production, September 2018 | Volume 9 | Article 2164 Frontiers in Microbiology | www.frontiersin.org 8 Scrophularia striata and the Rumen Microbiome in vitro Bagheri Varzaneh et al. FIGURE 4 | Comparison of the mean relative abundance of phyla between treatment groups supplemented with Scrophularia striata extract at 80 mg g−1 DM
(S. striata-High) and those given monensin at 0.83 mg g−1 DM. Differences in the P-value for least square means between monensin and S. striata-High are
Bacteroidetes (P < 0.001), Firmicutes (P < 0.001), Fusobacteria (P < 0.001), Proteobacteria (P = 0.001), Fibrobacteres (P = 0.001), Spirochaetes (P = 0.001). Other
includes Lentisphaerae (P = 0.04), Planctomycetes (P = 0.001), Actinobacteria (P = 0.96), Chloroflexi (P = 0.65), Elusimicrobia (P = 0.99), Synergistetes (P = 0.69),
Tenericutes (P = 0.37), TM7 (P = 0.99), WPS-2 (P = 0.28). ∗Individual phyla within the “Other” group do not have means which are significantly (P < 0.05). FIGURE 4 | Comparison of the mean relative abundance of phyla between treatment groups supplemented with Scrophularia striata extract at 80 mg g−1 DM
(S. striata-High) and those given monensin at 0.83 mg g−1 DM. Differences in the P-value for least square means between monensin and S. striata-High are
Bacteroidetes (P < 0.001), Firmicutes (P < 0.001), Fusobacteria (P < 0.001), Proteobacteria (P = 0.001), Fibrobacteres (P = 0.001), Spirochaetes (P = 0.001). Other
includes Lentisphaerae (P = 0.04), Planctomycetes (P = 0.001), Actinobacteria (P = 0.96), Chloroflexi (P = 0.65), Elusimicrobia (P = 0.99), Synergistetes (P = 0.69),
Tenericutes (P = 0.37), TM7 (P = 0.99), WPS-2 (P = 0.28). ∗Individual phyla within the “Other” group do not have means which are significantly (P < 0.05). Puniya et al., 2015). However, supplementation with S. striata,
despite decreasing methane production did not show the
same increases in Bacteroides, Fusobacterium, and Succinivibrio
and only minor decreases in Ruminococcus. Frontiers in Microbiology | www.frontiersin.org September 2018 | Volume 9 | Article 2164 DISCUSSION FIGURE 5 | Effect of supplementation of Scrophularia striata extract and monensin on percent relative abundance and taxonomic classification of 30 most abundant
operational taxonomic units (OTUs). 1Control: no additive; monensin: monensin, contained 0.83 mg g−1 of DM of substrate monensin sodium salt; S. striata-Low
and S. striata-High contained 40 and 80 mg g−1 of DM of substrate dried plant extract, respectively. 2OTUs listed in order of relative abundance. 3Name of the
closest relative based on 97% similarity in the sequence database at the class (c), order (o), family (f), genus (g), or species (s) level. †Adjusted mean separation
based on Dunnett-Hsu shows a significant variation from the Control. ∗Means are not significantly different (P < 0.05). FIGURE 5 | Effect of supplementation of Scrophularia striata extract and monensin on percent relative abundance and taxonomic classification of 30 most abundant
operational taxonomic units (OTUs). 1Control: no additive; monensin: monensin, contained 0.83 mg g−1 of DM of substrate monensin sodium salt; S. striata-Low
and S. striata-High contained 40 and 80 mg g−1 of DM of substrate dried plant extract, respectively. 2OTUs listed in order of relative abundance. 3Name of the
closest relative based on 97% similarity in the sequence database at the class (c), order (o), family (f), genus (g), or species (s) level. †Adjusted mean separation
based on Dunnett-Hsu shows a significant variation from the Control. ∗Means are not significantly different (P < 0.05). S. striata-High supplementation groups, Protozoa copy numbers
were significantly increased and yet Archaea numbers were
decreased. Since differences were seen in the copy numbers
in the S. striata-Low supplemented groups for both Protozoa
and Archaea, this indicated a dose dependant response in
these groups to S. striata supplementation. These results again
contradict previous research which demonstrated that monensin
does not directly inhibit methanogens (Russell and Houlihan,
2003). indirectly decreasing methane production (Russell and Houlihan,
2003). Despite differences in microbial abundance between
monensin and S. striata supplemented groups, both groups
had decreases in CH4 and increases in propionate productions,
with a dose dependant response being seen in the S. striata
supplementation. However, the mechanism of action on methane
production is probably different between monensin and S. striata. In contrast to S. striata, supplementation with monensin
decreased the copy numbers of both Archaea and Protozoa
populations. This supports previous research which noted a dose
dependant response in Protozoa to monensin (Dennis et al.,
1986). DISCUSSION striata supplementation and
is predominated by the genus Prevotella spp. Prevotella spp. is a well known group of bacteria with extremely diverse
metabolic capabilities (Bekele et al., 2010). It is possible that
a member of this group is able to convert succinate produced
by the Fibrobacter spp. to propionate, explaining the variation
between the monensin and S. striata groups. Interestingly, the
total number of Butyrivibrio also increased with increasing
dosages of S. striata, which is in direct contrast to the effects
of monensin supplemented groups found in this experiment. Similar to a previous study (Shen et al., 2017), we observed that
the relative abundance of Butyrivibrio and Pseudobutyrivibrio in the S. striata treatments was significantly higher than in the
monensin treatment. B. fibrisolvens (Stewart et al., 1997) and
Pseudobutyrivibrio spp. (Kopeˇcný et al., 2003) are important
butyrate-producing species in the rumen and their presence
may explain the higher butyrate levels in the S. striata groups
compared to monensin group. There is also some evidence of
growth promoting effects of phenolic compounds on several pure
cultures of cellulolytic and non-cellulolytic bacteria resulting in
enhanced acetate production in vitro (Borneman et al., 1986). In particular, the cellulolytic bacteria Treponema and Fibrobacter
increased by S. striata in the current study, which could
explain comparable acetate levels to the control. Accordingly,
the abundance of Fibrobacter positively correlated with acetate
concentration. The positive influence of S. striata on total SCFA
production is potentially of great benefit to performance animals
in order to meet the increased energy requirements of growth and
production. The potential of plant secondary metabolites to affect methane
production has varied extensively ranging from −30 to +30% in a
screening study with 450 plant species (Bodas et al., 2008). In the
present study, the extent of methane reduction was, respectively,
29–36% in S. striata-Low and S. striata-High which, based on
a study that reviewed enteric methane mitigation options, falls
within the range of medium to high decrement (Hristov et al.,
2013). Scrophularia extract supplemented treatments showed a
decreased overall abundance of Firmicutes, which are important
ruminal H2-producing microorganisms (Shen et al., 2017),
whereas monensin supplementation showed no effect. These
results contradict previous research which demonstrated that
monensin can directly inhibit H2-producing bacteria, thereby September 2018 | Volume 9 | Article 2164 9 Scrophularia striata and the Rumen Microbiome in vitro Bagheri Varzaneh et al. Frontiers in Microbiology | www.frontiersin.org CONCLUSION Addition of hydro-alcoholic extract of S. striata to rumen
fluid in vitro elicited similar propionigenic, and methanostatic
effects to monensin and decreased the acetate:propionate ratio. Microbial analysis, however, showed that S. striata and monensin
have distinctly different impacts on relative abundances of the
total populations of Protozoa and Archaea, as well as effects
on the diversity and taxonomy of the bacterial population. The potential of high level of S. striata to decrease ammonia
concentration and protein degradability at a greater extent
than monensin is promising as it could be translated to more
efficacious utilization of nitrogen by the host animal. However,
these promising results necessitate the investigation of using
S. striata in in vivo feeding trials. DISCUSSION Therefore, the antiprotozoal effect of monensin seen in
this experiment may have resulted indirectly in the reduction
of the methanogenic Archaea due to their attachment to and
dependency on Protozoa for hydrogen (Morgavi et al., 2010). In Since defaunation decreases rumen ammonia concentration
resulting in lower deamination, an increased uptake of ammonia
by the bacterial population and a smaller recycling of microbial
proteins, low Protozoa counts could explain the decreased
ammonia concentrations with monensin. However, in the
S. striata-High treatment, Protozoa populations were highest and September 2018 | Volume 9 | Article 2164 Frontiers in Microbiology | www.frontiersin.org 10 Scrophularia striata and the Rumen Microbiome in vitro Bagheri Varzaneh et al. ammonia concentrations lowest. Two major groups of bacteria
are responsible for ammonia production in the rumen; bacteria
with low activity and high number and those with low numbers
and high specific activity (Russell et al., 1991). Clostridium
aminophilum, Clostridium sticklandii (Russell et al., 1988), and
Peptostreptococcus anaerobius (McIntosh et al., 2003) have been
recognized as important hyper-ammonia producing bacteria. The
sensitivity of C. sticklandii and P. anaerobius to essential oils
has been previously reported (McIntosh et al., 2003). Therefore,
the lower ammonia production and CP degradation in S. striata
treatments could be attributed to lower abundance of genus
Clostridium spp. Ruminococcus also showed positive significant
correlations with ammonia concentration and their abundances
decreased with supplementation of monensin and S. striata. Lower ammonia concentrations in S. striata-High compared to
monensin suggests that a higher dose of S. striata had greater
impact on ammonia producing species. Inhibition of ruminal
protein degradation by S. striata plant extract has important
implications on improving post ruminal delivery of true protein
(Szumacher-Strabel and Cie´slak, 2010). TABLE 4 | Pearson correlation coefficients (r) between rumen fermentation parameters and genera significantly impacted by supplementation. DISCUSSION Genera
Rumen fermentation parameters
Bacteroides
Butyrivibrio
Clostridium
Fibrobacter
Fusobacterium
Lachnospira
Prevotella
Ruminococcus
Succinivibrio
Methane
−0.76
0.36
0.65
0.34
−0.76
−0.11
−0.2
0.8
−0.71
Ammonia
−0.32
−0.23
0.71
−0.24
−0.23
−0.71
−0.47
0.75
−0.35
Acetate
−0.66
0.72
−0.17
0.75
−0.74
0.53
0.33
0.2
−0.41
P-value
Propionate
0.74
−0.39
−0.47
−0.33
0.74
0.13
0.1
−0.78
0.64
>0.05
Isobutyrate
−0.45
0.05
0.77
−0.02
−0.41
−0.51
−0.51
0.83
−0.45
0.05–0.02
Butyrate
−0.73
0.44
0.73
0.47
−0.74
−0.03
−0.31
0.67
−0.58
0.01–0.001
Isovalerate
−0.82
0.56
0.26
0.79
−0.84
0.08
0.06
0.56
−0.46
<0.001
Valerate
0.83
−0.42
−0.53
−0.51
0.82
−0.02
0.04
−0.67
0.72
Total SCFA1
−0.67
0.72
0.06
0.86
−0.74
0.46
0.12
0.22
−0.32
1 ts (r) between rumen fermentation parameters and genera significantly impacted by supplementation. AUTHOR CONTRIBUTIONS MBV, FK, and QZ designed the experiments. MBV and FK
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data. RP and MBV drafted the paper. All authors read and
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physrev.1990.70.2.567 Goel, G., Makkar, H. P. S., and Becker, K. (2008). FUNDING This work has been supported by the Center for International
Scientific Studies and Collaboration (CISSC), Ministry of
Science, Research and Technology of Iran, and Institute
of
Animal
Nutrition
and
Functional
Plant
Compounds,
Department for Farm Animals and Veterinary Public Health,
University of Veterinary Medicine, Vienna, Austria. The authors
acknowledge the funding support of the Vetmeduni Post Doc
Program. September 2018 | Volume 9 | Article 2164 11 Bagheri Varzaneh et al. Scrophularia striata and the Rumen Microbiome in vitro ACKNOWLEDGMENTS The authors thank Prof. R. Chizzola (Institute of Animal
Nutrition and Functional Plant Compounds), S. Bauer (Institute
of Meat Hygiene, Meat Technology and Food Science), and
Dr. H. J. Busse (Institute of Bacteriology, Mycology and
Hygiene) for helping with extraction. The skillful assistance
of P. Pourazad, M. Qumar, A. Dockner, S. Sharma, M. Wild, S. Leiner, and A. Sener in this experiment is greatly
appreciated. The authors thank Prof. R. Chizzola (Institute of Animal
Nutrition and Functional Plant Compounds), S. Bauer (Institute
of Meat Hygiene, Meat Technology and Food Science), and
Dr. H. J. Busse (Institute of Bacteriology, Mycology and
Hygiene) for helping with extraction. The skillful assistance
of P. Pourazad, M. Qumar, A. Dockner, S. Sharma, M. Wild, S. Leiner, and A. Sener in this experiment is greatly
appreciated. The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fmicb. 2018.02164/full#supplementary-material FIGURE S1 | Mean fermenter pH for each treatment group, on each day of the
experimental period, averaged from all runs. FIGURE S1 | Mean fermenter pH for each treatment group, on each day of the
experimental period, averaged from all runs. TABLE S1 | Ingredients and chemical composition of the concentrate and basal
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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
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practice. No use, distribution or reproduction is permitted which does not comply
with these terms. Russell, J. B., Strobel, H. J., and Chen, G. J. (1988). Enrichment and isolation of a
ruminal bacterium with a very high specific activity of ammonia production. Appl. Environ. Microbiol. 54, 872–877. Schären, M., Drong, C., Kiri, K., Riede, S., Gardener, M., Meyer, U., et al. (2017). Differential effects of Monensin and a blend of essential oils on rumen September 2018 | Volume 9 | Article 2164 Frontiers in Microbiology | www.frontiersin.org 13
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| null |
Moderate intakes of soluble corn fibre or inulin do not cause gastrointestinal discomfort and are well tolerated in healthy children
|
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* These authors contributed equally to this work * These authors contributed equally to this work a Tate & Lyle PLC, 5 Marble Arch, London, W1H 7EJ, UK Supplementary Materials Table S1. AUC for individual GI symptoms for the mITT population1. Abbreviations: Max, maximum; Min,
minimum; n, sample size; Q1, quartile 1; Q3, quartile 3; SD, standard deviation. Moderate intakes of soluble corn fiber or inulin do not cause gastrointestinal discomfort and
are well tolerated in healthy children Davide Rissoa*, Melissa Kaczmarczyka*, Ieva Lauriea, Eunice Mahb, Traci M. Blonquistb, Linda
Derrigb, Kavita Karnika. Table S1. AUC for individual GI symptoms for the mITT population1. Abbreviations: Max, maximum; Min,
minimum; n, sample size; Q1, quartile 1; Q3, quartile 3; SD, standard deviation. External use permitted Table S2. AUC for individual GI symptoms by age groups for the mITT population
Abbreviations: Max, maximum; Min, minimum; n, sample size; Q1, quartile 1; Q3,
standard deviation. Table S2. AUC for individual GI symptoms by age groups for the mITT population1. Abbreviations: Max, maximum; Min, minimum; n, sample size; Q1, quartile 1; Q3, quartile 3; SD, standard deviation. Table S3. Stool frequency during the 10-day intervention periods for the mITT population1. Abbreviations: BM, bowel movement; Max, maximum; Min, minimum; n, sample size; Q1, quartile
1; Q3, quartile 3; SD, standard deviation. Abbreviations: BM, bowel movement; Max, maximum; Min, minimum; n, sample size; Q1, quartile
1; Q3, quartile 3; SD, standard deviation. External use permitted Table S4. Stool frequency during the 10-day intervention periods by age group for the mITT
population1. Abbreviations: BM, bowel movement; Max, maximum; Min, minimum; n, sample
size; Q1, quartile 1; Q3, quartile 3; SD, standard deviation. External use permitted Table S5. Stool consistency during the 10-day intervention period for the mITT population. Abbreviations:
CI, confidence interval; Max, maximum; Min, minimum; n, sample size; Q1, quartile 1; Q3, quartile 3; SD,
standard deviation. Table S6. Stool consistency during the 10-day intervention period by age group for the mIT y
g
y
p
y g g
p
p p
Abbreviations: CI, confidence interval; Max, maximum; Min, minimum; n, sample size; Q1, quartile 1; Q3,
quartile 3; SD, standard deviation. Footnote:
1Linear mixed model contained fixed effect terms for product, age stratification, and baseline/pre
intervention values and a random intercept for the subject nested within sequence Footnote: Abbreviations: CI, confidence interval; Max, maximum; Min, minimum; n, sample size; Q1, quartile 1; Q3,
quartile 3; SD, standard deviation. External use permitted ternal use permitted External use permitted Table S7. Alpha diversity indices for the mITT population. Max, maximum; Min, minimum; n, sample size;
Q1, quartile 1; Q3, quartile 3; SD, standard deviation. Measure
Source
Statistic
SCF
Inulin
High-dose
(HSCF)
Low-dose
(LSCF)
High-dose
(IH)
Low-dose
(IL)
Shannon
Summary
statistics
N
39
39
39
38
Mean (SD)
4.5 (0.3)
4.6 (0.3)
4.5 (0.31)
4.5 (0.30)
Median (Min, Max)
4.5 (3.9, 4.9)
4.7 (3.7, 4.9)
4.6 (3.8, 5)
4.6 (3.8, 5.1)
(Q1, Q3)
4.3, 4.8
4.4, 4.8
4.2, 4.7
4.3, 4.8
Model1
Estimate
4.5
4.6
4.5
4.5
Confidence interval
4.4, 4.6
4.5, 4.6
4.4, 4.5
4.5, 4.6
p-value HSCF vs IH
0.71
p-value LSCF vs IL
0.24
p-value LSCF vs
HSCF
0.09
Simpson
Summary
statistics
N
39
39
39
38
Mean (SD)
0.99 (0.006)
0.98 (0.007)
0.98 (0.008)
0.98 (0.009)
Median (Min, Max)
0.98 (0.96,
0.99)
0.98 (0.96,
0.99)
0.98 (0.96,
0.99)
0.98 (0.95,
0.99)
(Q1, Q3)
0.98, 0.99
0.98, 0.99
0.97, 0.99
0.98, 0.99
Model1
Estimate
0.98
0.98
0.98
0.98
Confidence interval
0.98, 0.98
0.98, 0.98
0.98, 0.98
0.98, 0.98
p-value HSCF vs IH
0.25
p-value LSCF vs IL
0.12
p-value LSCF vs
HSCF
0.24
Footnote:
1Linear mixed model contained fixed effect terms for product, age stratification, and baseline/pre
intervention values and a random intercept for the subject nested within sequence Footnote:
1Linear mixed model contained fixed effect terms for product, age stratification, and baseline/pre
intervention values and a random intercept for the subject nested within sequence External use permitted Figure S8. Alpha diversity by age group. Abbreviations: IH, high-dose inulin; IL, low-dose inulin; ns, not
significant; HSCF, high-dose soluble corn fibre; LSCF, low-dose soluble corn fibre; trt, treatment. Figure S9. Beta diversity by age group. Abbreviations: IH, high-dose inulin; IL, low-dose inulin; ns, not
significant; HSCF, high-dose soluble corn fibre; LSCF, low-dose soluble corn fibre. Figure S9. Beta diversity by age group. Abbreviations: IH, high-dose inulin; IL, low-dose inulin; ns, not
significant; HSCF, high-dose soluble corn fibre; LSCF, low-dose soluble corn fibre. External use permitted External use permitted Table S10. Relative abundance of specific phylum level bacterial phylotypes for the mITT population. Table S10. Relative abundance of specific phylum level bacterial phylotypes for the mITT population. Abbreviations: Max, maximum; Min, minimum; n, sample size; Q1, quartile 1; Q3, quartile 3; SD, standard
deviation. Abbreviations: CI, confidence interval; Max, maximum; Min, minimum; n, sample size; Q1, quartile 1; Q3,
quartile 3; SD, standard deviation. Abbreviations: Max, maximum; Min, minimum; n, sample size; Q1, quartile 1; Q3, quartile 3; SD, standard
deviation. External use permitted Figure S11. False-discovery rate-adjusted Wilcoxon signed rank test comparisons between products for the
mITT population. External use permitted External use permitted External use permitted
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|
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| null |
A Simplified Theory of Electroadhesion for Rough Interfaces
|
Frontiers in mechanical engineering
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| 7,772
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ORIGINAL RESEARCH
published: 12 June 2020
doi: 10.3389/fmech.2020.00027 Edited by:
Yang Xu,
University of Glasgow,
United Kingdom Keywords: electroadhesion, JKR model, DMT model, soft matter, roughness models Reviewed by:
Mehmet Ayyildiz,
Istanbul Bilgi University, Turkey
Witold Franciszek Habrat,
Rzeszów University of Technology,
Poland Reviewed by:
Mehmet Ayyildiz,
Istanbul Bilgi University, Turkey
Witold Franciszek Habrat,
Rzeszów University of Technology,
Poland
*Correspondence:
Michele Ciavarella
mciava@poliba.it
†These authors have contributed
equally to this work A Simplified Theory of
Electroadhesion for Rough Interfaces
Michele Ciavarella 1,2*† and Antonio Papangelo 1,2†
1 Department of Mechanics Mathematics and Management, Politecnico di Bari, Bari, Italy, 2 Department of Mechanical
Engineering, Hamburg University of Technology, Hamburg, Germany Electroadhesion is a very hot topic in tribology due to its implications for the science
of surface haptics. Building on theories of Persson, we develop a simpler theory for
electroadhesion between rough surfaces using the bearing area model of Ciavarella. The theory is derived for the cases of conducting and insulating bodies and shows
that pull-off forces depend mostly on the applied voltage, the surface root mean square
(RMS) height, and the longest wavelength in the surface representation. However, the
real contact area and friction additionally depend on rms slopes and hence on truncation
of the roughness spectrum. Two stickiness criteria are derived based on the present
theory and on the energy balance proposed by Persson and Tosatti. The coefficient of
friction decays with the normal pressure, tending to a plateau in a manner consistent with
existing experiments. 1. INTRODUCTION doi: 10.3389/fmech.2020.00027 Specialty section:
This article was submitted to
Tribology,
a section of the journal
Frontiers in Mechanical Engineering Received: 10 January 2020
Accepted: 21 April 2020
Published: 12 June 2020 1. INTRODUCTION An electric voltage applied between two contacting rough elastic bodies leads to accumulation of
charges of opposite signs on the surfaces, which obviously produces electrostatic attraction. The
electrostatic load adds to the external repulsive loads and possibly to van der Waals adhesive
interactions. Also, it influences the intimate contact area and therefore the frictional force when the
solids are sliding, which is of great interest for new touch screen applications (Vardar et al., 2017). It is therefore of utmost importance to understand the fundamentals of friction and adhesion,
particularly their relation with tactile perception. This is a very active field of research nowadays,
and electroadhesion is a promising technology in this area. The discovery of electroadhesion dates
back to 1923, when Johnsen and Rahbek (1923) described the physical phenomenon. †These authors have contributed
equally to this work †These authors have contributed
equally to this work p y
p
Persson (2018) has extended his theory of contact mechanics (Persson, 2001, 2007) and
developed a general mean-field theory that includes electroadhesion in a way similar to the Persson
and Scaraggi theory for van der Waals adhesion (Persson and Scaraggi, 2014). These theories use
the “Derjaguin-Muller-Toporov (DMT) assumption” (Derjaguin et al., 1975; Ciavarella et al., 2019),
namely that the contact can be considered similar to that in the absence of adhesion (repulsive
problem), and the effect of the adhesive forces is estimated based on the gap distribution by simply
convoluting the force-separation law. The theory of Persson (2018) is developed for two limiting
cases: (i) the case where an electric insulating film (actually two films in general) is interposed
between the two conducting bodies, and (ii) the case of two electric conducting solids, which results
in contact resistance and a voltage drop over a narrow region at the interface; in the latter case a
theorem of Barber (2003) on repulsive contact is used, which states that the contact conductance is
proportional to the mechanical contact stiffness. A simplified theory was also proposed by Popov
and Hess (2018). Specialty section:
This article was submitted to
Tribology,
a section of the journal
Frontiers in Mechanical Engineering
Received: 10 January 2020
Accepted: 21 April 2020
Published: 12 June 2020
Citation:
Ciavarella M and Papangelo A (2020)
A Simplified Theory of Electroadhesion
for Rough Interfaces. Front. Mech. Eng. 6:27. Citation: Ciavarella M and Papangelo A (2020)
A Simplified Theory of Electroadhesion
for Rough Interfaces. Front. Mech. Eng. 6:27. doi: 10.3389/fmech.2020.00027 June 2020 | Volume 6 | Article 27 Frontiers in Mechanical Engineering | www.frontiersin.org A Simplified Theory of Electroadhesion Ciavarella and Papangelo It is, however, unclear from the full Persson theories which
are the main parameters governing the problem, since Persson’s
theories were developed in very general settings, particularly
in terms of the full spectrum of roughness and the complex
details of the gap between the surfaces. This results in quite
elaborate theories which may not be easy to apply or interpret. A simpler theory was proposed for van der Waals adhesion by
Ciavarella (2018) and Ciavarella and Papangelo (2019), called the
bearing area model (BAM) as it provides a simple estimate of the
adhesion forces between rough hard surfaces using an extremely
simple and therefore closed-form geometric estimate of the
adhesive area. The BAM is also loosely a DMT model similar to
that of Persson and Scaraggi (2014), but thanks to the geometrical
estimate for the bearing area, it is completely expressible in
simple closed-form equations in the case of a power-law power
spectrum density (PSD) of the roughness. The main goal of the
present paper is to generalize this theory to electroadhesion. FIGURE 1 | Sketch of the geometry considered: A rough elastic solid is
squeezed against a flat rigid solid. Both solids are made of conducting
materials, with insulating coatings of thicknesses d1 and d2 and dielectric
constants ε1 and ε2. An electric voltage difference V is applied at the two
solids. The interfacial separation u = u(x) depends on the in-plane coordinate
x = (x, y). FIGURE 1 | Sketch of the geometry considered: A rough elastic solid is FIGURE 1 | Sketch of the geometry considered: A rough elastic solid is
squeezed against a flat rigid solid. Both solids are made of conducting
materials, with insulating coatings of thicknesses d1 and d2 and dielectric
constants ε1 and ε2. An electric voltage difference V is applied at the two
solids. The interfacial separation u = u(x) depends on the in-plane coordinate
x = (x, y). p
p p
g
y
As suggested in a recent paper by Dalvi et al. Citation: (2019), surface
topography can easily have more than seven orders of magnitude
of almost-power-law spectrum, including down to the Ångström
scale (see Figure S2 in Dalvi et al., 2019, which gives the 2D
isotropic PSD). But in the end, as originally suggested by Persson
and Tosatti (2001), the effective adhesive force depends only
on the large-wavelength components of the PSD; see also the
stickiness criteria derived by Ciavarella (2019). We shall also
extend the stickiness criteria to electroadhesion, and we find
surprisingly universal results, despite the very different origins
of the various proposals we compare. However, the real contact
area, and therefore the friction force, depends crucially on the
truncation of the PSD spectrum, implying a rather difficult choice
which will ultimately require a best-fit exercise. ε0
2 E2
z, i.e., ε0
2 E2
z, i.e., σzz(x) = ε0
2
V
u(x) + h0
2
,
(3) (3) where ε0 ≃8.8542 × 10−12 N/V2 is the vacuum permittivity. Hence, we have an adhesive mean pressure pad = ε0
2 V2
Z ∞
0
1
u(x) + h0
2
P(p|u) du,
(4) (4) where P(p|u) is the probability distribution of interfacial gaps at
pressure p for given u, including the contact area. For insulating
solids, the theory of Persson (2018) indeed accounts for the
adhesive interactions within the contact area [u(x)
=
0 in
Equation 3] and for those exerted outside the contact patches. The probability distribution of gaps is in fact Frontiers in Mechanical Engineering | www.frontiersin.org 2. ELECTROADHESION In
this figure, Z = 10−8 m0.6, with q0 = 102 1/m, q1 = 1010 1/m, and H = 0.7. FIGURE 3 | Typical electrostatic attraction force law replaced by a Maugis
step function law, plotted in dimensionless form with epad/eσ0 vs. eu = u/h0. 2019), i.e., neglecting the elastic deformations induced by the
adhesive interactions. In what follows, we shall assume for simplicity self-affine
fractal rough surfaces that have a typical power-law PSD
C( )
Z −2(1+H)
(8) 3.1. General Equations Determining the external load pext by Equation (7) requires in
general that the probability distribution P1(p|u) of interfacial
separations be known, which can be achieved by using Persson’s
theories (see Almqvist et al., 2011). Instead, we propose here a
simpler theory, which builds on the previous BAM (Ciavarella,
2018; Ciavarella and Papangelo, 2019) and gives a very simple
estimate of the adhesion of hard rough solids based on a bearing
area estimation of the adhesive contact area (Ciavarella, 2018). The theory is not an approximation of the DMT theory of
Persson, since it is based on a different approximation. We first
replace the electrostatic potential (3) with a Maugis–Dugdale
equivalent, for which the adhesive traction is defined as a function
of the gap u (see Figure 3): FIGURE 2 | Typical power-law PSD of a self-affine randomly rough surface. In
this figure, Z = 10−8 m0.6, with q0 = 102 1/m, q1 = 1010 1/m, and H = 0.7. pad(u) =
(
σ0,
u ≤h0,
0,
u > h0. (9) (9) FIGURE 3 | Typical electrostatic attraction force law replaced by a Maugis
step function law, plotted in dimensionless form with epad/eσ0 vs. eu = u/h0. Upon integration for nominally flat surfaces, the work of the
adhesion 1γ is given by 1γ = ε0
2 V2
Z ∞
0
1
u(x) + h0
2
du = ε0
2h0
V2. (10) (10) For this to be equal to the surface energy in the Maugis-Dugdale
approximation, i.e., 1γ =
R h0
0 σ0 du = σ0h0, we need to have σ0 = ε0
2h2
0
V2,
(11) (11) and hence the results in Ciavarella (2018) apply (BAM). For a nominally flat surface with Gaussian distribution
of heights, the main idea is to estimate the adhesive area1
analogously to a bearing area estimate which works very well for
the sphere problem (see Ciavarella, 2018); for the nominally flat
Gaussian roughness this results in FIGURE 3 | Typical electrostatic attraction force law replaced by a Maugis
step function law, plotted in dimensionless form with epad/eσ0 vs. eu = u/h0. Aad
A0
= 1
2
erfc
u −h0
√
2 hrms
−erfc
u
√
2 hrms
,
(12) (12) 2019), i.e., neglecting the elastic deformations induced by the
adhesive interactions. where u is the mean separation of the surfaces and hrms is the rms
amplitude of roughness. 1The adhesive area is defined as the portion of surface on which tensile stress
is applied. 3. BEARING AREA METHOD FOR
ELECTROADHESION FIGURE 2 | Typical power-law PSD of a self-affine randomly rough surface. In
this figure, Z = 10−8 m0.6, with q0 = 102 1/m, q1 = 1010 1/m, and H = 0.7. 2. ELECTROADHESION Let us consider the contact configuration shown in Figure 1. We have an elastic rough solid which is brought into contact
with a rigid flat surface. Both solids are made of conducting
materials, but they are coated with electric insulating (and rigid)
films of thicknesses d1 and d2, respectively, and relative dielectric
constants ε1 and ε2 (see Figure 1). A (remote) electric voltage
difference V is applied at the two conducting solids. As the elastic
solid is rough, the local interfacial separation u = u(x) depends
on the in-plane coordinate x = (x, y). P(p|u) = A
A0
δ(u) + P1(p|u),
(5) (5) where δ(u) is the delta function, A is the contact area, A0 is the
nominal contact area, and P1(p|u) is normalized so that Z ∞
0
P1(p|u) du = 1 −A
A0
. (6) (6) By writing the variation of the electric potential through the
contact pair, Persson (2018) obtained the z component of the
electric field at the interface: From the above analysis, the applied external pressure pext at
a given mean separation u can be obtained by summing the
repulsive p and the adhesive pressure pad (pressure is negative
when compressive), which results in Ez(x) = −
V
u(x) + h0
,
(1) (1) (2)
pext = p + pad = p + ε0
2
V
h0
2 A
A0
+ ε0
2 V2
Z ∞
0
1
u(x) + h0
2
P1(p|u) du,
(7) h0 = d1
ε1
+ d2
ε2
. (2)
pext = p + pad = p + ε0
2
V
h0
2 A
A0
+ ε0
2 V2
Z ∞
0
1
u(x) + h0
2
P1(p|u) du,
(7) where where where h0 = d1
ε1
+ d2
ε2
. (2) (7) (2) The normal pressure acting at the solid-vacuum interface is given
by the zz component of the Maxwell stress tensor, σzz(x)
= The normal pressure acting at the solid-vacuum interface is given
by the zz component of the Maxwell stress tensor, σzz(x)
= where the adhesive terms are estimated by following the
DMT method (Derjaguin et al., 1975; Ciavarella et al., June 2020 | Volume 6 | Article 27 Frontiers in Mechanical Engineering | www.frontiersin.org 2 Ciavarella and Papangelo A Simplified Theory of Electroadhesion FIGURE 2 | Typical power-law PSD of a self-affine randomly rough surface. 3.2. Insulating Solids In the
electroadhesion case, υ2 =
ε0V2
2E∗h2
0 replaces la/ǫ, and by changing
the voltage it can be varied by many orders of magnitude. Hence,
adhesion is favored at high applied voltage V, low elastic modulus
E∗, and thin insulating layer thickness. Clearly, if both the van
der Waals adhesion forces and the electroadhesive tractions are
p
t th
h
ld b
d where λL is the longest wavelength in the surface representation,
i.e., q0 = 2π/λL. Notice that in the standard case of van der
Waals forces, σ0
E∗≃
1γ/ǫ
E∗
=
la
ǫ with la = 1γ/E∗defines a
characteristic adhesion length, where ǫ is the characteristic length
of interaction of the order of the interatomic distance. In the
electroadhesion case, υ2 =
ε0V2
2E∗h2
0 replaces la/ǫ, and by changing where we have defineded0 = d0/h0. where we have defineded0 = d0/h0. 3.1. General Equations In what follows, we shall assume for simplicity self-affine
fractal rough surfaces that have a typical power-law PSD For intermediate mean separations, the repulsive pressure can
be obtained by using Persson’s theory (Persson, 2007; Papangelo
et al., 2017), which for self-affine fractal surfaces with power-
law PSD and fractal dimension D ≃2.2 gives (negative when
compressive) C(q) = Zq−2(1+H),
q0 < q < q1,
(8) (8) p(u) ≃−E∗q0hrms exp
−u
γ hrms
,
(13) where C(q) = 0 elsewhere, H = 3 −D is the Hurst exponent
with D being the fractal dimension of the surface, q =
2π
λ is
the wavenumber associated with the wavelength λ, q1, and q0 are
respectively the largest and smallest wavenumbers in the surface
representation, and Z =
H
π
hrms
q0
2 1
q0
−2(H+1) is a constant
multiplier, with hrms being the root mean square (rms) amplitude
of roughness (see Figure 2). (13) where γ ≃0.5 is a constant parameter and E∗is the composite
elastic modulus of the material. Frontiers in Mechanical Engineering | www.frontiersin.org June 2020 | Volume 6 | Article 27 3 Ciavarella and Papangelo A Simplified Theory of Electroadhesion 4. VOLTAGE NEAR FULL CONTACT As noted by Persson, adhesion increases very slowly as the voltage
increases, but above a certain threshold the increase becomes
very rapid as the surfaces approach each other. It may be useful
in certain applications to have an estimate of the voltage at
which the rough surfaces would be near full contact. Assuming
in our simplified theory that this occurs already at
u
hrms = 1, for
insulating bodies imposing a given pext results in the following
condition for υ: 0
the voltage it can be varied by many orders of magnitude. Hence,
adhesion is favored at high applied voltage V, low elastic modulus
E∗, and thin insulating layer thickness. Clearly, if both the van
der Waals adhesion forces and the electroadhesive tractions are
present, they should be summed. 3.2. Insulating Solids between electrical conduction and mechanical stiffness, finds
approximately that the actual voltage drop 1V(u) decreases with
the applied voltage V in a manner2 depending on the mean
separation u: between electrical conduction and mechanical stiffness, finds
approximately that the actual voltage drop 1V(u) decreases with
the applied voltage V in a manner2 depending on the mean
separation u: While for the spherical problem the bearing area estimate above
excludes the contact area itself, for Gaussian roughness the
expression (12) tends to 1 in the limit, so it would be inconsistent
to add separately the true contact area A/A0: hence, in our theory
there will be no effect of rms slopes, which in Persson’s (2018)
theory certainly enter the picture. Further, we sum the repulsive
(13) and attractive contributions to obtain an external applied
pressure (positive when tensile) 1V(u) =
V
1 + 4 d0
hrms
p(u)
E∗
,
(18) (18) where where d0 =
κ1d2 + κ2d1
κ1 + κ2
. (19) (19) pext(u)
E∗
≃−q0hrms exp
−u
γ hrms
+ υ2
2
erfc
u −h0
√
2 hrms
−erfc
u
√
2 hrms
,
(14) By Equation (11), for conducting materials the adhesive strength
will be dependent on the mean separation u. From Equation (11),
we define By Equation (11), for conducting materials the adhesive strength
will be dependent on the mean separation u. From Equation (11),
we define (14) σ0,cond(u) = ε0V2
2h2
0
1
1 + 4 d0
hrms
p(u)
E∗
2 =
σ0
1 + 4 d0
hrms
p(u)
E∗
2 ,
(20) where
we
have
defined
an
“electroadhesive”
dimensionless voltage “electroadhesive” υ =
r σ0
E∗=
r ε0
2E∗
V
h0
. (15) (20) (15) where σ0,cond(u) ≤σ0 and we recall that σ0 is the adhesive
strength for insulating bodies. Hence, for a fixed separation one
obtains For very low voltage the external load pext(u) coincides with the
repulsive load p, while for very high voltage the two surfaces will
stick together. 3.2. Insulating Solids By introducing the dimensionless parameters pext(u)
E∗
≃−q0hrms exp
−u
γ hrms
+ σ0
2E∗
h
erfc
u−h0
√
2 hrms
−erfc
u
√
2 hrms
i
1 + 4d0q0 exp
−u
γ hrms
2
(21) epext = pext
E∗,
eσ0 = σ0
E∗,
eu = u
h0
,
ehrms = hrms
h0
,
eλL = λL
h0
, (21) (16) or, in dimensionless form, or, in dimensionless form, Equation (14) can be written as epext
eu
≃−2π
eλL
ehrms exp
−eu
γehrms
+ υ2
2
h
erfc
eu−1
√
2ehrms
−erfc
eu
√
2ehrms
i
1 + 8π
eλL
ed0 exp
−eu
γehrms
2
,
(22) ext(eu) ≃−2π
eλL
ehrms exp
−eu
γehrms
+ υ2
2
erfc
eu −1
√
2ehrms
−erfc
eu
√
2ehrms
,
(17)
epext
eu
≃−2π
eλL
ehrms exp
−eu
γehrms
+ υ2
2
h
erfc
eu−1
√
2ehrms
−erfc
eu
√
2ehrms
i
1 + 8π
eλL
ed0 exp
−eu
γehrms
2
,
(22) epext(eu) ≃−2π
eλL
ehrms exp
−eu
γehrms
+ υ2
2
erfc
eu −1
√
2ehrms
−erfc
eu
√
2ehrms
,
(17) (22) (17) where λL is the longest wavelength in the surface representation,
i.e., q0 = 2π/λL. Notice that in the standard case of van der
Waals forces, σ0
E∗≃
1γ/ǫ
E∗
=
la
ǫ with la = 1γ/E∗defines a
characteristic adhesion length, where ǫ is the characteristic length
of interaction of the order of the interatomic distance. In the
electroadhesion case, υ2 =
ε0V2
2E∗h2
0 replaces la/ǫ, and by changing
the voltage it can be varied by many orders of magnitude. Hence,
adhesion is favored at high applied voltage V, low elastic modulus
E∗, and thin insulating layer thickness. Clearly, if both the van
der Waals adhesion forces and the electroadhesive tractions are
present, they should be summed. where λL is the longest wavelength in the surface representation,
i.e., q0 = 2π/λL. Notice that in the standard case of van der
Waals forces, σ0
E∗≃
1γ/ǫ
E∗
=
la
ǫ with la = 1γ/E∗defines a
characteristic adhesion length, where ǫ is the characteristic length
of interaction of the order of the interatomic distance. Frontiers in Mechanical Engineering | www.frontiersin.org 6. STICKINESS CRITERIA The above results show that the pull-offtraction is principally
determined by hrms, q0, and υ. Indeed, for a low contact area,
neither the repulsive nor the attractive tractions depend on
the surface “magnification” ζ = q1/q0 as in the adhesionless
load-separation relation (13). By adopting the BAM approach
to estimate the adhesive contact area and using the results in
Ciavarella (2019) for the case of van der Waals interactions, we (24) We see that the main parameters on which υfull depends are q0
and hrms. We see that the main parameters on which υfull depends are q0
and hrms. 3.3. Conducting Solids υfull =
s
ε0V2
full
2E∗h2
0
=
v
u
u
u
t2
epext + 2π
eλL
ehrms exp
−1
γ
erfc
1
√
2 −
1
√
2ehrms
−erfc
1
√
2
,
(23)
2Notice that we are following a DMT-like approach, so from Equation (13) we have
written the contact stiffness K⊥= p/(γ hrms); see Persson (2018). υfull =
s
ε0V2
full
2E∗h2
0
=
v
u
u
u
t2
epext + 2π
eλL
ehrms exp
−1
γ
erfc
1
√
2 −
1
√
2ehrms
−erfc
1
√
2
,
(23) If an electric voltage is applied between two contacting solids
with certain electric conductivity, then one has to account for the
electric current flow through the contact patches; hence, there
would be attraction only outside of the contact areas. However,
the voltage drop V across the (non-)contacting interface will
depend on the electric conductivities κ1 and κ2 of the solids. Barber’s (2003), using Persson (2018) theorem for the analogy (23) 2Notice that we are following a DMT-like approach, so from Equation (13) we have
written the contact stiffness K⊥= p/(γ hrms); see Persson (2018). Frontiers in Mechanical Engineering | www.frontiersin.org June 2020 | Volume 6 | Article 27 4 A Simplified Theory of Electroadhesion Ciavarella and Papangelo where we take γ = 0.5. For conducting solids, using Equation
(22) we obtain where we take γ = 0.5. For conducting solids, using Equation
(22) we obtain where we take γ = 0.5. For conducting solids, using Equation
(22) we obtain
υfull =
s
ε0V2
full
2E∗h2
0
=
v
u
u
u
u
t2
epext + 2π
eλL
ehrms exp
−1
γ
1 + 8π
eλL
ed0 exp
−1
γ
2
erfc
1
√
2 −
1
√
2ehrms
−erfc
1
√
2
. (24) 2, 048 and varying υ = [0.01, 0.02, 0.04, 0.08, 0.2]. Increasing the
dimensionless voltage υ increases the tensile traction needed to
separate the rough surfaces and makes the surfaces more prone to
adhesion. We infer from Figures 4–6 that conducting solids are
less inclined to exhibit macroscopic adhesion due to the smaller
voltage drop experienced at the interface. 5. NUMERICAL RESULTS ehrms (log-log scale), with a
constant voltage υ = 0.08 and increasing eλL = 2, 048 × [0.1, 1, 5, 10] for both
insulating (solid lines) and conducting (with ed0 = 1, 000, dashed lines) solids. FIGURE 5 | Pull-off mean pressure epmax
ext /eσ0 vs. ehrms (log-log scale), with a
constant voltage υ = 0.08 and increasing eλL = 2, 048 × [0.1, 1, 5, 10] for both
insulating (solid lines) and conducting (with ed0 = 1, 000, dashed lines) solids. FIGURE 4 | External pressure epext/eσ0 (> 0 when tensile) as a function of the
mean separation eu for eλL = 2, 048, ehrms = 1, and varying
υ = [0.01, 0.02, 0.04, 0.08, 0.2]. Solid lines correspond to insulating solids and
dashed lines to conducting solids with ed0 = 1, 000. FIGURE 6 | Pull-off mean pressure epmax
ext /eσ0 vs. ehrms (log-log scale) for
eλL = 2, 048 and varying υ = [0.01, 0.02, 0.04, 0.08, 0.2]. Solid lines
correspond to insulating solids and dashed lines to conducting solids with
ed0 = 1, 000. FIGURE 4 | External pressure epext/eσ0 (> 0 when tensile) as a function of the
mean separation eu for eλL = 2, 048, ehrms = 1, and varying
υ = [0.01, 0.02, 0.04, 0.08, 0.2]. Solid lines correspond to insulating solids and
dashed lines to conducting solids with ed0 = 1, 000. FIGURE 6 | Pull-off mean pressure epmax
ext /eσ0 vs. ehrms (log-log scale) for
eλL = 2, 048 and varying υ = [0.01, 0.02, 0.04, 0.08, 0.2]. Solid lines
correspond to insulating solids and dashed lines to conducting solids with
ed0 = 1, 000. FIGURE 6 | Pull-off mean pressure epmax
ext /eσ0 vs. ehrms (log-log scale) for
eλL = 2, 048 and varying υ = [0.01, 0.02, 0.04, 0.08, 0.2]. Solid lines
correspond to insulating solids and dashed lines to conducting solids with
ed0 = 1, 000. FIGURE 4 | External pressure epext/eσ0 (> 0 when tensile) as a function of the
mean separation eu for eλL = 2, 048, ehrms = 1, and varying
υ = [0.01, 0.02, 0.04, 0.08, 0.2]. Solid lines correspond to insulating solids and
dashed lines to conducting solids with ed0 = 1, 000. 5. NUMERICAL RESULTS Figure 4 shows the external pressure epext/eσ0 as a function of
the mean separation eu for eλL = 2, 048, ehrms = 1, and varying
υ = [0.01, 0.02, 0.04, 0.08, 0.2]. In Figure 4 solid lines refer to
insulating solids, while dashed lines are used for conducting
solids withed0 = 1, 000. Upon increasing the applied voltage, the
mean load vs. mean separation curve becomes more and more
attractive so that macroscopic adhesion becomes important. Clearly differences between the two cases of insulating and
conducting bodies are more evident at higher voltages υ (see
Equation 18). FIGURE 5 | Pull-off mean pressure epmax
ext /eσ0 vs. ehrms (log-log scale), with a
constant voltage υ = 0.08 and increasing eλL = 2, 048 × [0.1, 1, 5, 10] for both
insulating (solid lines) and conducting (with ed0 = 1, 000, dashed lines) solids. FIGURE 6 | Pull-off mean pressure epmax
ext /eσ0 vs. ehrms (log-log scale) for
eλL = 2, 048 and varying υ = [0.01, 0.02, 0.04, 0.08, 0.2]. Solid lines
correspond to insulating solids and dashed lines to conducting solids with
ed0 = 1, 000. FIGURE 5 | Pull-off mean pressure epmax
ext /eσ0 vs. ehrms (log-log scale), with a
constant voltage υ = 0.08 and increasing eλL = 2, 048 × [0.1, 1, 5, 10] for both
insulating (solid lines) and conducting (with ed0 = 1, 000, dashed lines) solids. If max[epext(eu ∈[0, +∞))] = epmax
ext
> 0, then the surfaces
are sticky and a positive external pressure is needed to separate
them. Figure 5 shows the abrupt decay in the pull-offpressure
epmax
ext /eσ0 withehrms, for a constant voltage υ = 0.08 and increasing
eλL = 2, 048 × [0.1, 1, 5, 10] for both insulating (solid lines) and
conducting (with ed0 = 1, 000, dashed lines) solids. It can be
seen that for a given ehrms, the pull-offpressure epmax
ext
increases
for surfaces with longer wavelengths eλL. In Figure 6, the decay
of the pull-offpressure with ehrms is plotted for constant eλL = FIGURE 5 | Pull-off mean pressure epmax
ext /eσ0 vs. ehrms (log-log scale), with a
constant voltage υ = 0.08 and increasing eλL = 2, 048 × [0.1, 1, 5, 10] for both
insulating (solid lines) and conducting (with ed0 = 1, 000, dashed lines) solids. FIGURE 5 | Pull-off mean pressure epmax
ext /eσ0 vs. 3Notice that we use the original convention and notation for C(q) of Persson and
not the definition of Dalvi et al. (2019), which is Ciso(q) = 4π2C(q). 5. NUMERICAL RESULTS FIGURE 4 | External pressure epext/eσ0 (> 0 when tensile) as a function of the
mean separation eu for eλL = 2, 048, ehrms = 1, and varying
υ = [0.01, 0.02, 0.04, 0.08, 0.2]. Solid lines correspond to insulating solids and
dashed lines to conducting solids with ed0 = 1, 000. Frontiers in Mechanical Engineering | www.frontiersin.org Frontiers in Mechanical Engineering | www.frontiersin.org Frontiers in Mechanical Engineering | www.frontiersin.org June 2020 | Volume 6 | Article 27 5 A Simplified Theory of Electroadhesion Ciavarella and Papangelo FIGURE 7 | Pull-off mean pressure epmax
ext /eσ0 as a function of υ and ehrms
(log-log scale) for eλL = 2, 048: (A) insulating solids; (B) conducting solids with
ed0 = 200. The thick black solid and dashed lines indicate the stickiness limits
obtained, respectively, with BAM (Equation 32) and by following the Persson
and Tosatti energy balance for H = 0.8 (Equation 31). easily obtain in our electroadhesion case that for stickiness, hrms <
0.6σ0h0
E∗λL
0.5
= 0.775υ
p
h0λL,
(25) (25) where we recognize that in the electroadhesive case the adhesive
energy depends on the applied dimensionless voltage υ. Alternatively, a stickiness criterion could be defined using
the energy balance proposed by Persson and Tosatti’s theory for
classical van der Waals adhesion. Persson and Tosatti (2001)
proposed that for a rough interface the effective adhesive energy
1γeffavailable at pull-offis 1γeff= A
A0
1γ −Uel
A0
,
(26) (26) where the term A
A0 (> 1) is the ratio between the contact area A in
full contact and the nominal contact area A0 and is greater than 1
because of a roughness-induced effect, and Uel is the elastic strain
energy stored to squeeze the roughness, with isotropic power
spectrum C(q) in full contact3 given by Uel (ζ)
A0
= πE∗
2
Z q1
q0
q2C(q) dq,
(27) (27) which depends on the magnification. With electroadhesion, we
replace 1γ with the term
ε0
2h0 V2, resulting in (omitting the van
der Waals contribution and for A/A0 ≃1) 1γeff= ε0
2h0
V2 −Uel
A0
. (28) (28) Using the results in Ciavarella (2019), a new stickiness criterion,
akin to that of “Persson and Tosatti", is derived by imposing
1γeff= 0 in (28), which can be cast in terms of the roughness
rms amplitude as hrms < υ
r
h0λL
2H −1
πH
. Frontiers in Mechanical Engineering | www.frontiersin.org 5. NUMERICAL RESULTS All plots are for a constant external pressure
pext = 100 Pa. In (A), the dot-dashed line represents the theoretical
electroadhesive strength σ0. In all panels the black circles and squares
represent estimates of the critical voltage υfull using Equations (23) and (24). FIGURE 9 | (A) Adhesive pressure pad = pext −p, (B) mean separation u, and
(C) normalized contact area A/A0 as functions of the applied voltage V for
E* = [1, 10, 100] MPa and for both insulating (solid lines) and conducting (with
d0 = 10 mm, dashed lines) solids. All plots are for a constant external pressure
pext = 100 Pa. In (A), the dot-dashed line represents the theoretical
electroadhesive strength σ0. In all panels the black circles and squares
represent estimates of the critical voltage υfull using Equations (23) and (24). FIGURE 9 | (A) Adhesive pressure pad = pext −p, (B) mean separation u, and
(C) normalized contact area A/A0 as functions of the applied voltage V for
E* = [1, 10, 100] MPa and for both insulating (solid lines) and conducting (with
d0 = 10 mm, dashed lines) solids. All plots are for a constant external pressure
pext = 100 Pa. In (A), the dot-dashed line represents the theoretical
electroadhesive strength σ0. In all panels the black circles and squares
represent estimates of the critical voltage υfull using Equations (23) and (24). 5. NUMERICAL RESULTS (29) FIGURE 7 | Pull-off mean pressure epmax
ext /eσ0 as a function of υ and ehrms
(log-log scale) for eλL = 2, 048: (A) insulating solids; (B) conducting solids with
ed0 = 200. The thick black solid and dashed lines indicate the stickiness limits
obtained, respectively, with BAM (Equation 32) and by following the Persson
and Tosatti energy balance for H = 0.8 (Equation 31). E 7 | Pull-off mean pressure epmax
ext /eσ0 as a function of υ and ehrms (29) For the typical Hurst exponent H
=
0.8 (Persson, 2014),
this becomes hrms < 0.5υ
p
h0λL,
(30) (30) which, except for the threshold (0.5 instead of 0.775), compares
well with the other criteria obtained with BAM. conducting solids (Equation 22, with ed0 = 200) in Figure 7B. The stickiness boundaries obtained with (32) and (31) for
H = 0.8 are represented by thick black solid and dashed lines,
respectively. Even though the two criteria have been obtained
from different arguments, it is noteworthy that they yield very
similar outcomes, which agree satisfactorily with the numerical
results obtained from Equations (17) and (22). In general the
pull-offforce increases with increasing voltage υ and decreasing
amplitude roughness ehrms. We have also distinguished a “no
sticky” region for epmax
ext /eσ0 < 10−4, which corresponds to very
rough hard solids and low voltage. We can rewrite the stickiness criteria in dimensionless form: ehrms < 0.5υ
q
eλL
(Persson-Tosatti),
(31)
ehrms < 0.775υ
q
eλL
(BAM). (32) (31) (32) Figure 7 shows two contour plots of the pull-offmean pressure
epmax
ext /eσ0 as a function of υ and ehrms (log-log scale) with eλL =
2, 048, for insulating solids (Equation 17) in Figure 7A and for Finally, in Figure 8 we provide a qualitative estimate of
the friction coefficient µ/eτ as a function of the external June 2020 | Volume 6 | Article 27 Frontiers in Mechanical Engineering | www.frontiersin.org 6 A Simplified Theory of Electroadhesion Ciavarella and Papangelo FIGURE 8 | Friction coefficient µ/eτ =
A/A0
(−epext) as a function of the external
pressure −pext for ehrms = 1, h′
rms = 2.5 and dimensionless electrical voltage
υ = [0.01, 0.02, 0.03, 0.04]. 5. NUMERICAL RESULTS pressure −epext: FIGURE 8 | Friction coefficient µ/eτ =
A/A0
(−epext) as a function of the external
pressure −pext for ehrms = 1, h′
rms = 2.5 and dimensionless electrical voltage
υ = [0 01 0 02 0 03 0 04] FIGURE 9 | (A) Adhesive pressure pad = pext −p, (B) mean separation u, and
(C) normalized contact area A/A0 as functions of the applied voltage V for
E* = [1, 10, 100] MPa and for both insulating (solid lines) and conducting (with
d0 = 10 mm, dashed lines) solids. All plots are for a constant external pressure
pext = 100 Pa. In (A), the dot-dashed line represents the theoretical
electroadhesive strength σ0. In all panels the black circles and squares
represent estimates of the critical voltage υfull using Equations (23) and (24). pressure −epext: µ/eτ =
A/A0
−epext
,
(33) (33) where we have defined a dimensionless shear strength of the
interface, eτ = τ/E∗. The normalized contact area is computed
according to Persson’s (2001) theory as where we have defined a dimensionless shear strength of the
interface, eτ = τ/E∗. The normalized contact area is computed
according to Persson’s (2001) theory as A
A0
= erf
√π
h′rms
2π
eλL
ehrms exp
−eu
γehrms
,
(34) (34) where h′
rms is the surface rms slope and A
A0 is corrected by a factor
of √π in the brackets to give it the correct slope at the origin
(see Putignano et al., 2012; Violano et al., 2019). In Figure 8, we
used eλL = 2, 048, ehrms = 1 and h′
rms = 2.5 and varied the
dimensionless electrical voltage as υ = [0.01, 0.02, 0.03, 0.04] for
both insulating (solid lines) and conducting (with ed0 = 250,
dashed lines) solids. Figure 8 shows that the friction coefficient
increases with increasing voltage and gets very high for low
normal forces, as a finite contact area is possible under a
vanishing normal load. The curves are consistent with what has
been observed in experiments (Shultz et al., 2015; Ayyildiz et al.,
2018; Sirin et al., 2019). FIGURE 9 | (A) Adhesive pressure pad = pext −p, (B) mean separation u, and
(C) normalized contact area A/A0 as functions of the applied voltage V for
E* = [1, 10, 100] MPa and for both insulating (solid lines) and conducting (with
d0 = 10 mm, dashed lines) solids. 7. FURTHER RESULTS external pressure pext = 100 Pa, and the normalized contact
area is computed according to Equation (34). For harder solids
we get lower adhesive contact pressure, higher mean separation,
and smaller contact area. Figure 9C effectively shows that A/A0
remains very small over the entire range of applied voltage,
indicating that the approximation we used is correct. In general,
for a given voltage, conducting bodies show less adhesion, higher
average separation, and smaller contact area. As in Persson
(2018), a critical voltage can be identified above which a sharp
increase in pad is observed and which can be estimated using
Equations (23) and (24); see the black circles and squares in
Figure 9. In this section we provide a possible example with dimensional
results based on a typical rough surface with the PSD shown in
Figure 2, for which Z = 10−8 m0.6 with q0 = 102 1/m, q1 =
1010 1/m and H = 0.7, so that the surface rms height is hrms =
8.43 µm and the surface rms slope is h′
rms = 0.324. Figure 9A
shows the adhesive pressure pad = pext−p, in Figure 9B the mean
separation u, and in Figure 9C the normalized contact area A/A0
as a function of the applied voltage for E∗= [1, 10, 100] MPa
and for both insulating (solid lines) and conducting (with d0 =
10 mm, dashed lines) solids. All the plots are for a constant June 2020 | Volume 6 | Article 27 Frontiers in Mechanical Engineering | www.frontiersin.org 7 A Simplified Theory of Electroadhesion Ciavarella and Papangelo DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the
article/supplementary material. where φ is defined by u(x) + h0(ω0) =
u(x) + h0(ω0)
exp(iφ). (37) (37) 8. DISCUSSION: ALTERNATING CURRENT surfaces. The model is derived for the cases of insulating and
conducting bodies, where in the latter case the voltage drop
near the interface needs to be evaluated by taking into account
the current flowing through the asperity contact regions. We
have shown that the friction coefficient increases with increasing
voltage and can reach very high values for low normal forces,
in agreement with experimental results. We have introduced a
new dimensionless parameter for electroadhesion, which governs
the behavior of the proposed model. Pull-offforces depend
mostly on well-defined quantities, such as the applied voltage,
the surface rms height, and the longest wavelength in the surface
representation. In the limit of small contact area, two stickiness
criteria have been derived based on the present theory and on
the energy balance proposed by Persson and Tosatti, which
turns out to give very similar results (except for a prefactor) for
pure-power-law power spectral densities. Electroadhesion with direct current is not as easy to detect as it
is when alternating current (AC) is applied. However, in terms of
the mathematical model, very few changes are needed to translate
the above results to the case of AC voltage. Indeed, when the
applied voltage oscillates in time, V(t), one should take into
account the variation of the dielectric properties of the media
with the applied frequency and replace h0 in Equation (2) with
a function h0(ω) =
d1
ε1(ω) +
d2
ε2(ω),
(35) (35) where εi(ω) are the dielectric functions of the two media. In the
classical case of sinusoidal voltage, V(t) = V0 cos(ω0t), we have
to change (3) to σzz(x, t) = ε0
4
V0
u(x) + h0 (ω0)
!2 1 + cos(2ω0t + 2φ)
, (36) AUTHOR CONTRIBUTIONS Notice that in this case we have that the adhesive traction σzz(x, t)
oscillates in time about its mean value All authors listed have made a substantial, direct and intellectual
contribution to the work, and approved it for publication. σzz(x) =
2π
ω0
Z
0
σzz(x, t) dt = ε0
4
V0
u(x) + h0 (ω0)
!2
,
(38) ACKNOWLEDGMENTS (38) (38) MC and AP acknowledge support from the Italian Ministry
of Education, University and Research under the Programme
Department of Excellence Legge 232/2016 (grant no. CUP-
D94I18000260001). AP is thankful to the DFG (German
Research Foundation) for funding the project PA 3303/1-1, and
acknowledges support from PON Ricerca e Innovazione 2014-
2020-Azione I.2 - D.D. n. 407, 27/02/2018, bando AIM (grant no. AIM1895471). MC and AP acknowledge support from the Italian Ministry
of Education, University and Research under the Programme
Department of Excellence Legge 232/2016 (grant no. CUP-
D94I18000260001). AP is thankful to the DFG (German
Research Foundation) for funding the project PA 3303/1-1, and
acknowledges support from PON Ricerca e Innovazione 2014-
2020-Azione I.2 - D.D. n. 407, 27/02/2018, bando AIM (grant no. AIM1895471). which coincides with Equation (3) for constant voltage. 9. CONCLUSIONS Using the bearing area model of Ciavarella, we have developed
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mechanics. Phys. Rev. Lett. 99:125502. doi: 10.1103/PhysRevLett.99.125502 Vardar, Y., Guclu, B., and Basdogan, C. (2017). Effect of waveform on tactile
perception by electrovibration displayed on touch screens. IEEE Trans. Haptics
10:488. doi: 10.1109/TOH.2017.2704603 Persson, B. N. J. (2014). On the fractal dimension of rough surfaces. Tribol. Lett. 54, 99–106. doi: 10.1007/s11249-014-0313-4 Violano,
G.,
Afferrante,
L.,
Papangelo,
A.,
and
Ciavarella,
M. (2019). On stickiness of multiscale randomly rough surfaces. J. Adhes. 1–19. doi: 10.1080/00218464.2019.1685384 Persson, B. N. J. (2018). The dependency of adhesion and friction on electrostatic
attraction. J. Chem. Phys. 148:144701. doi: 10.1063/1.5024038 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. 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. Persson, B. N. J., and Tosatti, E. (2001). The effect of surface roughness
on
the
adhesion
of
elastic
solids. J. Chem. Phys. 115,
5597–5610. doi: 10.1063/1.1398300 Popov, V. L., and Hess, M. (2018). Voltage induced friction in a contact of
a finger and a touchscreen with a thin dielectric coating. arXiv [Preprint]. arXiv:1805.08714. Available online at: https://arxiv.org/abs/1805.08714 Copyright © 2020 Ciavarella and Papangelo. 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. Copyright © 2020 Ciavarella and Papangelo. 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. Putignano, C., Afferrante, L., Carbone, G., and Demelio, G. (2012). A new efficient
numerical method for contact mechanics of rough surfaces. Int. J. Solids Struct. 49, 338–343. doi: 10.1016/j.ijsolstr.2011.10.009 Shultz, C. Frontiers in Mechanical Engineering | www.frontiersin.org June 2020 | Volume 6 | Article 27 REFERENCES D., Peshkin, M. A., and Colgate, J. E. (2015). “Surface haptics
via electroadhesion: Expanding electrovibration with Johnsen and Rahbek,” June 2020 | Volume 6 | Article 27 Frontiers in Mechanical Engineering | www.frontiersin.org 9
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https://zenodo.org/records/574484/files/BDJ_article_6459.pdf
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English
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Corrigendum: Three new species of Trigonospila Pokorny (Diptera: Tachinidae), from Area de Conservación Guanacaste, northwestern Costa Rica, with a key for their identification
|
Biodiversity data jurnal
| 2,015
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cc-by
| 761
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Biodiversity Data Journal 3: e6459
doi: 10.3897/BDJ.3.e6459
Corrigendum
Corrigendum: Three new species of Trigonospila
Pokorny (Diptera: Tachinidae), from Area de
Conservación Guanacaste, northwestern Costa
Rica, with a key for their identification
AJ Fleming , D. Monty Wood , Daniel Janzen , Winnie Hallwachs , M. Alex Smith
‡ Agriculture Agri-Food Canada, Ottawa, Canada
§ University of Pennsylvania, Philadelphia, United States of America
| Department of Integrative Biology and the Biodiversity Institute of Ontario, Guelph, Canada
Corresponding author: AJ Fleming (ajfleming604@gmail.com)
Academic editor: Pierfilippo Cerretti
Received: 02 Sep 2015 | Accepted: 28 Sep 2015 | Published: 07 Oct 2015
Citation: Fleming AJ, Wood DM, Janzen D, Hallwachs W, Smith MA (2015) Corrigendum: Three new species of
Trigonospila Pokorny (Diptera: Tachinidae), from Area de Conservación Guanacaste, northwestern Costa Rica,
with a key for their identification. Biodiversity Data Journal 3: e6459. doi: 10.3897/BDJ.3.e6459
In preparation of the manuscript (Fleming et al. 2015) the authors incorrectly misspelled
the patronym dedicated to Jose Mario Moraga. Therefore Based on Art. 32.5.1.1 of the
International Code of Zoological Nomenclature (International Commission on Zoological
Nomenclature 1999), and considering the etymology of the species we hereby act to
correct the incorrect original misspelling of the name Trigonospila josemariamoragai
Fleming & Wood, 2015, to the correct spelling Trigonospila josemariomoragai Fleming &
Wood, 2015. All details pertaining to original date of publication and original pagination
remain the same.
‡
‡
§
§
| Biodiversity Data Journal 3: e6459
doi: 10.3897/BDJ.3.e6459
Corrigendum © Fleming AJ 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. Corrigendum: Three new species of Trigonospila
Pokorny (Diptera: Tachinidae), from Area de
Conservación Guanacaste, northwestern Costa
Rica, with a key for their identification ‡ Agriculture Agri-Food Canada, Ottawa, Canada
§ University of Pennsylvania, Philadelphia, United States of America
| Department of Integrative Biology and the Biodiversity Institute of Ontario, Guelph, Canada Corresponding author: AJ Fleming (ajfleming604@gmail.com)
Academic editor: Pierfilippo Cerretti
Received: 02 Sep 2015 | Accepted: 28 Sep 2015 | Published: 07 Oct 2015
Citation: Fleming AJ, Wood DM, Janzen D, Hallwachs W, Smith MA (2015) Corrigendum: Three new species of
Trigonospila Pokorny (Diptera: Tachinidae), from Area de Conservación Guanacaste, northwestern Costa Rica,
with a key for their identification. Biodiversity Data Journal 3: e6459. doi: 10.3897/BDJ.3.e6459 In preparation of the manuscript (Fleming et al. 2015) the authors incorrectly misspelled
the patronym dedicated to Jose Mario Moraga. Therefore Based on Art. 32.5.1.1 of the
International Code of Zoological Nomenclature (International Commission on Zoological
Nomenclature 1999), and considering the etymology of the species we hereby act to
correct the incorrect original misspelling of the name Trigonospila josemariamoragai
Fleming & Wood, 2015, to the correct spelling Trigonospila josemariomoragai Fleming &
Wood, 2015. All details pertaining to original date of publication and original pagination
remain the same. In preparation of the manuscript (Fleming et al. 2015) the authors incorrectly misspelled
the patronym dedicated to Jose Mario Moraga. Therefore Based on Art. 32.5.1.1 of the
International Code of Zoological Nomenclature (International Commission on Zoological
Nomenclature 1999), and considering the etymology of the species we hereby act to
correct the incorrect original misspelling of the name Trigonospila josemariamoragai
Fleming & Wood, 2015, to the correct spelling Trigonospila josemariomoragai Fleming &
Wood, 2015. All details pertaining to original date of publication and original pagination
remain the same. © Fleming AJ 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. Fleming AJ et al. Fleming AJ et al. 2 2 References •
Fleming AJ, Wood DM, Janzen D, Hallwachs W, Smith MA (2015) Three new species
of Trigonospila Pokorny (Diptera: Tachinidae), from Area de Conservación Guanacaste,
northwestern Costa Rica, with a key for their identification. Biodiversity Data Journal 3:
e4595. DOI: 10.3897/bdj.3.e4595 j
•
International Commission on Zoological Nomenclature (1999) International code of
zoological nomenclature. [Code international de nomenclature zoologique]. Fourth
Edition. International Trust for Zoological Nomenclature, International Union of
Biological Sciences. c/o Natural History Museum, London, England, 306 pp. URL: http://
dx.doi.org/10.5962/bhl.title.50608 [ISBN 0 85301 006 4] DOI: 10.5962/bhl.title.50608 •
International Commission on Zoological Nomenclature (1999) International code of
zoological nomenclature. [Code international de nomenclature zoologique]. Fourth
Edition. International Trust for Zoological Nomenclature, International Union of
Biological Sciences. c/o Natural History Museum, London, England, 306 pp. URL: http://
dx.doi.org/10.5962/bhl.title.50608 [ISBN 0 85301 006 4] DOI: 10.5962/bhl.title.50608
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https://openalex.org/W2748379667
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http://jurnal.unmer.ac.id/index.php/jpkm/article/download/1292/854
|
Indonesian
| null |
PENYULUHAN HUKUM PERDATA DAN PIDANA BAGI APARAT KELURAHAN CEMPAKA BARU KEMAYORAN JAKARTA PUSAT
|
Abdimas
| 2,017
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cc-by-sa
| 4,206
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Jurnal ABDIMAS Unmer Malang Jurnal ABDIMAS Unmer Malang Vol. 2, Nomor 1, Juni 2017 1. Metode ceramah Metode ini digunakan untuk menyampaikan
materi penyuluhan yang bersifat kognitif seperti
sistem, azas, serta langkah-langkah dalam
peradilan perdata dan pidana. Pelaksanaan metode
ini digunakan waktu sebanyak 40% untuk
ceramah atau penyampaian materi, sedangkan
sisanya 60% digunakan untuk diskusi dan tanya
jawab. 1. PENDAHULUAN hukum Perdata dan hukum Pidana serta hukum
Acara.Berdasarkan uraian di atas maka rumusan
masalahnya
adalah
Bagaimana
melakukan
penyuluhan hukum khususnya hukum perdata dan
hukum pidana agar para aparat Kelurahan Cempaka
Baru Kecamatan Kemayoran meningkat kesadaran
hukumnya. Upaya peningkatan kesadaran hukum dapat
dilakukan
dengan
penyuluhan
hukum
secara
berkesinambungan
sehingga
menyadarkan
masyarakat akan pentingnya pemahaman terhadap
hukum yang berlaku (hukum positif) pada tata hukum
Indonesia. Rendahnya kesadaran hukum dapat
disebabkan oleh kurangnya sosialisasi hukum,
kurangnya akses masyarakat tentang informasi
hukum dan budaya masyarakat itu sendiri. Untuk itu
diperlukan
upaya
untuk
membuka
wawasan
pengetahuan hukum masyarakat agar lebih
memahami akan hukumnya sendiri, upaya hukum
yang dapat dilakukan apabila terjadi sengketa,
maupun untuk tujuan peningkatan kesadaran hukum
agar masyarakat makin taat hukum dan melek hukum. Pada hakikatnya upaya ini dilakukan dengan
memberi bekal materi pengetahuan hukum tentang
sistem hukum, kemudian dilanjutkan pada hukum
perdata dan hukum pidana, yang sangat penting untuk
diketahui oleh masyarakat, utamanya aparat sebagai
struktural pelayan masyarakat. 2. METODE KEGIATAN Kegiatan ini dilaksanakan dengan menggunakan
metode-metode berikut: Kegiatan ini dilaksanakan dengan menggunakan
metode-metode berikut: Derta Rahmanto1), Endang Purwaningsih2), Evie Rachmawati Nur Ariyanti3)
1,2,3)Fakultas Hukum Universitas YARSI, Jakarta
e-Mail : e.purwaningsih@yarsi.ac.id Derta Rahmanto1), Endang Purwaningsih2), Evie Rachmawati Nur Ariyanti3)
1,2,3)Fakultas Hukum Universitas YARSI, Jakarta
e-Mail : e.purwaningsih@yarsi.ac.id Abstract Promoting legal awareness to signify the importance of understanding the positive laws in Indonesia can be
done through sustainable counselling programs. The poor legal awareness might result from the lack of legal-
awareness raising programs, the lack of public access to legal-related issues, and the less-supporting local
culture. In attempts to better promote legal awareness, sustainable programs should start top-down, meaning
the programs are first addressed to sub-district officers so that they can set good examples to their people. The curriculum should first highlight the overview of Indonesia’s legal system, followed by civil law and
criminal law issues. These are fundamental for sub-district officers in Cempaka Baru Kemayoran Jakarta
Pusat. These legal-awareness raising programscame in the form of lectures and trainings. To see the
effectiveness, a pre-test and post-test were given to the participants. The conclusion suggests that—in attempts
to better promote legal awarenessand knowledge for Cempaka Baru sub-district officers—periodical
counselling on civil and criminal laws is essential. The curriculum should also include such topics as
preventive and repressive measures in law enforcement. Keywords: counselling, civil law, criminal law, sub-district officer Deskripsi data Posttest Setelah
dilakukan
penyuluhan,
dengan
menggunakan berbagai metode penyuluhan yang
meliputi metode ceramah interaktif, tanya jawab dua
arah, dan penyelesaian kasus hukum, pemahaman
para peserta penyuluhan dalam hal ini aparat
kelurahan Cempaka Baru semakin meningkat. Peningkatakan pemahaman mereka dapat dilihat pada
tabel berikut. 2. Metode Pelatihan Pada umumnya aparat Kelurahan Cempaka Baru
Kecamatan Kemayoran belum memahami secara
benar tentang Hukum Perdata maupun Hukum Pidana
dan bagaimana beracara melalui hukum acara perdata
dan hukum acara pidana. Para aparat belum pernah
mendapatkan penyuluhan atau pelatihan tentang Dalam metode ini, kegiatan utama yang
dilaksanakan
adalah
pembimbingan
dan
pelatihan bagi para peserta untuk membuat
pengaduan ataupun gugatan. Kegiatan ini antara
lain berupa pembimbingan dan pelatihan tentang
penulisan hukum, meskipun pada tingkat awal. 54 Jurnal ABDIMAS Unmer Malang Vol. 2, Nomor 1, Juni 2017 mayoritas peserta menjawab 2,9 atau dibulatkan
menjadi 3, yang berarti mayoritas peserta sebelum
dilakukan penyuluhan menjawab cukup paham. Setelah mengetahui pokok permasalahan aparat
kelurahan yang selama ini belum pernah menerima
penyuluhan sejenis, dalam rangka membantu para
aparat agar dapat menguasai materi hukum positif
Indonesia khususnya tentang Hukum Perdata dan
Pidana, penting sekali dilaksanakan kegiatan
penyuluhan
ini. Kegiatan
penyuluhan
ini
dilaksanakan 1x dengan peserta 138 RT yang diwakili
oleh 72 orang. Pada butir nomor 5 yang membahas tentang
istilah penyidikan, tersangka, terdakwa dan terpidana,
rata-rata peserta penyuluhan menjawab 2,9 atau
dibulatkan menjadi 3, yang berarti, mayoritas peserta
sebelum dilakukan penyuluhan menjawab cukup
memahami istilah penyidikan, tersangka, terdakwa
dan terpidana Pada awalnya dilakukan pre test untuk
mengungkap
pengetahuan
para
aparat
secara
brainstorming kemudian dilakukan pembekalan
materi dengan cara sosialisasi dan pelatihan, dengan
metode ceramah dan latihan. Dengan pendekatan
yang bersifat partisipastif, peserta diharapkan dapat
secara
maksimal
memahami
dan
turut
mempraktekkan langsung apa yang telah diperoleh
melalui penyuluhan dan pelatihan. Terakhir butir nomor 6 yang menanyakan
tentang subyek atau pihak yang menjadi penegak
hukum,
mayoritas peserta sebelum dilakukan
penyuluhan menjawab 3, yang berarti mayoritas
peseta baru dalam posisi cukup paham dengan
berbagai istilah tersebut. 3. HASIL DAN PEMBAHASAN Berdasarkan hasil Pretest yang telah dilakukan
kepada aparat kelurahan Cempaka Baru sebelum
pelaksanaan penyuluhan hukum perdata dan pidana
dapat dilihat pada tabel berikut. Tabel 1 Analisis Deskriptif Pretest No 1 s.d. 6 Tabel 1 Analisis Deskriptif Pretest No 1 s.d. 6 Tabel 1 Analisis Deskriptif Pretest No 1 s.d. 6
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Tabel 2 Analisis Deskriptif Posttest No 1 s.d. 6 Tabel 2 Analisis Deskriptif Posttest No 1 s.d. 6 Tabel 2 Analisis Deskriptif Posttest No 1 s.d. 6 p
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Berdasarkan tabel di atas dapat diketahui bahwa
rata-rata skor jawaban peserta penyuluhan untuk butir
no 1 hanya 2,8 artinya mereka hanya menjawab
cukup memahami perbedaan antara hukum perdata
dan pidana. Tabel di atas menunjukkan bahwa skor yang
diperoleh oleh para peserta pengabdian kepada
masyarakat yaitu para aparat kelurahan Cempaka
Baru sudah meningkat dengan rata-rata antara 3-4
(cukup paham hingga paham). Begitu juga butir soal no 2 tentang tahapan
dalam litigasi baik secara perdata maupun pidana,
rata-rata skor jawaba peserta sebelum dilakukan
penyuluhan hanya 2,3, artinya para peserta mayoritas
menjawab tidak paham. Pada butir nomor 1 hanya rata rata sebelum
penyuluhan 2,8 dan sesudah penyuluhan meningkat
menjadi 4,6 atau dibulatkan menjadi 6, artinya
mereka
mayoritas
sudah
sangat
memahami
perbedaan antara hukum perdata dan pidana. Pada butir nomor 3 tentang pembuktian dan alat
bukti, rata-rata jawaban peserta hanya 2,8 atau
dibulatkan menjadi 3, artinya mayoritas peserta
sebelum dilakukan penyuluhan cukup memahami
tentang konsep pembuktian dan alat bukti. Pada butir soal nomor 2 tentang tahapan dalam
litigasi baik secara perdata maupun pidana, rata-rata
skor jawabna peserta sebelum dilakukan penyuluhan
hanya 2,3 dan sesudah penyuluhan meningkat Butir nomor 4 berkaitan tentang istilah gugatan,
penggugat, dan tergugat, berdasarkan data di atas, 55 Jurnal ABDIMAS Unmer Malang Vol. 2, Nomor 1, Juni 2017 menjadi 4,2, artinya para peserta mayoritas menjawab
paham tentang litigasi baik acara perdata maupun
acara pidana. Diagram 1 :
Perbandingan rata-rata skor butir No 1 s.d. 7
antara pre dan posttest Pada butir nomor 3 yang menanyakan tentang
pembuktian dan alat bukti, rata-rata jawaban peserta
sebelum penyuluhan hanya 2,8 atau dibulatkan
menjadi 3, tetapi setelah penyuluhan meningkat
menjadi 4,6 atau dibulatkan menjadi 5, artinya
mayoritas peserta sesudah dilakukan penyuluhan
sangat memahami tentang konsep pembuktian dan
alat bukti. 3. HASIL DAN PEMBAHASAN Butir nomor 4 yang membahas tentang istilah
gugatan, penggugat, dan tergugat, berdasarkan
sebelum penyuluhan, mayoritas peserta menjawab
2,9 atau dibulatkan menjadi 3, dan setelah
penyuluhan, mayoritas peserta menjawab 4,6 atau
dibulatkan 5, yang berarti mayoritas peserta setelah
dilakukan penyuluhan menjawab sangat paham
tentang istilah gugatan, penggugat, dan tergugat. Diagram 1 :
Perbandingan rata-rata skor butir No 1 s.d. 7
antara pre dan posttest Pada butir nomor 5 yang membahas tentang
istilah penyidikan, tersangka, terdakwa dan terpidana,
rata-rata peserta sebelum mengikuti penyuluhan
menjawab 2,9 atau dibulatkan menjadi 3, dan setelah
mengikuti penyuluhan rata-rata peserta menjawab 4,7
atau dibulatkan menjadi 5, yang berarti mayoritas
peserta sesudah dilakukan penyuluhan menjawab
sangat memahami istilah penyidikan, tersangka,
terdakwa dan terpidana Data di atas menunjukkan bahwa: 1. Butir no 1 tetang perbedaan hukum perdata dan
pidana terjadi peningkatan hampir dua digit, dari
rata-rata skor 2,8 (3pembulatan) dalam arti cukup
paham menjadi rata-rata skor 4,6 (5 pembulatan)
dalam arti sangat paham. 2. Butir no 2 tentang litigasi baik secara perdata
maupun pidana juga terjadi peningkatan dari rata-
rata skor 2,3 (tidak paham) menjadi rata-rata skor
4,2 (paham). Pada butir nomor 6 yang menanyakan tentang
subyek atau pihak yang menjadi penegak hukum,
mayoritas peserta sebelum dilakukan penyuluhan
menjawab 3, dan sesudah mengikuti penyuluhan
menjawab 4,7 atau dibulatkan menjadi 5, yang berarti
mayoritas peserta setelah mengikuti penyuluhan
sangat paham dengan subyek atau pihak yang
menjadi penegak hukum. 3. Butir No 3 tentang pembuktian dan alat bukti juga
terjadi peningkatan dari rata-rata skor 2,8 (cukup
paham) menjadi rata-rata skor 4,6 (sangat paham) 4. Butir No 4 tentang istilah gugatan, penggugat,
dan tergugat juga terjadi peningkatan dari rata-
rata skor 2,9 (cukup paham) menjadi rata-rata
skor 4,7 (sangat paham) Perbandingan antara pretest dan postest 5. Butir No 5 tentang istilah penyidikan, tersangka,
terdakwa, dan terpidana juga terjadi peningkatan
dari rata-rata skor 2,9 (cukup paham) menjadi
rata-rata skor 4,7 (sangat paham) Perbandingan rata-rata mean antara pretest
dan posttest dapat dilihat pada tabel berikut : Tabel 3 rata-rata skor butir No 1 s.d.7
antara pretest dan Posttest
Sumber: Data hasil pengabdian Januari 2017 6. Butir No 6 tentang siapa saja yang menjadi
subyek penegak hukum juga terjadi peningkatan
dari rata-rata skor 3 (cukup paham) menjadi rata-
rata skor 3,7 (Sangat paham) Adapun perbandingan skor total hasil pretest
dan posttest dapat dilihat dari tabel berikut. Tabel di atas, apabila digambarkan dalam bentuk
diagram garis tampak sebagai berikut. 56 Jurnal ABDIMAS Unmer Malang Vol. 2, Nomor 1, Juni 2017 Tabel 4 Analisis Deskriptif Skor total Pre dan Tabel 4 Analisis Deskriptif Skor total Pre dan
posttest (2,8%), menjawab tidak paham sebanyak 24 orang
(33,3%), menjawab cukup paham sebanyak 11 orang
(15,3%), dan menjawab paham dan sangat paham
sebanyak 13 orang (18,1%). (2,8%), menjawab tidak paham sebanyak 24 orang
(33,3%), menjawab cukup paham sebanyak 11 orang
(15,3%), dan menjawab paham dan sangat paham
sebanyak 13 orang (18,1%). Tabel 4 Analisis Deskriptif Skor total Pre dan
posttest
Sumber: Data hasil pengabdian Januari 2017 Tabel 6 Analisis Deskriptif No_1_posttest Tabel 6 Analisis Deskriptif No_1_posttest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Pada saat sesudah penyuluhan, jumlah peserta
yang menjawab sangat tidak paham dan menjawab
tidak paham sudah tidak ada, menjawab paham
sebanyak 2 orang (2,8%), menjawab paham sebanyak
24 orang (33,3%), dan menjawab sangat paham
sebanyak 46 orang (63,9%). Sumber: Data hasil pengabdian Januari 2017 Tabel di atas menunjukkan bahwa rata-rata skor
total pretest hanya 15 dari skor total 30, sementara itu
setelah pemberian penyuluhan rata-rata skor total
mereka meningkat menjadi 16,9 dari skor total 30. Dengan demikian, terjadi peningkatan pemahaman
yang berarti antara sebelum dan sesudah penyuluhan
hukum perdata dan pidana. Dengan kata lain,
pemahaman aparat kelurahan Cempaka Baru tentang
hukum perdata dan pidana telah mengalami
peningkatan secara signifikan. Dengan kata lain,
penyuluhan hukum pidana dan perdata terhadap
aparat kelurahan Cempaka Baru memberikan
manfaat yang berarti. Rata-rata skor jawaban baik sebelum maupun
sesudah penyuluhan dapat digambarkan dalam
bentuk diagram sebagai berikut. Diagram 2 Perbandingan rata-rata skor butir No 1 Pemahaman tahapan dalam Ligitasi baik
Secara Perdata maupun Pidana Pemahaman tahapan dalam Ligitasi baik
Secara Perdata maupun Pidana
Tabel 7 Analisis Deskriptif No_2_pretest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Secara Perdata maupun Pidana
Tabel 7 Analisis Deskriptif No_2_pretest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Diagram 3 Perbandingan rata skor butir No 2 Berdasarkan diagram di atas dapat diketahui
bahwa sebelum penyuluhan, jumlah peserta yang
sangat tidak paham sebanyak 7 orang, sementara itu
setelah penyuluhan turun menjadi tidak ada seorang
pun yang menjawab sangat tidak paham tentang
litigasi baik secara perdata maupun pidana. Sebelum
penyuluhan, jumlah peserta yang menjawab tidak
paham tentang litigasi sebanyak 40 orang, dan setelah
penyuluhan turun menjadi tidak ada seorang pun yang
menjawab tidak paham tentang litigasi. Sebelum
penyuluhan, jumlah peserta yang menjawab cukup
paham tentang litigasi sebanyak 20 orang dan setelah
penyuluhan menurun menjadi 7 orang yang
menjawab cukup paham tentang litigasi. Sebelum
penyuluhan, jumlah peserta yang menjawab paham
tentang litigasi hanya 3 orang, tetapi setelah
penyuluhan meningkat menjadi 40 orang yang
menjawab
paham
tentang
litigasi. Sebelum
penyuluhan, jumlah peserta yang menjawab sangat
paham hanya 2 orang dan setelah penyuluhan
meningkat menjadi 25 orang yang sangat paham
tentang litigasi. Dengan demikian, pemberian
penyuluhan tentang litigasi memberikan makna yang
berarti bagi aparat kelurahan Cempaka Baru Pada saat sebelum penyuluhan, jumlah peserta
yang menjawab sangat tidak paham sebanyak 7 orang
(9,7%), menjawab tidak paham sebanyak 40 orang
(55,6%), menjawab cukup paham sebanyak 20 orang
(27,8%), menjawab paham sebanyak 3 orang (4,2%),
dan menjawab sangat paham sebanyak 2 orang
(2,8%). Tabel 8 Analisis Deskriptif No_2_posttest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Tabel 8 Analisis Deskriptif No_2_posttest Tabel 8 Analisis Deskriptif No_2_posttest Tabel 8 Analisis Deskriptif No_2_posttest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Pemahaman Perbedaan Hukum Perdata dan
Pidana Pemahaman aparat kelurahan Cempaka Baru
mengenai perbedaan antara hukum perdata dan
pidana pada saat sebelum dan sesudah penyuluhan
dapat dilihat pada tabel berikut. Diagram 2 Perbandingan rata-rata skor butir No 1 Berdasarkan diagram di atas dapat diketahui
bahwa sebelum penyuluhan, jumlah peserta yang
sangat tidak paham sebanyak 2 orang, sementara itu
setelah penyuluhan menjadi tidak ada seorang pun
yang menjawab sangat tidak paham. Sebelum
penyuluhan, jumlah peserta yang menjawab tidak
paham sebanyak 24 orang, setelah penyuluhan
menjadi tidak ada seorang pun yang menjawab tidak
paham. Sebelum penyuluhan, jumlah peserta yang
menjawab cukup paham sebanyak 33 orang dan
setelah penyuluhan menurun menjadi 2 orang. Sebelum penyuluhan, jumlah peserta yang menjawab
paham hanya 11 orang, tetapi setelah penyuluhan
meningkat menjadi 24 orang. Sebelum penyuluhan,
jumlah peserta yang menjawab sangat paham hanya 2
orang dan setelah penyuluhan meningkat menjadi 46 Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Pada saat sebelum penyuluhan, jumlah peserta
yang menjawab sangat tidak paham sebanyak 2 orang 57 Jurnal ABDIMAS Unmer Malang Vol. 2, Nomor 1, Juni 2017 orang. Dengan demikian, pemberian penyuluhan
tentang perbedaan antara hukum perdata dan pidana
memberikan manfaat yang berarti bagi aparat
kelurahan Cempaka Baru. Diagram 3 Perbandingan rata skor butir No 2 Pemahaman tahapan dalam Ligitasi baik
Secara Perdata maupun Pidana Pemahaman Pembuktian dan Alat Bukti Tabel 9 Analisis Deskriptif No_3_pretest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Tabel 9 Analisis Deskriptif No_3_pretest Sumber: data hasil pengabdian kepada masyarakat tahun 2017 umber: data hasil pengabdian kepada masyarakat tahun 201 Pada saat sesudah penyuluhan, jumlah peserta
yang menjawab sangat tidak paham dan menjawab
tidak paham sudah tidak ada, menjawab cukup paham
sebanyak 2 orang (2,8%), menjawab paham sebanyak
24 orang (33,3%), dan menjawab sangat paham
sebanyak 46 orang (63,9%). Kedua tabel di atas,apabula diringkas dalam
bentuk diagram akan tampak sebagai berikut. Pada saat sebelum penyuluhan, jumlah peserta
yang menjawab sangat tidak paham sebanyak 2 orang
(2,8%), menjawab tidak paham sebanyak 23 orang 58 Jurnal ABDIMAS Unmer Malang Vol. 2, Nomor 1, Juni 2017 (31,9%), menjawab cukup paham sebanyak 35 orang
(48,6%), menjawab paham sebanyak 10 orang
(13,9%), dan menjawab sangat paham sebanyak 2
orang (2,8%). hanya 10 orang, tetapi setelah penyuluhan meningkat
menjadi 23 orang yang menjawab paham tentang
pembuktian dan alat bukti. Sebelum penyuluhan, jumlah peserta yang
menjawab sangat paham tentang pembuktian dan alat
bukti hanya 2 orang dan setelah penyuluhan
meningkat menjadi 47 orang yang menjawab sangat
paham tentang pembuktian dan alat bukti. Tabel 10 Analisis Deskriptif No_3_posttest Tabel 10 Analisis Deskriptif No_3_posttest Tabel 10 Analisis Deskriptif No_3_posttest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Dengan
demikian,
pemberian
penyuluhan
tentang pembuktian dan alat bukti memberikan
manfaat yang cukup berarti bagi aparat kelurahan
Cempaka Baru. Pemahaman Istilah gugatan, Penggugat, dan
Tergugat er: data hasil pengabdian kepada masyarakat tahun 2017 Pada saat sesudah penyuluhan, sudah tidak ada
lagi peserta yang menjawab sangat tidak paham dan
menjawab tidak paham, jumlah peserta yang
menjawab cukup paham sebanyak 2 orang (2,8%),
menjawab paham sebanyak 23 orang (31,9%), dan
menjawab sangat paham sebanyak 47 orang (65,3%). Tabel 11 Analisis Deskriptif No_4_pretest Tabel 11 Analisis Deskriptif No_4_pretest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Untuk lebih jelasnya, perbedaan rata-rata skor
antara sebelum dan sesudah penyuluhan dapat dilihat
pada diagram berikut. Diagram 4 Perbandingan rata skor butir No 3 Pada saat sebelum penyuluhan, jumlah peserta
yang menjawab sangat tidak paham sebanyak 4 orang
(5,6%), menjawab tidak paham sebanyak 14 orang
(19,4%), menjawab cukup paham sebanyak 38 orang
(52,8%), menjawab paham sebanyak 13 orang
(18,1%), dan menjawab sangat paham sebanyak 3
orang (4,2%) Diagram 4 Perbandingan rata skor butir No 3 Berdasarkan diagram di atas dapat diketahui
bahwa sebelum penyuluhan, jumlah peserta yang
sangat tidak paham sebanyak 2 orang, sementara itu
setelah penyuluhan turun menjadi tidak ada seorang
pun yang menjawab sangat tidak paham tentang
pembuktian dan alat bukti. Sebelum penyuluhan,
jumlah peserta yang menjawab tidak paham tentang
pembuktian dan alat bukti sebanyak 23 orang, dan
setelah penyuluhan turun menjadi tidak ada seorang
pun yang menjawab tidak paham tentang pembuktian
dan alat bukti. Tabel 12 Analisis Deskriptif No_4_posttest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Tabel 12 Analisis Deskriptif No_4_posttest Tabel 12 Analisis Deskriptif No_4_posttest Sebelum penyuluhan, jumlah peserta yang
menjawab cukup paham tentang pembuktian dan alat
bukti sebanyak 35 orang dan setelah penyuluhan
menurun menjadi 2 orang yang menjawab cukup
paham tentang pembuktian dan alat bukti. Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Pada saat sesudah penyuluhan, sudah tidak ada
seoerang pun peserta yang menjawab sangat tidak
paham dan menjawab tidak paham, jumlah peserta
yang menjawab cukup paham sebanyak 4 orang
(5,6%), menjawab paham sebanyak 14 orang Sebelum penyuluhan, jumlah peserta yang
menjawab paham tentang pembuktian dan alat bukti 59 Jurnal ABDIMAS Unmer Malang Vol. 2, Nomor 1, Juni 2017 (19,4%), dan menjawab sangat paham sebanyak 54
orang (75%). memberikan manfaat yang cukup berarti bagi aparat
kelurahan Cempaka Baru. g
Diagram berikut merupakan ringkasan atas dua
tabel sebagaimana telah dikemukakan di atas. Pemahaman Istilah Penyidikan, Tersangka,
Terdakwa dan Terpidana 2, Nomor 1, Juni 2017 Pemahaman Subyek Penegak Hukum
Tabel 15 Analisis Deskriptif No_6_pretest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Pemahaman Subyek Penegak Hukum
Tabel 15 Analisis Deskriptif No_6_pretest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Pemahaman Subyek Penegak Hukum Diagram 6 Perbandingan rata skor butir No 5 Tabel 15 Analisis Deskriptif No_6_pretest Diagram 6 Perbandingan rata skor butir No 5 Berdasarkan diagram di atas dapat diketahui
bahwa sebelum penyuluhan, jumlah peserta yang
sangat tidak paham sebanyak 3 orang, sementara itu
setelah penyuluhan turun menjadi tidak ada seorang
pun yang menjawab sangat tidak paham tentang
istilah
penyidikan,
tersangka,
terdakwa,
dan
terpidana. Pada saat sebelum penyuluhan, jumlah peserta
yang menjawab sangat tidak paham sebanyak 5 orang
(6,9%), menjawab tidak paham sebanyak 10 orang
(13,9%), menjawab cukup paham sebanyak 40 orang
(55,6%), menjawab paham sebanyak 14 orang
(19,4%), dan menjawab sangat paham sebanyak 3
orang (4,2%) Sebelum penyuluhan, jumlah peserta yang
menjawab tidak paham tentang istilah penyidikan,
tersangka, terdakwa, dan terpidana sebanyak 14
orang, dan setelah penyuluhan turun menjadi tidak
ada seorang pun yang menjawab tidak paham tentang
istilah
penyidikan,
tersangka,
terdakwa,
dan
terpidana. Tabel 16 Analisis Deskriptif No_6_posttest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Tabel 16 Analisis Deskriptif No_6_posttest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Tabel 16 Analisis Deskriptif No_6_posttest Tabel 16 Analisis Deskriptif No_6_posttest Sebelum penyuluhan, jumlah peserta yang
menjawab cukup paham tentang istilah penyidikan,
tersangka, terdakwa, dan terpidana sebanyak 38
orang dan setelah penyuluhan menurun menjadi 3
orang yang menjawab cukup paham tentang istilah
penyidikan, tersangka, terdakwa, dan terpidana. Sebelum penyuluhan, jumlah peserta yang
menjawab paham tentang istilah penyidik, tersangka,
terdakwa, dan terpidana sebanyak 13 orang, tetapi
setelah penyuluhan menjadi 15 orang yang menjawab
paham
tentang
istilah
penyidikan,
tersangka,
terdakwa, dan terpidana. Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Pada saat sesudah penyuluhan, sudah tidak ada
seorang peserta pun yang menjawab sangat tidak
paham dan menjawab tidak paham, jumlah peserta
yang menjawab paham sebanyak 5 orang (6,9%),
menjawab paham sebanyak 10 orang (13,9%), dan
menjawab sangat paham sebanyak 57 orang (79,2%). Sebelum penyuluhan, jumlah peserta yang
menjawab sangat paham tentang istilah penyidikan,
tersangka, terdakwa, dan terpidana hanya 3 orang dan
setelah penyuluhan meningkat menjadi 54 orang yang
menjawab sangat paham tentang istilah penyidikan,
tersangka, terdakwa, dan terpidana. Untuk
memperjelas
pemahaman,
berikut
digambarkan diagram rata-rata pretest dan posttest. Pemahaman Istilah Penyidikan, Tersangka,
Terdakwa dan Terpidana Terdakwa dan Terpidana
Tabel 13 Analisis Deskriptif No_5_pretest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Tabel 13 Analisis Deskriptif No_5_pretest Diagram 5 Perbandingan rata skor butir No 4 Diagram 5 Perbandingan rata skor butir No 4 Diagram 5 Perbandingan rata skor butir No 4 Berdasarkan diagram di atas dapat diketahui
bahwa sebelum penyuluhan, jumlah peserta yang
sangat tidak paham sebanyak 4 orang, sementara
itu setelah penyuluhan turun menjadi tidak ada
seorang pun yang menjawab sangat tidak paham
tentang istilah gugatan, penggugat, dan tergugat. Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Pada saat sebelum penyuluhan, jumlah peserta
yang menjawab sangat tidak paham sebanyak 3 orang
(4,2%), menjawab tidak paham sebanyak 15 orang
(20,8%), menjawab cukup paham sebanyak 38 orang
(52,8%), menjawab paham sebanyak 13 orang
(18,1%), dan menjawab sangat paham sebanyak 3
orang (4,2%). Sebelum penyuluhan, jumlah peserta yang
menjawab tidak paham tentang istilah gugatan,
penggugat, dan tergugat sebanyak 14 orang, dan
setelah penyuluhan turun menjadi tidak ada
seorang pun yang menjawab tidak paham tentang
istilah gugatan, penggugat, dan tergugat. Tabel 14 Analisis Deskriptif No_5_posttest Tabel 14 Analisis Deskriptif No_5_posttest Tabel 14 Analisis Deskriptif No_5_posttest
Sumber: data hasil pengabdian kepada masyarakat tahun 2017 Sebelum penyuluhan, jumlah peserta yang
menjawab cukup paham tentang istilah gugatan,
penggugat, dan tergugat sebanyak 38 orang dan
setelah penyuluhan menurun menjadi 4 orang
yang menjawab cukup paham tentang istilah
gugatan, penggugat, dan tergugat. Sebelum penyuluhan, jumlah peserta yang
menjawab paham tentang istilah gugatan,
penggugat, dan tergugat sebanyak 15 orang,
tetapi setelah penyuluhan menjadi 14 orang yang
menjawab paham tentang istilah gugatan,
penggugat, dan tergugat. Pada saat sesudah penyuluhan, sudah tidak ada
seorang peserta pun yang menjawab sangat tidak
paham dan menjawab tidak paham, jumlah peserta
yang menjawab paham sebanyak 3 orang (4,2%),
menjawab paham sebanyak 15 orang (20,8%), dan
menjawab sangat paham sebanyak 54 orang (75%). Sebelum penyuluhan, jumlah peserta yang
menjawab sangat paham tentang istilah gugatan,
penggugat, dan tergugat hanya 3 orang dan
setelah penyuluhan meningkat menjadi 54 orang
yang menjawab sangat paham tentang istilah
gugatan, penggugat, dan tergugat. Dalam
rangka
menyederhanakan
data
sebagaimana telah ditampilkan pada dua tabel di atas
maka dapat digambarkan dalam bentuk diagram
berikut. Dengan
demikian,
pemberian
penyuluhan
tentang istilah gugatan, penggugat, dan tergugat 60 Jurnal ABDIMAS Unmer Malang Vol. 5. REFERENSI Salman, Otje dan Anton F. Susanto. 2004. Teori
Hukum, Bandung: Refika Aditama BPHN, 1994.Seminar Hukum Nasional Keenam
Buku I II, Jakarta: Depkeh BPHN Buku II
Kitab Undang-Undang Hukum Pidana
Kitab Undang-Undang Hukum Perdata
Kitab Undang-Undang Hukum Acara Pidana
Kitab Undang-Undang Hukum Acara Perdata
Undang-Undang Nomor 18 Tahun 2002 tentang
Sistem Nasional Penelitian, Pengembangan, dan
Penerapan Ilmu Pengetahuan dan Teknologi
(Sisnas P3 Ipteks) Dengan
demikian,
pemberian
penyuluhan
tentang siapa saja yang menjadi penegak hukum
memberikan manfaat yang cukup berarti bagi aparat
kelurahan Cempaka Baru. 4. KESIMPULAN Berdasarkan hasil dan pembahasan, maka dapat
disimpulkan bahwa dalam upaya meningkatkan
pengetahuan dan kesadaran hukum aparat Kelurahan
Cempaka Baru perlu dilakukan penyuluhan hukum
khususnya hukum perdata dan hukum pidana secara
berkala dengan ditambah muatan materi tentang
penegakan hukum baik preventif maupun represif. Sebelum penyuluhan, jumlah peserta yang
menjawab paham tentang siapa saja yang menjadi
penegak hukum sebanyak 14 orang, tetapi setelah
penyuluhan menjadi 10 orang yang menjawab paham
tentang siapa saja yang menjadi penegak hukum. Sebelum penyuluhan, jumlah peserta yang
menjawab sangat paham tentang siapa saja yang
menjadi penegak hukum hanya 3 orang dan setelah
penyuluhan meningkat menjadi 57 orang yang
menjawab sangat paham tentang siapa saja yang
menjadi penegak hukum. Berikut gambar model yang dihasilkan: Berikut gambar model yang dihasilkan: Diagram 7 Perbandingan rata skor butir No 6 Berikut gambar model yang dihasilkan: Berdasarkan diagram di atas dapat diketahui
bahwa sebelum penyuluhan, jumlah peserta yang
sangat tidak paham sebanyak 5 orang, sementara itu
setelah penyuluhan turun menjadi tidak ada seorang
pun yang menjawab sangat tidak paham tentang siapa
saja yang menjadi penegak hukum. Sebelum penyuluhan, jumlah peserta yang
menjawab tidak paham tentang siapa saja yang
menjadi penegak hukum sebanyak 10 orang, dan
setelah penyuluhan turun menjadi tidak ada seorang
pun yang menjawab tidak paham tentang siapa saja
yang menjadi penegak hukum. Sebelum penyuluhan, jumlah peserta yang
menjawab cukup paham tentang siapa saja yang
menjadi penegak hukum sebanyak 40 orang dan
setelah penyuluhan menurun menjadi 5 orang yang
menjawab cukup paham tentang siapa saja yang
menjadi penegak hukum. Pemahaman Istilah Penyidikan, Tersangka,
Terdakwa dan Terpidana Dengan
demikian,
pemberian
penyuluhan
tentang istilah penyidik, tersangka, terdakwa, dan
terpidana memberikan manfaat yang cukup berarti
bagi aparat kelurahan Cempaka Baru 61 Jurnal ABDIMAS Unmer Malang Vol. 2, Nomor 1, Juni 2017 terkait juga sangat penting dalam upaya membantu
penyadaran para aparat kelurahan, agar senantiasa
menjadi aparat yang ber-tata kelola baik dan jauh dari
KKN. Untuk
itu
perlu
pemberdayaan
yang
mensinergikan antara motivasi aparat itu sendiri
terhadap penegakan hukum, edukasi untuk
peningkatan pemahaman hukum serta intervensi
positif dari pihak terkait baik kampus maupun
birokrasi atasan. Perlu terus menerus (secara berkala)
dilakukan update pemahaman hukum agar aparat
makin berdaya dan makin memahami penegakan
hukum baik secara preventif maupun represif. Diagram 7 Perbandingan rata skor butir No 6 Berikut gambar model yang dihasilkan: Model Pemberdayaan Untuk meningkatkan pengetahuan hukum yang
tujuan akhirnya adalah kepatuhan yang diharapkan
menjadi cikal bakal kesadaran hukum, perlu
sosialisasi, edukasi dan teladan. Uluran tangan pihak 62
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https://peerj.com/articles/6599.pdf
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Resource-dependent dispersal and the irrelevance of extra-patch information for Daphnia carinata
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bioRxiv (Cold Spring Harbor Laboratory)
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cc-by
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How to cite this article Erm P, Hall MD, Phillips BL. 2019. Anywhere but here: local conditions motivate dispersal in Daphnia.
PeerJ 7:e6599 DOI 10.7717/peerj.6599 ABSTRACT Dispersal is fundamental to population dynamics. However, it is increasingly
apparent that, despite most models treating dispersal as a constant, many organisms
make dispersal decisions based upon information gathered from the environment. Ideally, organisms would make fully informed decisions, with knowledge of
both intra-patch conditions (conditions in their current location) and extra-patch
conditions (conditions in alternative locations). Acquiring information is
energetically costly, however, and extra-patch information will typically be costlier
to obtain than intra-patch information. As a consequence, theory suggests that
organisms will often make partially informed dispersal decisions, utilising
intra-patch information only. We test this proposition in an experimental two-patch
system using populations of the aquatic crustacean, Daphnia carinata. We
manipulated conditions (food availability) in the population’s home patch, and in its
alternative patch. We found that D. carinata made use of intra-patch information
(resource availability in the home patch induced a 10-fold increase in dispersal
probability) but either ignored or were incapable of using of extra-patch information
(resource availability in the alternative patch did not affect dispersal probability). We also observed a small apparent increase in dispersal in replicates with higher
population densities, but this effect was smaller than the effect of resource constraint,
and not found to be significant. Our work highlights the considerable influence
that information can have on dispersal probability, but also that dispersal decisions
will often be made in only a partially informed manner. The magnitude of the
response we observed also adds to the growing chorus that condition-dependence
may be a significant driver of variation in dispersal. Submitted 15 November 2018
Accepted 11 February 2019
Published 12 March 2019
Corresponding author
Philip Erm,
philip.erm@unimelb.edu.au
Academic editor
Andrew Byrne
Additional Information and
Declarations can be found on
page 11
DOI 10.7717/peerj.6599
Copyright
2019 Erm et al. Distributed under
Creative Commons CC-BY 4.0 Submitted 15 November 2018
Accepted 11 February 2019
Published 12 March 2019
Corresponding author
Philip Erm,
philip.erm@unimelb.edu.au
Academic editor
Andrew Byrne
Additional Information and
Declarations can be found on
page 11
DOI 10.7717/peerj.6599
Copyright
2019 Erm et al. Di t ib t d
d Subjects Animal Behavior, Ecology, Zoology, Freshwater Biology
Keywords Condition-dependent dispersal, Information use, Daphnia carinata, Biological invasion Anywhere but here: local conditions
motivate dispersal in Daphnia
Philip Erm1, Matthew D. Hall2 and Ben L. Phillips1
1 School of BioSciences, University of Melbourne, Parkville, VIC, Australia
2 School of Biological Sciences, Monash University, Clayton, VIC, Australia 1 School of BioSciences, University of Melbourne, Parkville, VIC, Australia
2 School of Biological Sciences, Monash University, Clayton, VIC, Australia Subjects Animal Behavior, Ecology, Zoology, Freshwater Biology
Keywords Condition-dependent dispersal, Information use, Daphnia carinata, Biological invasion INTRODUCTION Dispersal, like survival and reproduction, is a fundamental facet of life history (Bonte &
Dahirel, 2017). Behaviours which govern dispersal can have profound consequences for a
variety of biological and ecological phenomena, such as individual fitness, metapopulation
dynamics, and evolutionary outcomes across a species’ range (Bonte & Dahirel, 2017;
Clobert et al., 2001). For reasons of simplicity, the majority of spatially-explicit ecological
models assume that dispersal is both uninformed and unchanging; that individuals disperse
at fixed rates, and that they do so without recourse to information about environmental Distributed under
Creative Commons CC-BY 4.0 conditions (e.g. by default in models utilising reaction-diffusion or integrodifference
equations; Fisher, 1937; Skellam, 1951; Kot, Lewis & Van Den Driessche, 1996;
but see Fronhofer, Nitsche & Altermatt, 2016). There is now considerable evidence, however,
that dispersal decisions are routinely informed by aspects of the environment (Clobert et al.,
2009), with such information use expected to have non-negligible effects on ensuing
population and evolutionary dynamics (Delgado et al., 2014; Ponchon et al., 2015;
Urban et al., 2016). The most common form of informed (or condition-dependent) dispersal is
density-dependent dispersal (Bowler & Benton, 2005). Here, individuals acquire
information about population density, and, if conditions more favourable to survival and
reproduction are likely to be found elsewhere, make the decision to disperse. When all else
is equal, high density—with its greater competition for resources, greater rates of
disease transmission and so on—will be associated with poorer conditions (Bowler &
Benton, 2005). Many species have been shown to acquire information on density and act
upon it, such as the spider Erigone atra (De Meester & Bonte, 2010), protozoa like
Paramacium caudatum and Tetrahymena thermophila (Fellous et al., 2012; Fronhofer, Gut &
Altermatt, 2017), and the plant Heterosperma pinnatum (Martorell & Martínez-López, 2014). In many species, this information is acquired through food availability; when local
resources are limited, individuals tend to be more dispersive, regardless of taxa. This was
demonstrated by Fronhofer et al. (2018), where resource limitation (and to a lesser extent,
predator presence) was shown to induce higher dispersal rates in organisms as varied
as, amongst others, protists, slugs, crustaceans, crickets, newts, and fish. Information on the
relative merits of different alternative locations can also be acquired in numerous ways,
including prospecting (e.g. Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 INTRODUCTION actively assessing potential breeding sites, as in collared
flycatchers (Ficedula albicollis) monitoring which locations are producing well-provisioned
nests; Pärt & Doligez, 2003) and by observing immigrating conspecifics (as in the common
lizard Zootoca vivipara; Cote & Clobert, 2007). Several models have now been constructed to examine the evolution of informed
dispersal (Bocedi, Heinonen & Travis, 2012; Delgado et al., 2014). However, while they are
in general agreement that informed dispersal will often evolve, much hinges on the
ease with which information is acquired, along with its value (Poethke et al., 2016). In an
ideal world, we would expect dispersal decisions to be made on a balance of ‘push’ factors,
such as local patch conditions, and ‘pull’ factors, such as the quality of other patches. The quality of the new patch should be high enough (relative to the home patch) that it
offsets the fitness costs of moving. But organisms do not live in an ideal world: information
can be costly to acquire, both in terms of time and energy (Bonte et al., 2012), with a
likely asymmetry of cost such that information about alternative patches is harder to
obtain than information about an individual’s home patch. Thus, it may often be
the case that individuals act on the limited information that is most easily acquired:
intra-patch information. Conversely, ecological and population dynamics may also be influenced by the relative
strength of the pull exerted by extra-patch information. In metapopulations with
dispersers that can take advantage of this kind of information, patch persistence may be Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 expected to increase if patches with perilously low population sizes—but abundant
resources as a result—become more appealing to dispersers. Indeed, simply being able
to detect other conspecifics in these low-density patches may have the same effect
(Clobert et al., 2009), although patches with suitable habitat could also become
overpopulated if they attract a disproportionate number of migrants. In biological
invasions, invasion speed may be boosted if colonisers are able to use extra-patch
information to select suitable habitats, or hindered if it instead causes them to favour
ecological traps (Kokko, 2006). INTRODUCTION If intra-patch information is dominant in motivating
dispersal on the other hand, invaders may be expected to distribute themselves
indiscriminately, rendering the invasion highly sensitive to both the proportion of suitable
habitat in the landscape and any temporal fluctuations in its quality (Neubert, Kot &
Lewis, 2000; Schreiber & Lloyd-Smith, 2009). Gauging the relative strength of the
push caused by intra-patch information and the pull caused by extra-patch information
may help to resolve such questions. Here, we examine the relative influence of intra- and extra-patch information on
dispersal by manipulating food resource levels in experimental populations of the aquatic
crustacean, Daphnia carinata. Dispersal in Daphnia is usually characterised as being
driven by the passive transport of ephippia (long-lived resting eggs) by water fowl
or other vectors (Allen, 2007; Frisch, Green & Figuerola, 2007; Van De Meutter, Stoks &
De Meester, 2008); however, individuals can also actively disperse between permanently
or temporarily interconnected water bodies (Michels et al., 2001; Cottenie et al.,
2003). Although it has been demonstrated that Daphnia do boost ephippia production in
response to information cues indicating low local resource availability (Carvalho &
Hughes, 1983; Hobaek & Larsson, 1990; Kleiven, Larsson & Hobaek, 1992), a greater
range of responses has been observed regarding its effects on active movement. Here, we examine the relative influence of intra- and extra-patch information on
dispersal by manipulating food resource levels in experimental populations of the aquatic
crustacean, Daphnia carinata. Dispersal in Daphnia is usually characterised as being
driven by the passive transport of ephippia (long-lived resting eggs) by water fowl
or other vectors (Allen, 2007; Frisch, Green & Figuerola, 2007; Van De Meutter, Stoks &
De Meester, 2008); however, individuals can also actively disperse between permanently
or temporarily interconnected water bodies (Michels et al., 2001; Cottenie et al.,
2003). Although it has been demonstrated that Daphnia do boost ephippia production in
response to information cues indicating low local resource availability (Carvalho &
Hughes, 1983; Hobaek & Larsson, 1990; Kleiven, Larsson & Hobaek, 1992), a greater
range of responses has been observed regarding its effects on active movement. Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 MATERIALS AND METHODS Laboratory population of D. carinata y p p
All D. carinata used were genetically identical members of a single clone line. The founding
member of this lineage was collected at 3810′34.3″S, 14421′14.1″E (a lake in Geelong,
VIC, Australia) in October 2016. Its offspring were used to establish laboratory stock
cultures, which were housed in glass jars containing 300 ml of ADaM zooplankton medium
(according to the recipe of Klüttgen et al., 1994; as modified by Ebert, Zschokke-Rohringer &
Carius, 1998 using only 5% of the recommended SeO2 concentration) and kept within
growth chambers maintained at 22 C on a 12.30 light:11.30 dark photoperiod. Stocks were
fed the non-motile green algae Scenedesmus. In order to reduce any potential impact of
maternal effects, all individuals used in the experiment were taken from stocks that
were maintained under these conditions for at least two generations. Experimental materials and conditions We set up two-patch microcosms within which to measure dispersal of Daphnia. Each patch was a 950 ml plastic Cryovac XOP-0980 container filled with 600 ml of ADaM
and kept on bench tops in an open-air laboratory. The laboratory was maintained at 22 C
and each container was covered with a transparent plastic sheet that was only
removed during feeding and data collection. Each container measured 90 75 mm at the
base, was 110 mm high and widened gradually towards the top to 100 90 mm. A circular
hole with a diameter of 15 mm was centrally located 35 mm above the base on one
of the long sides of each container. This was connected to plastic PVC piping of an
identical internal diameter that linked one container to the next, acting as a 117 mm long
tunnel through which D. carinata could disperse between the two containers. For Daphnia
generally, such a length would be easily traversable within less than a minute for an
individual swimming in a straight line (O’Keefe, Brewer & Dodson, 1998). At the
commencement of the experimental trials, dispersal between containers was prevented
by inserting cotton balls into the openings of the connecting tunnel. INTRODUCTION Environments with relatively higher food concentrations have been shown to increase
Daphnia movement behaviours like swimming speed and sinking rate (Dodson et al.,
1997); however, in other instances, they have been shown to slow movement, with
much depending on the Daphnia species or clone line under examination (Young &
Getty, 1987; Larsson & Kleiven, 1996; Roozen & Lürling, 2001). Daphnia have also been
seen to adhere to ideal free distributions under ordinary circumstances, with individuals
favouring regions of high food concentration so long as they fall within natural
ranges (Jakobsen & Johnsen, 1987; Neary, Cash & McCauley, 1994; Jensen, Larsson &
Högstedt, 2001). It would appear likely then, that Daphnia exploit information to
regulate their dispersal efforts between patches. It is less clear however, if this behaviour
is governed entirely by intra-patch information, or if extra-patch information also
influences dispersal propensity. Environments with relatively higher food concentrations have been shown to increase
Daphnia movement behaviours like swimming speed and sinking rate (Dodson et al.,
1997); however, in other instances, they have been shown to slow movement, with
much depending on the Daphnia species or clone line under examination (Young &
Getty, 1987; Larsson & Kleiven, 1996; Roozen & Lürling, 2001). Daphnia have also been
seen to adhere to ideal free distributions under ordinary circumstances, with individuals
favouring regions of high food concentration so long as they fall within natural
ranges (Jakobsen & Johnsen, 1987; Neary, Cash & McCauley, 1994; Jensen, Larsson &
Högstedt, 2001). It would appear likely then, that Daphnia exploit information to
regulate their dispersal efforts between patches. It is less clear however, if this behaviour
is governed entirely by intra-patch information, or if extra-patch information also
influences dispersal propensity. Using D. carinata, we determine if individuals modify their rates of active
dispersal between patches in small multi-patch mesocosms when exposed to different
intra-patch resource levels. We also ask whether this response is contingent upon
extra-patch conditions; the presence or absence of ad libitum food in the
neighbouring patch. Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 3/15 Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 Food availability experiment Within this two-patch system, we examined the effects of intra- and extra-patch food
availability on the dispersal rate of D. carinata. We seeded one half of each two-patch
system with 10 adult females taken from stock cultures, and allowed this population
to grow for 9 days in the experimental system while dispersal was blocked. This resulted in
each population containing individuals of a variety of age and size classes when dispersal
commenced (D. carinata have a lifespan of 1–2 months depending on the conditions
at which they are maintained, and generally reach reproductive maturity when between
5 and 10 days old; Venkataraman & Job, 1980). On the 10th day, we then unblocked
the dispersal tunnels and made one exhaustive count by eye of the number of adult
(i.e. individuals large enough to reproduce) and juvenile D. carinata in each patch every
24 h thereafter for 4 days. Our patch pairs were allocated to four treatment combinations (n ¼ 5 per combination)
according to a two factor crossed design in which we independently modified food Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 4/15 availability in the two patches. Factor 1 was intra-patch food availability: once the dispersal
tunnel was unblocked, half of the populations no longer received food in their starting
patch. Factor 2 was extra-patch food availability: here we either daily added food to
the second patch (commencing on day 7, 3 days before dispersal was allowed) or withheld
food altogether from this patch. This meant that half of the populations were dispersing
into patches that contained no food at all, and the other half into patches with an
abundance of food. Food in this case was a daily fed mixture of 8 million Scenedesmus sp. cells (an unidentified Australian Scenedesmus) and 12 million Scenedesmus obliquus cells. We examined the effect of feeding regimes on absolute population sizes using
ANOVA. There were two response variables: the total population size at 96 h (summed
across both patches); and the population size in patch 2 at 96 h. We verified that they did not
violate standard ANOVA assumptions by testing each for normality and homogeneity
of variances using the Shapiro–Wilk test and Levene’s test. Food availability experiment Neither assumption was violated
for population size at 96 h (Shapiro–Wilk: W ¼ 0.92659, P ¼ 0.133; Levene’s: F ¼ 0.910,
P ¼ 0.458) nor population size in patch 2 at 96 h (Shapiro–Wilk: W ¼ 0.93031,
P ¼ 0.157; Levene’s: F ¼ 1.620, P ¼ 0.224). We next compared the proportion of
individuals that had reached patch 2 after 96 h between treatment combinations using a
generalised linear model with binomial errors and a logit link, with each individual
in each patch being characterised as a trial in which either success (dispersing into patch 2)
or failure (remaining in patch 1) had resulted. We likewise used a generalised
linear model with binomial errors and a logit link to see if adults or juveniles were
over-represented as dispersers after 96 h. Finally, we also examined the relationship
between the proportion of individuals dispersing and density using a generalised linear
model with quasibinomial errors and a logit link. A quasibinomial error distribution was
selected because there was a large difference between the residual deviance of the
binomial model (61.287) and its degrees of freedom (16). We examined the effect of feeding regimes on absolute population sizes using
ANOVA. There were two response variables: the total population size at 96 h (summed
across both patches); and the population size in patch 2 at 96 h. We verified that they did not
violate standard ANOVA assumptions by testing each for normality and homogeneity
of variances using the Shapiro–Wilk test and Levene’s test. Neither assumption was violated
for population size at 96 h (Shapiro–Wilk: W ¼ 0.92659, P ¼ 0.133; Levene’s: F ¼ 0.910,
P ¼ 0.458) nor population size in patch 2 at 96 h (Shapiro–Wilk: W ¼ 0.93031,
P
0 157 L
’
F
1 620 P
0 224) W
t
d th
ti
f All statistical tests were performed in R version 3.5.0 (R Development Core Team,
2018). All experimental data and scripts are available in the figshare repository at
https://doi.org/10.4225/49/5b0f62dc23b4c. Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 RESULTS Over the course of the dispersal phase, total population sizes across both patches generally
increased or decreased according to whether patch 1 was fed or not, with fed treatments
overall growing in size and unfed treatments shrinking (Fig. 1). At 96 h, we found a
significant effect of food availability in patch 1 on total population size across both patches
(F1,16 ¼ 10.826, P < 0.01; Table 1), but not of food availability in patch 2 (F1,16 ¼ 0.013,
P ¼ 0.912; Table 1). The interaction between feeding treatments in the two patches
was also not significant with regard to total population size (F1,16 ¼ 0.481, P ¼ 0.498;
Table 1). Examining the proportion of individuals dispersing after 96 h, we found no significant
effect of the interaction between intra- and extra-patch feeding treatments (z ¼ 1.073,
P ¼ 0.283; Table 2), and likewise no significant effect of food availability in patch 2
(z ¼ 0.138, P ¼ 0.890; Table 2). We did however find a significant effect of food availability Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 5/15 Patch 2 not fed
Patch 2 well fed
Patch 1 not fed
Patch 1 well fed
0
1
2
3
4
0
1
2
3
4
0
50
100
150
200
0
50
100
150
200
Time (days of dispersal)
Mean population size
Patch 1
Patch 2
Figure 1 D. carinata population size over time and space across distinct feeding regimes. The effect
of feeding regime on mean population size across both patch 1 and patch 2 over the dispersal phase
(n ¼ 5 container pairs per treatment combination). Bars are stacked, such that both patch 1 (black) and
patch 2 (grey) population sizes combine to indicate mean population size. Error bars show SE for each
patch’s mean population size, rather than for stacked mean population size. Replicates where patch 1 was
not fed produced higher numbers of dispersers despite having lower total population sizes (Table 1). Full-size
DOI: 10.7717/peerj.6599/fig-1 Patch 2 not fed
Patch 2 well fed
Patch 1 not fed
Patch 1 well fed
0
1
2
3
4
0
1
2
3
4
0
50
100
150
200
0
50
100
150
200
Time (days of dispersal)
Mean population size Patch 1 well fed Figure 1 D. carinata population size over time and space across distinct feeding regimes. Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 RESULTS Parameter
Estimate (SE)
z stat
P-value
Intercept
-0.938 (0.100)
9.371
<0.001
Food available in patch 1
-2.872 (0.265)
10.84
<0.001
Food available in patch 2
-0.0206 (0.150)
0.138
0.890
Food available in patch 1 Food available in patch 2
0.375 (0.350)
1.073
0.283
Note:
Test results for differences in the proportion of D. carinata dispersing after 96 h depending on food availability in patch 1
and food availability in patch 2. A generalised linear model was used with parameter estimates on the logit scale and
binomial errors. Food being available in patch 1 led a significantly lower proportion (P < 0.001) of individuals dispersing. Model variance was checked for overdispersion and did not violate standard GLM assumptions. tatistical results for proportion of individuals dispersing after 96 h of dispersal. ote:
Test results for differences in the proportion of D. carinata dispersing after 96 h depending on food availability in patch 1
and food availability in patch 2. A generalised linear model was used with parameter estimates on the logit scale and
binomial errors. Food being available in patch 1 led a significantly lower proportion (P < 0.001) of individuals dispersing. Model variance was checked for overdispersion and did not violate standard GLM assumptions. ote:
Test results for differences in the proportion of D. carinata dispersing after 96 h depending on food availability in patch 1
and food availability in patch 2. A generalised linear model was used with parameter estimates on the logit scale and
binomial errors. Food being available in patch 1 led a significantly lower proportion (P < 0.001) of individuals dispersing. Model variance was checked for overdispersion and did not violate standard GLM assumptions. between patch 1 food availability and density (t ¼ 0.856, P ¼ 0.404; Table A1) despite the
seemingly steeper increase in dispersal when patch 1 was not fed (Fig. A1). An analysis based on absolute numbers in patch 2, rather than proportions, yielded the
same overall trends in dispersal across our feeding treatments. Here, we found a significant
difference in the total number of individuals in patch 2 according to whether patch 1
had been fed or not (F1,16 ¼ 13.605, P < 0.01; Table 1), but no significant effect of food
availability in patch 2 (F1,16 ¼ 0.102, P ¼ 0.754; Table 1). RESULTS The effect
of feeding regime on mean population size across both patch 1 and patch 2 over the dispersal phase
(n ¼ 5 container pairs per treatment combination). Bars are stacked, such that both patch 1 (black) and
patch 2 (grey) population sizes combine to indicate mean population size. Error bars show SE for each
patch’s mean population size, rather than for stacked mean population size. Replicates where patch 1 was
not fed produced higher numbers of dispersers despite having lower total population sizes (Table 1). Full-size
DOI: 10.7717/peerj.6599/fig-1 in patch 1 (z ¼ 10.843, P < 0.001; Table 2), with intra-patch food deprivation resulting
in an approximately 10-fold higher proportion of the total population dispersing
(food-deprived patch 1, mean ¼ 0.259, SE ¼ 0.0374; well-fed patch 1, mean ¼ 0.0218,
SE ¼ 0.00718; Fig. 2). Since total population size did differ based on food availability in patch 1, we examined
its relationship with the proportion of D. carinata dispersing. Although the proportion
of dispersers in both the patch 1 fed and unfed groups appeared to increase with
higher densities (food-deprived patch 1, r2 ¼ 0.234; well-fed patch 1, r2 ¼ 0.401; Fig. A1),
we did not find a significant effect of density on the proportion of dispersers (t ¼ 1.273,
P ¼ 0.221; Table A1). We likewise found no significant effect of the interaction Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 6/15 Table 1 Statistical results for total population sizes and absolute number of dispersers after 96 h of
dispersal. Source
df
F stat
P-value
Total population sizes
Food available in patch 1
1
10.826
<0.01
Food available in patch 2
1
0.013
0.912
Food available in patch 1 Food available in patch 2
1
0.481
0.498
16
Absolute number of dispersers
Food available in patch 1
1
13.605
<0.01
Food available in patch 2
1
0.102
0.754
Food available in patch 1 Food available in patch 2
1
0.408
0.532
16
Note:
ANOVA test results for differences in the total D. carinata population sizes and absolute number of dispersers after 96 h
of dispersal, depending on food availability in patch 1 and food availability in patch 2. Replicates with food available in
patch 1 produced a significantly higher number of dispersers (P < 0.01) despite having significantly lower total population
sizes (P < 0.01). Standard ANOVA assumptions were not violated. RESULTS Table 2 Statistical results for proportion of individuals dispersing after 96 h of dispersal. Parameter
Estimate (SE)
z stat
P-value
Intercept
-0.938 (0.100)
9.371
<0.001
Food available in patch 1
-2.872 (0.265)
10.84
<0.001
Food available in patch 2
-0.0206 (0.150)
0.138
0.890
Food available in patch 1 Food available in patch 2
0.375 (0.350)
1.073
0.283
Note:
Test results for differences in the proportion of D. carinata dispersing after 96 h depending on food availability in patch 1
and food availability in patch 2. A generalised linear model was used with parameter estimates on the logit scale and
binomial errors. Food being available in patch 1 led a significantly lower proportion (P < 0.001) of individuals dispersing. Model variance was checked for overdispersion and did not violate standard GLM assumptions. Table 1 Statistical results for total population sizes and absolute number of dispersers after 96 h of
dispersal. Source
df
F stat
P-value
Total population sizes
Food available in patch 1
1
10.826
<0.01
Food available in patch 2
1
0.013
0.912
Food available in patch 1 Food available in patch 2
1
0.481
0.498
16
Absolute number of dispersers
Food available in patch 1
1
13.605
<0.01
Food available in patch 2
1
0.102
0.754
Food available in patch 1 Food available in patch 2
1
0.408
0.532
16
Note:
ANOVA test results for differences in the total D. carinata population sizes and absolute number of dispersers after 96 h
of dispersal, depending on food availability in patch 1 and food availability in patch 2. Replicates with food available in
patch 1 produced a significantly higher number of dispersers (P < 0.01) despite having significantly lower total population
sizes (P < 0.01). Standard ANOVA assumptions were not violated. Table 1 Statistical results for total population sizes and absolute number of dispersers after 96 h of
dispersal. Note:
ANOVA test results for differences in the total D. carinata population sizes and absolute number of dispersers after 96 h
of dispersal, depending on food availability in patch 1 and food availability in patch 2. Replicates with food available in
patch 1 produced a significantly higher number of dispersers (P < 0.01) despite having significantly lower total population
sizes (P < 0.01). Standard ANOVA assumptions were not violated. Table 2 Statistical results for proportion of individuals dispersing after 96 h of dispersal. RESULTS Indeed, patch 1 unfed groups had
a far higher number of individuals in patch 2 despite their significantly lower total
population sizes (individuals in patch 2: food-deprived patch 1, mean ¼ 25.1, SE ¼ 5.12;
well-fed patch 1, mean ¼ 4.3, SE ¼ 1.73). Dispersers were also overwhelmingly juveniles
(Fig. A2), although not disproportionately so (z ¼ 2.068, P ¼ 0.331; Table A2). Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 DISCUSSION In our system, there was a significant increase in inter-patch dispersal when D. carinata
were deprived of food (Fig. 2), indicating that D. carinata exploited intra-patch
information to inform their dispersal decisions. By contrast, extra-patch conditions In our system, there was a significant increase in inter-patch dispersal when D. carinata
were deprived of food (Fig. 2), indicating that D. carinata exploited intra-patch
information to inform their dispersal decisions. By contrast, extra-patch conditions Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 NS
NS
***
0.0
0.2
0.4
Proportion of D. carinata dispersing into patch 2
Patch 1
Patch 2
not fed
not fed
well fed
well fed
not fed
well fed
Figure 2 Proportion of D. carinata dispersersing after 96 h across distinct feeding regimes. The effect
of 96 h of food deprivation on the proportion of D. carinata that had dispersed into patch 2, both with
and without food available in patch 2 (n ¼ 5 container pairs per treatment combination). indicates a
significant difference between treatment combinations (at P < 0.001), NS indicates a non-significant
difference. Each point and line is given by the mean number of D. carinata individuals in patch 2 as a
proportion of the total population size between the two patches ± SE. SE was calculated as
SE=
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
p 1 p
ð
Þ=n
q
, where p is the proportion of dispersers and n is the number of individuals in the
container pair. Food availability in patch 1 alone was found to have a significant effect on the proportion
of the population that dispersed (Table 2). Full-size
DOI: 10.7717/peerj.6599/fig-2 NS
NS
***
0.0
0.2
0.4
Proportion of D. carinata dispersing into patch 2
Patch 1
Patch 2
not fed
not fed
well fed
well fed
not fed
well fed
Fi
2 P
i
f D
i
di
i
f
96 h
di i
f
di
i
Th
ff Figure 2 Proportion of D. carinata dispersersing after 96 h across distinct feeding regimes. The effect
of 96 h of food deprivation on the proportion of D. carinata that had dispersed into patch 2, both with
and without food available in patch 2 (n ¼ 5 container pairs per treatment combination). indicates a
significant difference between treatment combinations (at P < 0.001), NS indicates a non-significant
difference. Each point and line is given by the mean number of D. Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 DISCUSSION carinata individuals in patch 2 as a
proportion of the total population size between the two patches ± SE. SE was calculated as
SE=
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
p 1 p
ð
Þ=n
q
, where p is the proportion of dispersers and n is the number of individuals in the
container pair. Food availability in patch 1 alone was found to have a significant effect on the proportion
of the population that dispersed (Table 2). Full-size
DOI: 10.7717/peerj.6599/fig-2 (food either abundant or entirely absent) had no effect on dispersal rates. Thus it appears
that D. carinata either did not use, or were incapable of using, extra-patch information to
inform their dispersal decisions. Our first result—that animals increase dispersal propensity when faced with local
resource shortages—has been well established empirically. Studies on taxa ranging from
plants to invertebrates and vertebrates either imply, or experimentally demonstrate, that
resource shortage is a powerful piece of information motivating dispersal (Bowler &
Benton, 2005; Martorell & Martínez-López, 2014; Fronhofer et al., 2018). Our study adds
D. carinata to the long list of organisms that exploit this piece of intra-patch information. Due to the seeming generality of this phenomenon across taxa (as established by
Fronhofer et al. (2018)), it also appears likely that comparable results would be seen for
other species of Daphnia, although additional studies using different clone lines or species
of Daphnia may be prudent. Indeed, our results can only strictly be said to apply to
the single clone line of D. carinata that we tested. Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 8/15 That the dispersal we observed was largely driven by resource shortage, rather than
density, becomes obvious when examining patch 1 population sizes across treatments. Density, independent of resource shortage, has been demonstrated to cause changes
in life-history in Daphnia spp. (Matveev, 1993; Burns, 1995, 2000); but here, intra-patch
resource shortage proved to be a far more powerful driver of dispersal than density in
and of itself. Since the nature of our experimental design precluded any attempt to control
density, those treatments that were well fed in patch 1 kept growing over time compared
to those that were not, manifesting in a significantly higher total population sizes by
the end of the experiment (Fig. 1). DISCUSSION Despite these higher densities, which would not only
have created more potential dispersers but also exacerbated any density-driven push
effect, a significantly greater number of individuals dispersed in the treatments
experiencing lower densities. Density did possibly drive some dispersal, with higher
total population sizes increasing the proportion of the population dispersing regardless of
whether patch 1 was fed or not (Fig. A1), although this effect was not found to be
statistically significant (Table A1). Likewise, no significant effect was found for the
interaction between density and food availability in patch 1, despite the apparent difference
in the slopes of the patch 1 fed and unfed groups seen in Fig. A1. The quasibinomial error
structure used in our GLM and the relatively low number of replicates means that
any conclusions concerning the effects of density should only be drawn with caution;
further investigation of the specific effects of density may be warranted. The surprising result that food availability in patch 2 had no effect on final patch 2
population size (which should have been influenced by both births and deaths within
the patch) was likely due a combination of factors. Since almost all dispersers were
juveniles (Fig. A2), there was limited potential for births to occur in patch 2, regardless of
food availability. Moreover, even if coming from a starved patch 1, those individuals
that migrated to patch 2 had to be healthy enough to disperse in the first place, presumably
depressing the death rate there. Nonetheless, had we recorded population sizes in patch 2
beyond 96 h, it seems likely that some difference would have soon become apparent
between fed and unfed patch 2 populations. The magnitude of the dispersal increase we observed also indicates that the effect of local
resource information on dispersal rates may be pronounced. In terms of the proportion
of dispersers, 25.9% of individuals dispersed into patch 2 under food deprivation,
whereas less than a tenth of that (2.18%) did so under well-fed conditions (Fig. 2). Although this particular measure may have been inflated by the population growth that
continued to occur in the well-fed treatments, the large difference in the absolute number
of dispersers (food-deprived patch 1, 25.1 dispersers; well-fed patch 1, 4.3 dispersers)
despite the afore-mentioned higher density in the well-fed treatments reiterated the
strength of the effect. Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 DISCUSSION This suggests that ecological models may benefit substantially by
accounting for conditional factors, like resource availability, that may have a large effect
on dispersal behaviour. We caution, however, that parameterising models using our or similar results should
only be undertaken with great care. Such findings are likely to be influenced by the
size and arrangement of the experimental set-up. Additionally, this study and comparable Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 efforts using Daphnia or other organisms typically rely upon highly unnatural
environments within which to measure dispersal and movement (Young & Getty, 1987;
Dodson et al., 1997; Larsson & Kleiven, 1996; Roozen & Lürling, 2001; Fronhofer
et al., 2018), which may prompt aberrant behaviours. In our set-up in particular, it is
possible that some individuals may not have recognised the dark tunnel opening as a
dispersal avenue, or that individuals in general may have only encountered the
opening infrequently. The length and width of the connections between our patches were
likewise considerably smaller and narrower in scale than dispersal avenues might be
expected to be in natural settings (Michels et al., 2001). Conducting equivalent experiments
in natural environments and at scales relevant to the dispersal of the organisms under
examination is likely to be useful, although admittedly difficult in practice. Our second result—that favourability of conditions in the second patch had no effect on
dispersal—highlights the apparent importance of push vs pull factors in driving a
population’s movement. In our study, to obtain information that would draw D. carinata
into the second patch individuals either had to engage in prospecting within the
inter-patch tunnel and the second patch, or to sense extra-patch conditions remotely. We found no evidence to suggest that D. carinata was capable of exploiting either source of
information. In terms of more direct means of gathering information, extra-patch
information gathering behaviours like prospecting are predicted to be costly due to the
threat of predation that comes from moving into novel environments (Bonnet, Naulleau &
Shine, 1999; Hiddink, Kock & Wolff, 2002; Bonte et al., 2012), or the simple energetic
cost of having to move to assess new patches (Delgado et al., 2014). In D. DISCUSSION carinata
specifically, it seemed much more likely that chemoreception would serve as the primary
means of ascertaining extra-patch conditions, as chemical signals from both conspecifics
and other organisms have been demonstrated to have a multitude of effects on
Daphnia growth and behaviour (Larsson & Dodson, 1993; Dodson et al., 1994). Indeed, it
has been previously shown that D. magna and D. pulex are unaffected by olfactory cues
from algae (Roozen & Lürling, 2001), but that a D. galeata and D. hyalina hybrid responds
to them (Van Gool & Ringelberg, 1996). Here, however, the dominance of resource
limitation in pushing dispersal from the local patch indicated that the pull to move into
new patches was relatively weak in comparison, either because obtaining more information
was costly, or because that information was in some way imperceptible or ignored. Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 CONCLUSIONS In conclusion, our results add to the growing body of evidence that condition-dependent
dispersal is the norm amongst taxa, and that it is capable of generating substantial differences
in dispersal behaviour (Legrand et al., 2015; Fronhofer et al., 2018). This growing
empirical consensus warns against the simplifying assumption—used in the majority of
ecological and evolutionary models—that dispersal rate is constant with respect to
conditions. Relaxing that assumption is now well justified on empirical grounds, and the
magnitude of shift in dispersal resulting from condition dependence suggests that it will
have non-trivial effects when incorporated into mechanistic models of evolution,
population dynamics, invasion spread, and so on. Amongst others, these effects may Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 include increased local adaptation within populations (Armsworth, 2008; Armsworth &
Roughgarden, 2008), heightened risk of overcrowding (Armsworth, 2008), greater
metacommunity stability (Fronhofer et al., 2018), and differences in metapopulation and
invasion dynamics (Neubert, Kot & Lewis, 2000; Kokko, 2006; Clobert et al., 2009;
Schreiber & Lloyd-Smith, 2009). In this light, the relative use of extra- vs intra-patch
information is important because, when we move to a conditional dispersal model, the
obvious simplifying assumption is that organisms exploit only intra-patch information. Our results suggest that intra-patch information is dominant in D. carinata, but the
degree to which this is true generally will determine how complex our models of
dispersal really need to be. Author Contributions Philip Erm conceived and designed the experiments, performed the experiments,
analysed the data, prepared figures and/or tables, authored or reviewed drafts of the
paper, approved the final draft. Matthew D. Hall analysed the data, authored or reviewed drafts of the paper, approved
the final draft. Ben L. Phillips conceived and designed the experiments, analysed the data, authored or
reviewed drafts of the paper, approved the final draft. Competing Interests Competing Interests The authors declare that they have no competing interests. ACKNOWLEDGEMENTS We thank Katrina-Lee Ware and Hannah Edwards for their invaluable assistance in the
preparation of the experimental set-up. We also thank Pepijn Luijckx, Christina
Buesching, and Nicolas Schtickzelle for their comments on drafts of the manuscript. Funding This work was supported by funding provided by the Australian Research Council
(DP160101730; FT160100198). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript. Grant Disclosures The following grant information was disclosed by the authors: Australian Research Council: DP160101730; FT160100198. Erm et al. (2019), PeerJ, DOI 10.7717/peerj.6599 REFERENCES Allen MR. 2007. Measuring and modeling dispersal of adult zooplankton. Oecologia
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Self-face Captures, Holds, and Biases Attention
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Self-face Captures, Holds, and
Biases Attention
Michał J. Wójcik 1, 2*, Maria M. Nowicka 1, Ilona Kotlewska 1, 3 and Anna Nowicka 1
1 Laboratory of Psychophysiology, Department of Neurophysiology, Nencki Institute of Experimental Biology, Warsaw, Poland,
2 Wroclaw Faculty of Psychology, SWPS University of Social Sciences and Humanities, Wroclaw, Poland, 3 Faculty of
Humanities, Nicolaus Copernicus University, Torun, Poland 1 Laboratory of Psychophysiology, Department of Neurophysiology, Nencki Institute of Experimental Biology, Warsaw, Poland,
2 Wroclaw Faculty of Psychology, SWPS University of Social Sciences and Humanities, Wroclaw, Poland, 3 Faculty of
Humanities, Nicolaus Copernicus University, Torun, Poland The implicit self-recognition process may take place already in the pre-attentive stages
of perception. After a silent stimulus has captured attention, it is passed on to the
attentive stage where it can affect decision making and responding. Numerous studies
show that the presence of self-referential information affects almost every cognitive level. These effects may share a common and fundamental basis in an attentional mechanism,
conceptualized as attentional bias: the exaggerated deployment of attentional resources
to a salient stimulus. A gold standard in attentional bias research is the dot-probe
paradigm. In this task, a prominent stimulus (cue) and a neutral stimulus are presented
in different spatial locations, followed by the presentation of a target. In the current study
we aimed at investigating whether the self-face captures, holds and biases attention
when presented as a task-irrelevant stimulus. In two dot-probe experiments coupled
with the event-related potential (ERP) technique we analyzed the following relevant ERPs
components: N2pc and SPCN which reflect attentional shifts and the maintenance
of attention, respectively. An inter-stimulus interval separating face-cues and probes
(800 ms) was introduced only in the first experiment. In line with our predictions, in
Experiment 1 the self-face elicited the N2pc and the SPCN component. In Experiment
2 in addition to N2pc, an attentional bias was observed. Our results indicate that
unintentional self-face processing disables the top-down control setting to filter out
distractors, thus leading to the engagement of attentional resources and visual short-term
memory. Edited by:
Sebastian Loth,
Bielefeld University, Germany
Reviewed by:
Eva Wiese,
George Mason University,
United States
Paola Sessa,
Università degli Studi di Padova, Italy
*Correspondence: Edited by:
Sebastian Loth,
Bielefeld University, Germany Reviewed by:
Eva Wiese,
George Mason University,
United States
Paola Sessa,
Università degli Studi di Padova, Italy *Correspondence:
Michał J. Wójcik
mwojcik13@st.swps.edu.pl Specialty section:
This article was submitted to
Cognitive Science,
a section of the journal
Frontiers in Psychology
Received: 08 September 2017
Accepted: 28 December 2017
Published: 11 January 2018 Specialty section:
This article was submitted to
Cognitive Science,
a section of the journal
Frontiers in Psychology Keywords: self-recognition, self-face, attention, ERP, attentional bias, N2pc, SPCN ORIGINAL RESEARCH
published: 11 January 2018
doi: 10.3389/fpsyg.2017.02371 INTRODUCTION Although being yourself is a profound and undeniable belief, it is not obvious from a functional
perspective. The unconscious machinery that is the cornerstone of the self works perpetually in
order to recognize as well as consolidate different and temporally separated pieces of self-related
information into one coherent whole (McAdams, 2001; Conway, 2005). Without it, there would be
no concept of “me” as distinct from “you,” no self-conscious thought, no identity. Although being yourself is a profound and undeniable belief, it is not obvious from a functional
perspective. The unconscious machinery that is the cornerstone of the self works perpetually in
order to recognize as well as consolidate different and temporally separated pieces of self-related
information into one coherent whole (McAdams, 2001; Conway, 2005). Without it, there would be
no concept of “me” as distinct from “you,” no self-conscious thought, no identity. Citation: The ability to cognitively identify oneself as an object in the environment, which is self-
recognition (Platek et al., 2004), is the central process that enables maintaining the coherence of the
self. It can be described on two levels: (1) implicit as the preference of self-related information, and
(2) explicit, as identification of one’s own image (Ross et al., 2011). As the Self Attention Network
model states, the implicit self-recognition process may take place already in the pre-attentive stages Wójcik MJ, Nowicka MM, Kotlewska I
and Nowicka A (2018) Self-face
Captures, Holds, and Biases
Attention. Front. Psychol. 8:2371. doi: 10.3389/fpsyg.2017.02371 Wójcik MJ, Nowicka MM, Kotlewska I
and Nowicka A (2018) Self-face
Captures, Holds, and Biases
Attention. Front. Psychol. 8:2371. doi: 10.3389/fpsyg.2017.02371 January 2018 | Volume 8 | Article 2371 Frontiers in Psychology | www.frontiersin.org Wójcik et al. Attention and Self-face of perception, assigning a saliency property to self-related
information that includes the image of one’s own face
(Humphreys and Sui, 2016). posterior contralateral negativity component (SPCN). Because
this component reflects later stages of information processing,
a prolonged time of cue presentation and delay between the
cue and target onsets were introduced in Experiment 1. Finally,
faster responses to targets preceded by self-face in comparison
to targets appearing contralateral to the self-face will indicate an
attentional bias (Experiment 2). Existing evidence related to the attentional capture effect of
self-face stimuli is rather inconclusive. For example, Tong and
Nakayama (1999) showed that the self-face was detected faster
among distractors than an other-face even if presented in an
unusual orientation. An attention-grabbing property of the self-
face was also shown by Brédart et al. (2006). A flanking self-face
caused a stronger interference in the detection of a classmate’s
name in comparison to the reversed condition. In contrast,
Devue et al. (2009) reported that if the self-face was presented
outside of the participant’s focus of attention, it failed in capturing
attention. The N2pc component consists of a greater negativity at the
contralateral sites than the ipsilateral sites to an attended stimuli. It is typically detected at posterior scalp sites, approximately
200–300 ms after stimulus onset, with a maximum voltage at
the parietal-occipital region (Luck and Hillyard, 1994; Eimer,
1996). N2pc is used to determine whether the focus of attention
has covertly been shifted to the location of a silent stimulus. Citation: It relies on the exaggerated deployment of attentional
resources to a salient stimulus that is present in a person’s external
environment. A gold standard in attentional bias research is the dot-probe
paradigm, which also enables investigation of attentional capture
effects (Pfabigan and Tran, 2015). In this task, a prominent
stimulus (cue) and a neutral stimulus are presented at the same
time in different spatial locations (e.g., one to the left and
one to the right of the central fixation point), followed by the
presentation of a target at cued or not cued locations. Reaction
times (RTs) to targets that appear at the prior location of the
prominent stimulus (i.e., cue-congruent trials) are compared
with RTs to targets that appear at the prior location of the neutral
stimulus (i.e., cue-incongruent trials). Faster responses typically
observed in cue-congruent trials are interpreted as evidence of
an attentional bias to the location of the prominent stimulus. It is worth noting that biases caused by different cue
presentation times reflect biases in different stages of processing
(Cisler and Koster, 2010). Longer cue durations may lead to
the difficulty in disengaging attention from prominent stimuli,
which may be caused by the failure of the dorsal fronto-parietal
network to control the deployment of attention (Corbetta and
Shulman, 2002; De Raedt and Koster, 2010). In turn, shorter
cue duration times (typically less than 500 ms) reflect rapid
orienting to the silent stimulus. This exaggerated sensitivity is
caused by a stimulus-driven detection mechanism that is likely
to involve the amygdala, accompanied by a failure of control
mechanisms in the left lateral prefrontal cortex, which normally
filters out the to-be-ignored stimulus (Bishop et al., 2004, 2007). Therefore, we hypothesized that the extended cue presentation
time in Experiment 1 should produce an SPCN component. In
order to prevent the influence of ongoing perceptual processing
on this component, we separated the self-face cue from the
target with a black-screen. It should be clarified that this In the current study, we aimed at investigating whether the
self-face captures, holds and biases attention when presented as
a task-irrelevant, to-be-ignored stimulus. To achieve these goals,
we conducted two experiments using the dot-probe task coupled
with electrophysiological measurements (EEG). Both of them
were intended to reveal attention-grabbing properties of self-face
as operationalized by the emergence of a lateralized event-related
potentials (ERPs) component N2-posterior-contralateral, N2pc
(Eimer and Kiss, 2008; Sawaki and Luck, 2010). Citation: It
reflects the allocation of a limited-capacity process to a relevant
object (Ester et al., 2012). Previous dot-probe studies reported
the presence of N2pc for prominent stimuli such as emotional
faces (Holmes et al., 2009; Grimshaw et al., 2014). To the best
of our knowledge, none of the studies used the self-face as
N2pc-evoking stimulus. Theeuwes (2010), as well as Itti and Koch (2001), have
proposed that after a silent stimulus has captured attention, it
is passed on to the attentive stage where it is identified. Because
the information about this stimulus is now available for the top-
down processes, it can affect decision making and responding. Numerous studies show that the presence of self-referential
information affects almost every cognitive level (Humphreys and
Sui, 2016). Self-related information alters perception (Sui et al.,
2015), attention (Tong and Nakayama, 1999; Brédart et al., 2006;
Devue et al., 2009), memory (Symons and Johnson, 1997) and
even meta-cognition (Pronin et al., 2002). Moreover, self-bias
exerts influence also on social perception (Ross et al., 1977). In tasks that engage visual short-term memory (Jolicœur et al.,
2008) an SCPN component is often observed. It begins about
300–400 ms after stimulus onset and persists for the duration
of the retention interval. The SPCN is also present in tasks that
are not defined as memory tasks, but that engage visual short-
term memory as an intermediate processing buffer (Jolicœur
et al., 2008). This component is computed as the difference
between contralateral and ipsilateral activity time-locked to a
lateralized stimulus. Its amplitude increases with visual working
memory (VWM) informational load (e.g., Vogel and Machizawa,
2004; Jolicœur et al., 2008; Perron et al., 2009; Robitaille et al.,
2009) and with increased number (Vogel and Machizawa, 2004)
and the complexity (Luria et al., 2010) of stimuli to be held
in VWM. Sessa et al. (2011, 2012) have further demonstrated
that SPCN amplitude is modulated by emotional expressions
of faces and varies proportionally to the resolution of the
faces’ representations in VWM, such that representations of
high-resolution faces elicit larger SPCN amplitudes relative to
representations of low-resolution faces. One may suppose that these effects share a common and
fundamental basis in an attentional mechanism described by
Theeuwes (2010), which could be conceptualized as attentional
bias. This term refers to the tendency for people’s perception
to be affected by previously processed information (Bar-Haim
et al., 2007). Frontiers in Psychology | www.frontiersin.org January 2018 | Volume 8 | Article 2371 Experiment 2 The face-cues were presented for 50 and 150 ms and were not
separated from the probe by a black-screen. All other details
were the same as in Experiment 1. The experimental procedure
is shown in Figure 3. Participants Twenty-one subjects (10 female) between the ages of 24 and 35
(M = 27.6, SD = 3) participated in the study. All participants had
normal or corrected-to-normal vision and reported no history
of mental or neurological disorders. Twenty of these subjects
were right-handed, as assessed by the Edinburgh Handedness
Inventory (Oldfield, 1971). The study was conducted with
the approval of the Human Ethics Committee of the SWPS
University of Social Sciences and Humanities (Warsaw, Poland). All participants gave written informed consent prior to the
experiments. Dot-Probe Task
Experiment 1 inter-stimulus interval reduces the influence of the cue on the
target, thus probably eliminating the facilitation effect regarding
reaction times. In Experiment 2 we focused on investigating the
attentional bias toward the self-face, thus the cue and the target
were not separated. Each trial started with a fixation cross (subtending 0.4◦× 0.4◦
of visual angle; positioned in the center of the screen) which
remained onscreen for the duration of the trial. After 1,000 ms, a
pair of faces was presented bilaterally. In order to minimalize the
occurrence of readiness potentials, the face-cues were presented
for either 150 or 600 ms (Libet et al., 1983). Cues were followed by
a black-screen which remained on screen for 800 ms. The probe
(an asterisk subtending 0.3◦× 0.3◦of visual angle) was then
displayed in the visual field previously occupied by the self-face
(congruent condition), in the opposed visual field (incongruent
condition) or in the visual field previously occupied by one of
two bilaterally presented other-faces, i.e., non-aligned condition
(see Figure 2). In the first two conditions, an other-face was
presented contralateral to the self-face. Then the participants
were instructed to indicate (as quickly and as accurately as
possible) the side on which the probe appeared, by pressing with
their left or right index finger the button corresponding to the
probe’s location. The participants were requested to maintain
fixation on the cross and ignore the face cues. Stimuli The stimuli consisted of bilaterally presented pairs of gray-scaled
face photographs. Depending on the condition, stimuli pairs
contained either a self-face and an other-face or two other-faces. Twenty-six (13 male and 13 female) other-face photographs were
taken from the A series of the Karolinska Directed Emotional
Faces database (Lundqvist et al., 1998). Figure 1 presents example
stimuli used in the current study. In order to avoid effects
of facial expressions, the photos were selected based on the
unbiased hit-rates for neutral expression detection (Goeleven
et al., 2008). Self-face photographs were taken prior to the
experiments. All stimuli were cropped to include only the face,
resized to subtend 6.9◦× 8.9◦of visual angle and equaled for
mean luminance using Photoshop R⃝(Adobe, San Jose, CA). Cue-faces appeared on the screen with their inner edge 3◦left
and right from the fixation. This distance is sufficient to detect
horizontal eye movements and, as a consequence, to reject trials
contaminated with these artifacts (Meyberg et al., 2017). The
gender of other-faces was matched to each participant’s gender
in order to control the between-category variability in attentional
effects. EEG and EOG Data The electroencephalogram (EEG) was continuously recorded
with 64 Ag-AgCl electrically shielded electrodes mounted on
an elastic cap (ActiCAP, Munich, Germany) and positioned
according to the extended 10–20 system. For ocular artifact
scoring, vertical and horizontal electrooculograms (EOGs) were
recorded from bipolar electrodes placed at the supra- and
suborbit of the right eye and at external canthi of eyes. EEG
electrode impedances were kept below 10 k. The data were
amplified using a 128-channel amplifier (QuickAmp, Brain
Products, Enschede, Netherlands) and digitized at a 500 Hz
sampling rate. The EEG signal was recorded against an average
of all channels calculated by the amplifier hardware. Offline, the
62 channels were re-referenced to the algebraic average of the left
and right earlobes, notch filtered at 50 Hz and digitally band-pass
filtered from 1 to 30 Hz using a zero-phase Butterworth filter (12
dB/oct). Procedure and Apparatus After electrode cap placement (ActiCAP, Brain Products,
Munich, Germany), participants were seated in a comfortable
chair in a dimly lit and sound-attenuated room. During the
task, an adjustable chin rest maintained head position and a
constant viewing distance of 72 cm. The dot-probe task was
presented on a Flex Scan EV-2450 (Hakusan, Ishikawa, Japan)
computer screen through an Intel Core i3 computer running
Presentation R⃝software (Neurobehavioral Systems, Albany, CA,
USA). The screen was specially calibrated for correction to
black in order to not exhaust eyes with intense background
illumination. EEG signal was amplified using QuickAmp and
digitized using BrainVision Recorder R⃝software (Brain Products,
Munich, Germany). Citation: The presence of
attentional hold effects can be, in turn, evidenced by a sustained January 2018 | Volume 8 | Article 2371 Frontiers in Psychology | www.frontiersin.org 2 Wójcik et al. Attention and Self-face ERP Analysis Occipital-temporal channels PO8 and PO7 were chosen for
the ERPs analysis. These electrodes are frequently reported by
many authors as disclosing maximal N2pc amplitudes (Eimer
and Kiss, 2008; Jolicœur et al., 2008; Burra and Kerzel, 2013). Moreover, CSD topographic maps (obtained by subtracting non-
aligned conditions from those containing the self-cue) showed
a clear distribution of N2pc that is maximal over the parietal-
occipital region (see Figures 4, 5). The EEG signal was segmented
into 800 ms epochs from 200 ms before to 600 after cue onset. These epochs were baseline-corrected against the mean voltage January 2018 | Volume 8 | Article 2371 Frontiers in Psychology | www.frontiersin.org 3 Wójcik et al. Attention and Self-face FIGURE 1 | Set of example other-faces stimuli (F01, F03, F05, F07, F13, F23, F24, F26, F29 and M01, M06, M07, M08, M10, M11, M14, M18, M25, M31) taken
from the A series of the Karolinska Directed Emotional Faces database (Lundqvist et al., 1998). FIGURE 1 | Set of example other-faces stimuli (F01, F03, F05, F07, F13, F23, F24, F26, F29 and M01, M06, M07, M08, M10, M11, M14, M18, M25, M31) taken
from the A series of the Karolinska Directed Emotional Faces database (Lundqvist et al., 1998). FIGURE 1 | Set of example other-faces stimuli (F01, F03, F05, F07, F13, F23, F24, F26, F29 and M01, M06, M07, M08, M10, M11, M14, M18, M25, M31) taken
from the A series of the Karolinska Directed Emotional Faces database (Lundqvist et al., 1998). FIGURE 1 | Set of example other-faces stimuli (F01, F03, F05, F07, F13, F23, F24, F26, F29 and M01, M06, M07, M08, M10, M
from the A series of the Karolinska Directed Emotional Faces database (Lundqvist et al., 1998). presentation time (M = 61, SD = 14), 50 ms cue presentation
time (M = 66, SD = 12). The number of epochs used to compute
ERPs did not differ significantly between conditions (ipsilateral
and contralateral waveforms were computed based on the same
datasets). during the 200 ms pre-stimulus period. N2pc A two (laterality: contralateral vs. ipsilateral) × two (cue
presentation time: 150 ms vs. 600 ms) repeated measures
ANOVA yielded a significant main effect of laterality, F(1, 17)
= 17.618, p < 0.001, ηp2 = 0.509. This indicates a clear N2pc
for both cue presentation times quantified as more negative
mean amplitudes of the contralateral (M = 1.455 µV) than the
ipsilateral (M = 2.242 µV) waveform. The main effect of “cue
presentation time” also reached significance, F(1, 17) = 16.835,
p < 0.001, ηp2 = 0.498, showing more negative amplitudes for
150 ms than 600 ms of self-face presentation. The interaction
between these two factors was non-significant. ERP Analysis FIGURE 2 | Experiment 1: a sequence of events in a single experimental trial in 3 experimental conditions: congruent, incongruent, and non-aligned. The example
self-face is a photograph of one of the co-authors; other-faces stimuli (F19 and F34) were taken from the A series of the Karolinska Directed Emotional Faces
database (Lundqvist et al., 1998). FIGURE 2 | Experiment 1: a sequence of events in a single experimental trial in 3 experimental conditions: congruent, incongruent, and non-aligned. The example
self-face is a photograph of one of the co-authors; other-faces stimuli (F19 and F34) were taken from the A series of the Karolinska Directed Emotional Faces
database (Lundqvist et al., 1998). in response when the self-face was presented for 600 ms (M =
370.5 ms) than for 150 ms (M = 383.4 ms). Other effects and
interactions were non-significant. analysis. These quantifications of the N2pc component are
in line with previous studies on electrophysiological markers
of attentional capture (Eimer and Kiss, 2007; Buodo et al.,
2009; Holmes et al., 2009; Grimshaw et al., 2014). After the
contralateral and ipsilateral curves converged in Experiment 1,
indicating the end of the N2pc component, another contralateral
negativity emerged, corresponding to the onset of the SPCN
component (Jolicœur et al., 2008). It was analyzed in 450–550 ms
time window. Non-aligned trials were not taken into account
in this analysis, as they lacked a laterally-located cue that could
serve as a reference. Behavioral Analyses Behavioral Analyses All trials with RTs shorter than 100 ms and longer than 1,000 ms
were excluded from analysis. Mean RTs for correct trials were
computed for each participant for each condition (see Table 1
for descriptive statistics). The same three subjects as in the
ERP analysis were excluded from the analysis because of artifact
contamination. A three (condition: congruent vs. incongruent vs. non-aligned) × two (cue presentation time: 150 ms vs. 600 ms)
repeated measures analysis of variance was computed. A main
effect of “cue presentation time” was found, F(1, 17) = 27.738,
p < 0.000, ηp2 = 0.620, showing that the subjects were faster ERP Analysis Epochs contaminated
with vertical eye movements and blinks (a change in voltage in
the VEOG channel exceeding 100 µV within a 200 ms period),
horizontal eye movements (a change in voltage in the HEOG
channel exceeding 50 µV within any 200 ms period) or other
artifacts (in all channels: voltage steps exceeding 50 µV, voltage
change exceeding 100 µV within any 200 ms period, amplitudes
greater than 200 µV and lower than −200 µV, activity in 100 ms
intervals lower than 0.5 µV) were rejected. Because of excessive
artifact contamination, three participants were excluded from the
analyses (82.5, 49.2, and 48% rejected trials) in Experiment 1
and three (93.8, 84.4, and 63.1% rejected trials) in Experiment
2. An average of 15 and 20.5% of trials per subject was rejected in
Experiment 1 and Experiment 2, respectively. The mean number
of segments in Experiment 1 which passed the artifact rejection
procedure was as follows: 600 ms cue presentation time (M =
138, SD = 14), 150 ms cue presentation time (M = 136, SD
= 16). The mean number of segments in Experiment 2 which
passed the artifact rejection procedure was as follows: 150 ms cue The ipsilateral waveform was calculated as the average of
signals recorded at PO7 electrode to the left-sided self-face
stimulus and at PO8 electrode to the right-sided self-face
stimulus. The contralateral waveform was computed as the
average of signals recorded at PO7 electrode to the right-sided
self-face stimulus and at PO8 electrode to the left-sided self-
face stimulus. In order to clearly visualize the N2pc and SPCN
components and isolate them from overlapping components
a difference between collapsed contralateral and ipsilateral
waveforms was calculated (Luck, 2014). On
the
basis
of
visual
inspection
of
grand
average
contralateral-ipsilateral waveforms obtained in Experiment
1 and Experiment 2 (see Figures 4, 5), mean amplitude in
240–360 ms time window was chosen for successive statistical Frontiers in Psychology | www.frontiersin.org January 2018 | Volume 8 | Article 2371 4 Wójcik et al. Attention and Self-face FIGURE 2 | Experiment 1: a sequence of events in a single experimental trial in 3 experimental conditions: congruent, incongruent, and non-aligned. The example
self-face is a photograph of one of the co-authors; other-faces stimuli (F19 and F34) were taken from the A series of the Karolinska Directed Emotional Faces
database (Lundqvist et al., 1998). N2pc In order to assess the presence of the N2pc component a two
(laterality: contralateral vs. ipsilateral) × two (cue presentation
time: 150 ms vs. 50 ms) repeated measures analysis of variance
was performed. It yielded a significant main effect of laterality,
F(1, 17) = 28.552, p < 0.001, ηp2 = 0.627, indicating that overall
the mean amplitudes of the contralateral waveform were more
negative (M = 0.423 µV), than the mean amplitudes of the
ipsilateral waveform (M = 1.397 µV). The main effect of “cue
presentation time” also reached significance, F(1, 17) = 7.916,
p < 0.05, η2
p = 0.318, showing more negative amplitudes for
50 ms than 150 ms of self-face presentation (see Figure 5). The
interaction between these two factors was non-significant. In order to assess the presence of the N2pc component a two
(laterality: contralateral vs. ipsilateral) × two (cue presentation
time: 150 ms vs. 50 ms) repeated measures analysis of variance
was performed. It yielded a significant main effect of laterality,
F(1, 17) = 28.552, p < 0.001, ηp2 = 0.627, indicating that overall
the mean amplitudes of the contralateral waveform were more
negative (M = 0.423 µV), than the mean amplitudes of the
ipsilateral waveform (M = 1.397 µV). The main effect of “cue
presentation time” also reached significance, F(1, 17) = 7.916,
p < 0.05, η2
p = 0.318, showing more negative amplitudes for
50 ms than 150 ms of self-face presentation (see Figure 5). The
interaction between these two factors was non-significant. y
Based on the mean percentage of trials contaminated with
ocular artifacts per participant, the same three participants
as in the ERP analysis were excluded from the sample. Descriptive statistics regarding every experimental condition
are presented in Table 2. The data structure was analyzed
with a three (condition: congruent vs. incongruent vs. non-
aligned) × two (cue presentation time: 150 ms vs. 50 ms)
repeated measures ANOVA. The main effect of “condition,”
F(2, 34) = 3.996, p < 0.05, ηp2 = 0.190, indicated a significant
difference in reaction times. Post-hoc tests revealed that
overall participants were faster in response when the target
appeared in the visual field previously occupied by the self-
face (M = 368.9 ms) than in the incongruent condition (M
= 375.6 ms), pb < 0.05. N2pc The main effect of “cue presentation
time” also reached significance, F(1, 17) = 18.915, p < 0.001,
η2
p = 0.527, showing faster responses in trials with cues
displayed for 150 ms (363.7 ms) than 50 ms (380.8 ms). The
interaction between the aforementioned factors was non-
significant. SPCN The repeated measures ANOVA performed on “laterality” and
“cue presentation time” yielded a significant interaction between
these two factors, F(1, 17) = 9.424, p < 0.05, ηp2 = 0.357,
indicating that an SPCN was only present when the cue was
displayed for 600 ms. The factor of “cue presentation time” also
obtained a significant main effect, F(1, 17) = 5.320, p < 0.05, ηp2 =
0.238, showing more negative amplitudes for 600 ms than 150 ms
of cue presentation time. January 2018 | Volume 8 | Article 2371 Frontiers in Psychology | www.frontiersin.org 5 Wójcik et al. Attention and Self-face FIGURE 3 | Experiment 2: a sequence of events in a single experimental trial in 3 experimental conditions: congruent, incongruent, and non-aligned. The example
self-face is a photograph of one of the co-authors; other-faces stimuli (F19 and F34) were taken from the A series of the Karolinska Directed Emotional Faces
database (Lundqvist et al., 1998). FIGURE 3 | Experiment 2: a sequence of events in a single experimental trial in 3 experimental conditions: congruent, incongruent, and non-aligned. The example
self-face is a photograph of one of the co-authors; other-faces stimuli (F19 and F34) were taken from the A series of the Karolinska Directed Emotional Faces
database (Lundqvist et al., 1998). DISCUSSION The aim of the current study was to examine whether the self-
face captures automatic attention and as a consequence produces
an attentional bias. Additionally, we assessed the attentional hold
effects of this stimulus. The time course of the attentional effects January 2018 | Volume 8 | Article 2371 Frontiers in Psychology | www.frontiersin.org 6 Wójcik et al. Attention and Self-face E 4 | Grand average ERPs time-locked to the onset of the cue-faces (self vs. other) at electrodes PO7/PO8 and difference waves for 150 ms (A) and 600 ms
presentation time obtained in Experiment 1. (C) Presents topographical CSD maps of activity distribution in N2pc time window obtained by subtracting the
gned condition from the conditions containing cues. Two maps on the left (150 ms cue presentation) and two maps on the right (600 ms cue presentation)
mplitude distributions for self-face presented in the right and left visual field, respectively. (D) Presents topographical CSD maps of activity distribution in SPCN
ndow obtained by subtracting the non-aligned condition from the conditions containing cues. The map on the left and the right show amplitude distributions for
e presented in the right and left visual field, respectively. FIGURE 4 | Grand average ERPs time-locked to the onset of the cue-faces (self vs. other) at electrodes PO7/PO8 and difference waves for 150 ms (A) and 600 ms
(B) cue presentation time obtained in Experiment 1. (C) Presents topographical CSD maps of activity distribution in N2pc time window obtained by subtracting the
non-aligned condition from the conditions containing cues. Two maps on the left (150 ms cue presentation) and two maps on the right (600 ms cue presentation)
show amplitude distributions for self-face presented in the right and left visual field, respectively. (D) Presents topographical CSD maps of activity distribution in SPCN
time window obtained by subtracting the non-aligned condition from the conditions containing cues. The map on the left and the right show amplitude distributions for
self-face presented in the right and left visual field, respectively. In line with our predictions, the self-face elicited a clear
N2pc component in both dot-probe tasks: with and without
a time delay between onsets of the cue (involving bottom-
up processes) and target (involving top-down processes). The
emergence of N2pc reflects the self-face’s attention-grabbing
properties on a neural level. DISCUSSION Previous ERP studies on self-
face recognition suggested such enhanced attentional processing;
however, without specifying the stage of processing in which this
effect occurs (Sui et al., 2006; Tacikowski and Nowicka, 2010). was investigated using the ERP technique, which allowed us to
trace the neural basis and the time-course of cognitive processes
that occur very fast. Our analysis was focused on two ERPs
components, N2pc and SPCN which reflect attentional shift and
the engagement of visual short-term memory, respectively. On
the behavioral level, attentional bias was evidenced by comparing
RTs to targets preceded by self-face in comparison to RTs to
targets preceded by other-faces. In general, the findings of this
study supported all our predictions. was investigated using the ERP technique, which allowed us to
trace the neural basis and the time-course of cognitive processes
that occur very fast. Our analysis was focused on two ERPs
components, N2pc and SPCN which reflect attentional shift and
the engagement of visual short-term memory, respectively. On
the behavioral level, attentional bias was evidenced by comparing
RTs to targets preceded by self-face in comparison to RTs to
targets preceded by other-faces. In general, the findings of this
study supported all our predictions. January 2018 | Volume 8 | Article 2371 Frontiers in Psychology | www.frontiersin.org 7 Wójcik et al. Attention and Self-face RE 5 | Grand average ERPs time-locked to the onset of the cue-faces (self vs. other) at electrodes PO7/PO8 and difference waves for 50 ms (A) and 150 ms
e presentation time obtained in Experiment 2. (C) Presents topographical CSD maps of activity distribution in N2pc time window obtained by subtracting the
gned condition from the conditions containing cues. Two maps on the left (50 ms cue presentation) and two maps on the right (150 ms cue presentation) show
ude distributions for self-face presented in the right and left visual field, respectively. FIGURE 5 | Grand average ERPs time-locked to the onset of the cue-faces (self vs. other) at electrodes PO7/PO8 and difference waves for 50 ms (A) and 150 ms
(B) cue presentation time obtained in Experiment 2. (C) Presents topographical CSD maps of activity distribution in N2pc time window obtained by subtracting the
non-aligned condition from the conditions containing cues. January 2018 | Volume 8 | Article 2371 DISCUSSION Two maps on the left (50 ms cue presentation) and two maps on the right (150 ms cue presentation) show
amplitude distributions for self-face presented in the right and left visual field, respectively. visual stimuli (cue and probe) were processed in a serial
manner. The only study—to our best knowledge—that did not confirm
prioritized visual selection of the self-face was a behavioral
study of (Devue et al., 2009). Such discrepancy may be related
to the issue of parallel vs. serial processing. It seems that
bottom-up processes in the visual search paradigm applied
by Devue and colleagues took place parallel to the ongoing
top-down processing. Due to the attentional bottleneck that
occurs at the advanced stages of the top-down processing
(indicated by the delayed time to arrive at the self-face), the
self-preference effects could be inhibited (Sigman and Dehaene,
2008). This was not the case in the present study as all The only study—to our best knowledge—that did not confirm
prioritized visual selection of the self-face was a behavioral
study of (Devue et al., 2009). Such discrepancy may be related
to the issue of parallel vs. serial processing. It seems that
bottom-up processes in the visual search paradigm applied
by Devue and colleagues took place parallel to the ongoing
top-down processing. Due to the attentional bottleneck that
occurs at the advanced stages of the top-down processing
(indicated by the delayed time to arrive at the self-face), the
self-preference effects could be inhibited (Sigman and Dehaene,
2008). This was not the case in the present study as all An important and novel finding of the present dot-probe
study is the attentional bias toward self-face, whereas previous
studies reported such bias only for emotional faces, especially
threatening ones (Bar-Haim et al., 2007). The facilitation effect
(faster RTs) observed in our study may result from the enhanced
neural sensitivity at locations that were previously in the focus
of attention. This notion is based on findings showing that
directing attention to a specific area of the visual field enhances
contrast sensitivity and spatial resolution at the attended location January 2018 | Volume 8 | Article 2371 Frontiers in Psychology | www.frontiersin.org 8 Wójcik et al. Attention and Self-face TABLE 1 | Descriptive statistics of behavioral results in Experiment 1. FUNDING This work was funded by the National Science Centre, Poland
(2015/19/B/HS6/01258 to AN). AUTHOR CONTRIBUTIONS MW and AN developed the study concept. All authors
contributed to the study design. Testing and data collection were
performed by all authors. MW and MN performed the data
analysis and interpretation under the supervision of AN. MW
and AN wrote the manuscript. All authors approved the final
version of the manuscript for submission. DISCUSSION Moreover, this attentional bias may be
caused by a difficulty in disengaging attention from self-face, a
phenomenon that was reported by Devue et al. (2009). These two
interpretations may even complement each other. (Carrasco et al., 2004). Moreover, this attentional bias may be
caused by a difficulty in disengaging attention from self-face, a
phenomenon that was reported by Devue et al. (2009). These two
interpretations may even complement each other. Another unique feature of the present study is the presence
of SPCN that was found exclusively for longer (600 ms) self-
face cue presentations, indicating an attentional hold effect. This
component has been typically observed when the task required
encoding the peripheral stimulus and is associated with visual
short-term memory (Jolicœur et al., 2008). Taking into account
that the self-face could be viewed as a silent, prominent stimulus,
the emergence of SPCN in our study is in line with an EEG
study that analyzed this component for emotional faces (Stout
et al., 2013). Despite the fact that in the present study cues were
supposed to be ignored by participants a clear SPCN emerged. This indicates that unintentional self-face processing disables the
top-down control setting to filter out distractors, thus leading
to the engagement of visual short-term memory. Apparently,
the visual system treats self-referential stimuli as prioritized,
thus neglecting their status as distractors. As Holmes et al. (2009) propose, SPCN may also reflect mechanisms involved
in the maintenance of attention. In their opinion retaining in
visual short-term memory information about the silent stimuli
is thought to drive top-down template signals that facilitate the
maintenance of visual spatial attention, even though they are not
task-relevant. DISCUSSION Cue presentation time
Condition
Mean
SD
N
600 ms
Congruent
370.2
48.20
18
Incongruent
370.2
49.80
18
Non-aligned
371.3
51.60
18
150 ms
Congruent
384.8
54.29
18
Incongruent
385.8
55.25
18
Non-aligned
379.7
52.21
18
TABLE 2 | Descriptive statistics of behavioral results in Experiment 2. Cue presentation time
Condition
Mean
SD
N
150 ms
Congruent
359.1
38.37
18
Incongruent
367.3
38.05
18
Non-aligned
364.8
35.47
18
50 ms
Congruent
378.8
36.13
18
Incongruent
384.0
34.54
18
Non-aligned
379.7
32.12
18 Overall, the reported results provide novel evidence pointing
to
the
similarities
between
self-face
and
emotional
face
processing, i.e., similar effects are obtained in the same processing
stages of these two types of stimuli. First of all, both of
them capture involuntary attention (quantified by the N2pc
component) as revealed by our and Grimshaw et al.’s experiments
(2014). As a consequence of the above, self-face and emotional-
face (Fox et al., 2002) produce attentional bias. Finally, visual
short-term memory is engaged in the processing of these stimuli
as revealed by the emergence of SPCN (Holmes et al., 2009). However, this highly interesting resemblance should be further
investigated through direct comparison of these two types
of stimuli, in order to reveal plausible common mechanisms
involved in their processing. p
g
At the end, we would like to comment on future directions
in the field of the self-research. The neural basis of self-face
recognition has been extensively investigated, resulting in various
neuroanatomical network models (Kircher et al., 2000; Uddin
et al., 2005; Platek et al., 2006) and electrophysiological correlates
(Keyes et al., 2010; Tacikowski and Nowicka, 2010; Kotlewska
and Nowicka, 2015) associated with processing of this unique
stimulus. However, as Devue and Brédart (2011) postulate,
the understanding of neural correlates of self-face recognition
will not substantially improve without specifying more clearly
the cognitive operations induced by self-perception. It seems
that discussions in many studies in this field—including our
previously published papers (Tacikowski and Nowicka, 2010;
Kotlewska and Nowicka, 2015)—are mainly devoted to the
description of results in light of other findings, either supporting
or contradicting the currently reported ones. Unfortunately,
underlying cognitive mechanisms are not sufficiently elaborated,
therefore impeding and complicating the interdisciplinary
research of self (Zahavi and Roepstorff, 2011). In the present
study, we attempted to solve this problem by referring directly
to very basic attentional mechanisms, well-documented in
experimental paradigms with stimuli other than the self-face. (Carrasco et al., 2004). Bishop, S., Duncan, J., Brett, M., and Lawrence, A. D. (2004). Prefrontal cortical
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Neurosci. 7, 184–188. doi: 10.1038/nn1173
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article distributed under the terms of the Creative Commons Attribution License (CC
BY). January 2018 | Volume 8 | Article 2371 Frontiers in Psychology | www.frontiersin.org REFERENCES The use, distribution or reproduction in other forums is permitted, provided the
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Series in Audiovisual Arts and Production Bulletin of Kyiv National University of Culture and Arts. Series in Audiovisual Arts and Production DOI: 10.31866/2617-2674.2.2.2019.185693
УДК 791.221.9 2019 · 2(2) · pp.165-172 ДО ПИТАННЯ ХОРРОР
ЯК ЖАНРУ СУЧАСНОГО ЕКРАННОГО ПРОСТОРУ Ганна Чміль1a, Марина Войцещук2a 1 доктор філософських наук, доцент;
e-mail: gannachmil@gmail.com; ORCID: 0000-0001-6569-1066
2 асистент кафедри кіно-, телемистецтва;
e-mail: dekanat25kino@ukr.net; ORCID: 0000-0001-8164-012X
a Київський національний університет культури і мистецтв, Ки Ключові слова:
мистецтво;
жахи;
кінематограф;
фільм;
хоррор;
страх Анотація
Мета дослідження полягає у розкритті особливостей жанру та констатуванні
його значення для вирішення художніх завдань як окремої галузі
аудіовізуального мистецтва. Методологія дослідження. У нашому дослідженні
було застосовано наступні методи: аналітичний – для виокремлення певних
частин загальної проблеми та вивчення для повного уявлення про предмет
дослідження; комплексний підхід – для цілісного та різнобічного висвітлення
теми, зокрема під час розгляду теоретичного розділу з досліджувальної
проблеми; мистецтвознавчий – застосовувався під час аналізу кінотворів
у художньому дискурсі; порівняльний – у процесі дослідження жанрів та
піджанрів різних років; описовий – під час дослідження особливостей
жанрових категорій та окремих фільмів; діалектичний – під час аналізу
поглядів, думок різних кінокритиків та науковців стосовно жанру та
піджанрів. Наукова новизна одержаних результатів полягає в тому, що
завдяки аналізу та синтезу з’ясовано характер впливу жанру жахів на
усвідомлюване світорозуміння людини, а також характер побудови такого
кінопродукту, принципів звернення авторів до внутрішнього світу глядачів
через співвідношення різних рівнів розвитку світогляду режисера та глядача. Висновки. У роботі досліджено хоррор як жанр сучасного екранного простору. Доведено, що сьогодні фільм жахів – один з найвідоміших і стабільних жанрів
голлівудського кінематографа. Його засобами активно користуються й інші
творці, а деякі кінострічки викликають серйозний культурний резонанс. Конкретна категорія мистецтва відіграє визначальну роль у формуванні
нового кінематографічного майбутнього. Також у роботі наведено приклади
традиційних меж жанру та його кризового становища. Виклад основного матеріалу Такий вид мистецтва, як кінематограф,
займає своє почесне місце на п’єдесталі
за значущістю для розвитку суспільства
загалом й кожної особистості зокрема. Говорити про те, як впливає кіно на лю
дину, не варто, досить сказати, що такі
культові стрічки, як «Бійцівський клуб»,
«Втеча із Шоушенка» та ряд інших – на
завжди залишаться в пам’яті людини
завдяки тому, як вони позначились на
те або інше покоління. РЕЖИСУРА ТА ЗВУКОРЕЖИСУРА КІНО І ТЕЛЕБАЧЕННЯ
2019 · 2(2) · с.165-172
Вісник Київського національного університету культури і мистецтв.
Серія: Аудіовізуальне мистецтво і виробництво 2019 · 2(2) · с.165-172 Постановка проблеми А. Піотровський, А. Мачерет, С. Фрейлих,
Я. Маркулан, А. Рутковський, М. Бах
тін, В. Пропп, Д. Лихачов, Ю. Лотман,
М. Каган, С. Лазарук, Д. Салинський,
Л. Нехорошев. Питання жанрів у кіно періодично ви
являється в центрі мистецтвознавчих
досліджень. Попри те, що перші серйозні
спроби осмислення жанрових систем кі
нематографа відносяться до 20-х років
минулого століття, багато з кінознав
ців вважають, що теорія жанрів кіно
на даний момент досі не розроблена. Підтвердженням цьому служить те, що
в закордонному кінознавстві постійно
публікуються роботи й захищаються
дисертації присвячені цій темі. У відношенні хоррора поширені автор
ський (Є. Бансак, М. Макдона, Дж. Мьюір),
психоаналітичний або фрейдистський
(Ч. Деррі, Б. Крід, Дж. Айаччіно), ген
дерний (К. Кловер, Г. Беншофф, Р. Бе
ренстайн, І. Пінедо), історико-детермі
ністський (У. Діксон), расовий (Р. Мінс
Колман), історико-культурологічний
(Д. Скель, А. Кернер, М. Фостер, А. Ле
венштейн), соціокультурний (К. Іган,
Т. Джонсон), ринково-маркетинговий
(К. Хеффернен, Р. Ноувелл), морфоло
гічний (В. Діка) і ряд інших підходів. Тема жанрів знаходиться й у центрі
уваги вітчизняної мистецтвознавчої
думки, але міркування здобувають ді
аметрально протилежні напрямки, зво
дяться до повної відмови від необхід
ності розглядати цю жанрову систему. Як цитувати: Чміль, Г. та Войцещук, М. (2019). До питання хоррор як жанру сучасного екранного простору. Вісник
Київського національного університету культури і мистецтв. Серія: Аудіовізуальне мистецтво і виробництво,
2(2), с.165-172. Надійшла 22.01.2019 © Ганна Чміль, Марина Войцещук, 2019 ISSN 2617-2674 (Print),
ISSN 2617-4049 (Online) Мета дослідження Уявлення про класифікацію кіно про
тягом ХХ століття змінювалось, висува
лися різні концепції трактування жанру
й розуміння його ролі в художньому про
цесі. Точки зору відносно хоррору були
кардинально протилежними, а саме: від
вкрай радикальних міркувань, які вва
жаються певною науковою оманою,
до теорій, в яких жанр розглядався як
центральна художня категорія, як ро
бочий інструмент у творчій діяльності. Не дивлячись на відмінність вчень, до
слідницький інтерес до проблеми не
слабшає й має циклічний характер. Ця
проблематика як і раніше залишається
у центрі уваги теоретиків і практиків. Мета дослідження – проаналізувати,
висвітлити та вивчити жанр хоррор у су
часному екранному наративі. 166 FILM AND TELEVISION DIRECTING AND SOUND ENGINEERING
2019 · 2(2)
Bulletin of Kyiv National University of Culture and Arts.
Series in Audiovisual Arts and Production 2019 · 2(2) · pp.165-172 нерви. Стрічки цього стилю поклика
ні створити атмосферу напруги, жаху,
томливого очікування перед здійснен
ням якоїсь страшної події, котра значно
вплине на розвиток сюжету на екрані
(Артемьева, 2010, с.121). з самого його зародження, наприкінці
XIX століття, а першим подібним філь
мом заведено вважати «Замок диявола»
1896 року – 12-хвилинне оповідання
про Мефистофеля. До речі, ця робота
стала також першою з використанням
спецефектів. З того часу режисери по
стійно експериментували, намагаючись
вразити глядачів, які ще недавно були
шоковані кадрами «прибуття поїзда». Далі були «Франкенштейн», «Кабінет
доктора Калігарі», «Носферату», «Сим
фонія жахів» і багато інших шедеврів
чорно-білих хоррорів. ( р
)
Страх, який передається глядачам,
утворюється різними сюжетними по
воротами, моторошними образами, по
діями тощо. Тло для розвитку страху
може бути найрізноманітнішим. Роль
цього тла можуть відіграти які-небудь
фантастичні події, психічні патології, що
виражаються героями фільму, сценами
насильства, крові, людськими нутро
щами й іншими варіантами розвитку
подій. Втім, багато кінокритиків вказу
ють, що такі стрічки можуть самих жахів
і не містити: через перетворення кіне
матографа в конвеєр, багато фахівців
підкреслюють, що в цьому жанрі стає
все більше шаблонів і штампів відносно
розвитку подій, сюжетів і так далі. Спочатку кінематограф не тільки ди
вував, але й лякав демонстрацією ре
чей, досі небачених або малоймовірних. І коли людей на екрані запросто погли
нав кіноапарат, загрожуючи проковтну
ти усіх, це зрозумілим чином викликало
насамперед жах і лише потім полегшене
замилування технічними можливостями
нового мистецтва. І можна припусти
ти, що Жорж Мел’єс зі своїм фільмом
«Людина з головою, що збільшується» –
законний першопроходець жанру жахів:
на екрані божевільний учений знімає із
плечей власну голову й, поклавши на
стіл, водружає на її місце гумову по
добу, що роздувається до гігантських
розмірів, загрожує вибухнути в будь-яку
хвилину (Скал, 2009, с.98). 167 Звичайно, як і в багатьох інших тво
рах мистецтва, основною проблемою
у фільмі жахів є боротьба добра зі злом. Зло можна представити в узагальне
ному образі ката, у вигляді чудовись
ка, неконтрольованої природної стихії,
будь-якого монстра або просто людини
з девіантними психічними відхилен
нями. Добро виступає в ролі жертви –
найчастіше звичайної, середньоста
тистичної людини. І якщо в більшості
фільмів добро все ж таки перемагає
зло, то у фільмах жахів на такий роз
виток подій можна сподіватися далеко
не завжди. Аналіз основних досліджень
і публікацій Не можна упускати з уваги такий жанр
кіно, як хоррор, завдяки якому у крові
глядачів утворюється більше адрена
ліну, пробігає хвиля страху й тривоги. Такі фільми прекрасно підходять для
всіх тих, кому хочеться перевірити свої Жанрова специфіка фільму жахів
привернула увагу багатьох дослідни
ків кіно, таких як: О. Нечай, М. Власов,
Г. Ратніков, В. Шкловський, Ю. Тинянов, Чміль, Г., Войцещук, М. До питання хоррор
як жанру сучасного екранного простору Чміль, Г., Войцещук, М. До питання хоррор
як жанру сучасного екранного простору ISSN 2617-2674 (Print),
ISSN 2617-4049 (Online) РЕЖИСУРА ТА ЗВУКОРЕЖИСУРА КІНО І ТЕЛЕБАЧЕННЯ
Вісник Київського національного університету культури і мистецтв.
Серія: Аудіовізуальне мистецтво і виробництво 2019 · 2(2) · с.165-172 або забавляти, або годувати, або шо
кувати (Артемьева, 2010, с.78). У цьому
у свій час довелося особисто переко
натися письменнику Френсісу Скотту
Фіцджеральду. симфонія жаху». Місіс Стокер попросту
подала на творців картини до суду. Саме
з «Носферату» розпочав свій шлях євро
пейський хоррор, визначився з напрям
ком розвитку й пішов убік суб’єктивної
реальності, туди, де жах зовнішній зрів
нюється з жахом внутрішнім (Самутина,
2006, c.50). р
у
Відносно пошуків відповідних сюже
тів, то найбільш очевидним вирішенням
було звертання до історій, що вже заре
комендували себе у публіки. Кінемато
графісти з ентузіазмом ринулися вику
повувати права на відомі твори в жанрі
жахів. Стівен Кінг, який розуміє сутність
хоррору, у своєму програмному дослі
дженні жанру «Танець смерті» виділяє
три основні твори. Як нескладно здога
датися, три магічні книги – це «Дракула»
Брема Стокера, «Франкенштейн» Мері
Шеллі й «Дивна історія доктора Джекіла
й містера Хайда» Роберта Луїса Стівен
сона. Четвертою книгою Кінг називає
«Поворот гвинта» Генрі Джеймса, обмо
вившись, що формат «історій з приви
дами» більшою мірою розповсюджений
у літературі й не надто добре прижи
вається на екрані (Артемьева, 2010,
с.26). У ході дослідження слід зауважити, що
розквіт класичних хоррорів Universal
прийнято прямо зв’язувати з Великою
депресією, яка, у черговий раз нагадав
ши про «одинадцяту заповідь» Уайль
да, гнала людей у кінотеатри у пошу
ках розради й заспокійливого забуття
(Артемьева, 2010, с.16). І ні, звичайно,
нічого дивного в тому, що трагічна доля
Віктора (який перетворився на екрані
в романтичного Генрі) Франкенштейна
в більшості випадків виявлялася для
глядача переважнішою оптимістичних
мюзиклів Басбі Берклі. Замість того,
щоб говорити з екрана: «усе буде до
бре», фільми жахів утішали іншим, пові
домляючи, що «буває й гірше». І можна
скільки завгодно іронізувати над наїв
ними «страшилками», які пропонували
глядачу ці фільми, факт залишається
фактом: класичні хоррори Universal були
найбільш непристойним видовищем,
на яке можна було потрапити в ті роки
у звичайному кінотеатрі. 168 Отже, у розпорядженні кінематогра
фістів були такі неоднозначні, але по
тенційно конвертовані у валюту персо
нажі, як Вампір, Монстр, Перевертень
і Примара. На жаль, щирій запопадли
вості починаючих режисерів і сцена
ристів сильно заважали різноманітні
бюрократичні двозначності – зазвичай,
пов’язані з такою неприємною річчю,
як купування авторських прав на твір. FILM AND TELEVISION DIRECTING AND SOUND ENGINEERING
2019 · 2(2)
Bulletin of Kyiv National University of Culture and Arts.
Series in Audiovisual Arts and Production Коли поточні досягнення новітньої
техніки стали більш-менш очевидними
й звичними, навіть глядачам з остан
ніх рядів – показувати фокуси стало
трохи складніше, піонерам молодого
мистецтва довелося вигадувати нові
способи веселити, дивувати й лякати
публіку. Тому що, вже де-де, а у світі
кінематографа практично відразу без
застережною «одинадцятою заповіддю»
закріпилося відоме висловлення Оскара
Уайльда про те, що людей доводиться Доцільно зазначити, що концепції
жахіть змінювались з кожним десяти
літтям, а режисерів, що вводили у цей
жанр нові визначення, вважали дійсно
культовими майстрами. Жанр хоррор
з’явився в кінематографі практично Chmil, H., Voitseshchuk, M. To the question of horror
as a genre of modern screen space ISSN 2617-2674 (Print),
ISSN 2617-4049 (Online) Новизна дослідження Новизна дослідження полягає у тому,
що вперше проаналізовано здатність
осмислення жанрових систем кіне
матографа; чіткої уяви про жанрову
структуру стилю «хоррор» в екранному
наративі; проблематику жанрової дифе
ренціації в кінематографі й принципи
класифікації представленого жанру. ф р
уд
у,
р
р
На відміну від іншого знакового режи
сера того часу, короля зовнішніх ефектів
Вільяма Касла, Клейтон не захоплю
ється можливістю пред’явити гляда
чу привидів в «повний зріст». Питання
про те, чи дійсно вони живуть у будинку,
згідно з першоджерелом, залишається
відкритим, боротьба між добром і злом
відбувається не в затемнених коридо
рах стародавнього будинку, а в голові
набожної героїні Деборі Керр. РЕЖИСУРА ТА ЗВУКОРЕЖИСУРА КІНО І ТЕЛЕБАЧЕННЯ
Вісник Київського національного університету культури і мистецтв.
Серія: Аудіовізуальне мистецтво і виробництво Питання
про те, чи дійсно вони живуть у будинку,
згідно з першоджерелом, залишається
відкритим, боротьба між добром і злом
відбувається не в затемнених коридо
рах стародавнього будинку, а в голові
набожної героїні Деборі Керр. простір – нескінченні лабіринти кори
дорів тихого будинку, де кожний крок
віддається в повітрі тривожним гучним
звуком, і дах, де про щось напівголос
но переговорюються голуби, і сад, де
скрекіт цвіркунів і спів птахів (Делёз,
2004, c.94). Головним героєм стає сам
будинок, і він – насправді, персонаж
з багатою традицією. Відтоді, як при
мара батька Гамлета з’явилася в стінах
замка Ельсінор, у товарні асортименти
творів у жанрі романтичних жахів міцно
ввійшли мешканці, обставини й атмос
фера будинку, населеного примарами. зовсім ірраціонально прагне знищити
весь світ, і є чоловік, головний герой,
який повинен боротися з відьмами, але
при цьому вони не можуть зробити про
сто героя чоловіка-борця з відьмами,
оскільки тоді їх розкритикують (Зенкин,
2006, c.84). РЕЖИСУРА ТА ЗВУКОРЕЖИСУРА КІНО І ТЕЛЕБАЧЕННЯ
Вісник Київського національного університету культури і мистецтв.
Серія: Аудіовізуальне мистецтво і виробництво Вдова Флоренс Стокер відомого пись
менника сприймала в багнети будь-які
спроби екранізації роману, фактично
приватизувавши такого цікавого анта
гоніста, як Дракулу, і ввійшла в історію
як єдина людина в цивілізованому світі,
що не оцінила естетичне новаторство
фільму Фрідріха Мурнау «Носферату – Недарма згаданий роман Генрі Джейм
са «Поворот гвинта» тільки в 60-ті роки
минулого століття екранізувався три
чі. Але однойменну адаптацію Джона
Франкенхаймера й маловідому версію
Пітера Морлі повністю затьмарила
картина Джека Клейтона «Безневинні». Цей фільм цікавим чином представив
глядачу головним героєм не стриману,
релігійну дівчину-гувернантку у вико
нанні Дебори Керр, і не дітей, одержи
мих примарами, а внутрішньокадровий Чміль, Г., Войцещук, М. До питання хоррор
як жанру сучасного екранного простору Чміль, Г., Войцещук, М. До питання хоррор
як жанру сучасного екранного простору ISSN 2617-2674 (Print),
ISSN 2617-4049 (Online) 2019 · 2(2) · pp.165-172 простір – нескінченні лабіринти кори
дорів тихого будинку, де кожний крок
віддається в повітрі тривожним гучним
звуком, і дах, де про щось напівголос
но переговорюються голуби, і сад, де
скрекіт цвіркунів і спів птахів (Делёз,
2004, c.94). Головним героєм стає сам
будинок, і він – насправді, персонаж
з багатою традицією. Відтоді, як при
мара батька Гамлета з’явилася в стінах
замка Ельсінор, у товарні асортименти
творів у жанрі романтичних жахів міцно
ввійшли мешканці, обставини й атмос
фера будинку, населеного примарами. На відміну від іншого знакового режи
сера того часу, короля зовнішніх ефектів
Вільяма Касла, Клейтон не захоплю
ється можливістю пред’явити гляда
чу привидів в «повний зріст». Питання
про те, чи дійсно вони живуть у будинку,
згідно з першоджерелом, залишається
відкритим, боротьба між добром і злом
відбувається не в затемнених коридо
рах стародавнього будинку, а в голові
набожної героїні Деборі Керр. простір – нескінченні лабіринти кори
дорів тихого будинку, де кожний крок
віддається в повітрі тривожним гучним
звуком, і дах, де про щось напівголос
но переговорюються голуби, і сад, де
скрекіт цвіркунів і спів птахів (Делёз,
2004, c.94). Головним героєм стає сам
будинок, і він – насправді, персонаж
з багатою традицією. Відтоді, як при
мара батька Гамлета з’явилася в стінах
замка Ельсінор, у товарні асортименти
творів у жанрі романтичних жахів міцно
ввійшли мешканці, обставини й атмос
фера будинку, населеного примарами. На відміну від іншого знакового режи
сера того часу, короля зовнішніх ефектів
Вільяма Касла, Клейтон не захоплю
ється можливістю пред’явити гляда
чу привидів в «повний зріст». Висновки Підсумовуючи вище зазначене, слід
сказати, що на даний момент існує до
сить багато «субжанрів» хоррора й су
міжних з ним напрямків. Це слешер,
вірд, бодіхоррор, бізарро, сплаттерпанк,
екстрімпанк і так далі. Їх намагаються
вивчити, визначити характерні ознаки
й рамки, класифікувати у той час, як
деякі із цих «субжанрів» формувалися
саме як спроба вийти «за рамки». І це
не дивно: рано чи пізно будь-якому на
прямку стає тісно в традиційних межах
жанру. Поступово він починає прагнути
до порятунку від кліше й штампів. Од
ним зі способів подолання парадигми
служить залучення суміжних напрямків. У першу чергу сучасний хоррор «пара
нормальної традиції» характеризуєть
ся заграванням із християнством. Це
можуть бути відьми, демони, демони
у відьмах, демони в ляльках і взагалі
найрізноманітніші варіації. Адже хрис
тиянство – це не тільки культурне ко Підсумовуючи вище зазначене, слід
сказати, що на даний момент існує до
сить багато «субжанрів» хоррора й су
міжних з ним напрямків. Це слешер,
вірд, бодіхоррор, бізарро, сплаттерпанк,
екстрімпанк і так далі. Їх намагаються
вивчити, визначити характерні ознаки
й рамки, класифікувати у той час, як
деякі із цих «субжанрів» формувалися
саме як спроба вийти «за рамки». І це
не дивно: рано чи пізно будь-якому на
прямку стає тісно в традиційних межах
жанру. Поступово він починає прагнути
до порятунку від кліше й штампів. Од
ним зі способів подолання парадигми
служить залучення суміжних напрямків 169 Отже, у мейнстрімному кіно є зобра
ження страху перед швидкою жіночою
емансипацією. Дуже цікаво дивитися, як
з однієї сторони творці фільмів намага
ються не бути сексистами, а з іншого –
чітко втілюють старі міфи. Наприклад,
кіно про відьом завжди цікаво тим, що
тема відьмівства сама по собі відобра
жає прадавній страх перед жінками. Режисери розуміють, що фільм будуть
дивитися багато критиків і вчених, а на
Заході дуже розповсюджений підхід
дивитися з погляду гендерних стерео
типів, раси й класу. Вони зрозуміють, що
зачіпаючи тему відьмівства, вони відра
зу стають об’єктом пильного погляду
феміністок і людей, які зацікавлені саме
в такому аналізі фільму. Тому почина
ють показувати, що є зла відьма, яка У першу чергу сучасний хоррор «пара
нормальної традиції» характеризуєть
ся заграванням із християнством. Це
можуть бути відьми, демони, демони
у відьмах, демони в ляльках і взагалі
найрізноманітніші варіації. Адже хрис
тиянство – це не тільки культурне ко Chmil, H., Voitseshchuk, M. To the question of horror
as a genre of modern screen space Chmil, H., Voitseshchuk, M. СПИСОК ПОСИЛАНЬ 170 170 Делёз, Ж., 2004. Кино. Москва: Ад Маргинем. Д
д
р
Зенкин, С., 2006. Эффект фантастики в кино. В: Фантастическое кино. Эпизод первый. Москва: Новое литературное обозрение. Зенкин, С., 2006. Эффект фантастики в кино. В: Фантастическое кино. Эпизод первый. Москва: Новое литературное обозрение. Комарова, А., 2011. Есть ли будущее у фильмов ужасов? Newsland [online]. Доступно: <http://
newsland.com/user/4297684659/content/est-li-budushchee-u-filmov-uzhasov/4224874>
[Дата обращения 16 сентября 2018]. Комарова, А., 2011. Есть ли будущее у фильмов ужасов? Newsland [online]. Доступно: <http://
newsland.com/user/4297684659/content/est-li-budushchee-u-filmov-uzhasov/4224874>
[Дата обращения 16 сентября 2018]. Муромова, Ю.В., 2014. Вплив фільмів жахів на психіку людини. Психологія, соціологія
і
педагогіка. [online]
Доступно:
<http://psychology.snauka.ru/2014/05/2957>
[Дата
звернення 18 серпня 2018]. Самутина, Н., 2006. Реальный фантастический вариант, или Что хотел сказать редактор-
составитель. В: Фантастическое кино. Эпизод первый. Москва: Новое литературное
обозрение. Самутина, Н., 2006. Реальный фантастический вариант, или Что хотел сказать редактор-
составитель. В: Фантастическое кино. Эпизод первый. Москва: Новое литературное
обозрение. Самутина, Н., 2006. Реальный фантастический вариант, или Что хотел сказать редактор-
составитель. В: Фантастическое кино. Эпизод первый. Москва: Новое литературное
обозрение. Скал, Д.Дж., 2009. Книга ужаса. История хоррора в кино (The Monster Show: A Cultural History
of Horror. Санкт-Петербург: Амфора. Скал, Д.Дж., 2009. Книга ужаса. История хоррора в кино (The Monster Show: A Cultural History
of Horror. Санкт-Петербург: Амфора. РЕЖИСУРА ТА ЗВУКОРЕЖИСУРА КІНО І ТЕЛЕБАЧЕННЯ
Вісник Київського національного університету культури і мистецтв.
Серія: Аудіовізуальне мистецтво і виробництво 2019 · 2(2) · с.165-172 дування, що запекло втискається в мо
ральний апарат з дитинства. Із давніх
часів, коли воно приходило на заміну
поганству, можна було спостерігати ці
кавий момент. З появою кінематографа
багато творчих задумів стали можли
ві до реалізації їх на екрані. Основою
кожного фільму в жанрі хоррор є тема
смерті – за різними причинами, почи
наючи від укусу вампіра й закінчуючи
суїцидом героя, так само й будь-яка
моторошна ситуація в картині, зміст
якої змушує глядача відчувати страх
від непоясненого й неминучого. Якщо
впакувати стару книгу, яку ви читали,
у нову обкладинку, книга однаково буде
вам знайома. Режисерам необхідні нові,
зовсім незнайомі глядачеві сюжети
й напрямки хоррора, а не зйомки на дування, що запекло втискається в мо
ральний апарат з дитинства. Із давніх
часів, коли воно приходило на заміну
поганству, можна було спостерігати ці
кавий момент. З появою кінематографа
багато творчих задумів стали можли
ві до реалізації їх на екрані. Основою
кожного фільму в жанрі хоррор є тема
смерті – за різними причинами, почи
наючи від укусу вампіра й закінчуючи
суїцидом героя, так само й будь-яка
моторошна ситуація в картині, зміст
якої змушує глядача відчувати страх
від непоясненого й неминучого. Якщо
впакувати стару книгу, яку ви читали,
у нову обкладинку, книга однаково буде
вам знайома. Режисерам необхідні нові,
зовсім незнайомі глядачеві сюжети
й напрямки хоррора, а не зйомки на ступних частин фільму або ж римейків. А якщо ні, то, жанр жахів перебуває під
загрозою зникнення (Муромова, 2014). Відповідно, формули фільмів жахів не
існує, але можна зазначити, що дефі
ніція подібна до сеансів психотерапії. Є таємниця (психологічна проблема),
яку потрібно розгадати (вилікувати),
щоб в результаті врятуватися (одужати)
(Комарова, 2011). ступних частин фільму або ж римейків. А якщо ні, то, жанр жахів перебуває під
загрозою зникнення (Муромова, 2014). Відповідно, формули фільмів жахів не
існує, але можна зазначити, що дефі
ніція подібна до сеансів психотерапії. Є таємниця (психологічна проблема),
яку потрібно розгадати (вилікувати),
щоб в результаті врятуватися (одужати)
(Комарова, 2011). З усього вище зазначеного, ми бачимо,
що глядачі почали втрачати інтерес до
того чи іншого виду жахіть, тому й спо
стерігається криза жанру. Хоч режисери
й знімають яскраві фільми з колосаль
ними бюджетами й величезною кіль
кістю спецефектів, однак, це загалом
плагіат або римейк, яким глядацьку
аудиторію не обдуриш. Artemeva, O.E., 2010. Evoliutciia esteticheskoi modeli zhanra ″khorror″ v amerikanskom kino [The
evolution of the aesthetic model of the horror genre in American cinema]. Moscow. Delez, Zh., 2004. Kino [Cinema]. Moscow: Ad Marginem. Артемьева, О.Э., 2010. Эволюция эстетической модели жанра «хоррор» в американском
кино. Москва. Висновки To the question of horror
as a genre of modern screen space ISSN 2617-2674 (Print),
ISSN 2617-4049 (Online) Bulletin of Kyiv National University of Culture and Arts.
Series in Audiovisual Arts and Production 2019 · 2(2) · pp.165-172 Zenkin, S., 2006. Effekt fantastiki v kino. In: Fantasticheskoe kino. Epizod pervyi [Fantastic
Cinema. Episode One]. Moscow: Novoe literaturnoe obozrenie. Komarova, A., 2011. Est li budushchee u filmov uzhasov? Newsland [online]. Available at: <http://
newsland.com/user/4297684659/content/est-li-budushchee-u-filmov-uzhasov/4224874>
[Accessed 16 September 2018]. Muromova, Yu.V., 2014. Vplyv filmiv zhakhiv na psykhiku liudyny. Psykholohiia, sotsiolohiia
i pedahohika [Psychology, sociology and pedagogy]. [online] Available at: <http://psychology. snauka.ru/2014/05/2957> [Accessed 18 August 2018]. Samutina, N., 2006. Realnyi fantasticheskii variant, ili Chto khotel skazat redaktor-sostavitel. In: Fantasticheskoe kino. Epizod pervyi [Fantastic Cinema. Episode One]. Moscow: Novoe
literaturnoe obozrenie. Skal, D.Dzh., 2009. Kniga uzhasa. Istoriia khorrora v kino (The Monster Show: A Cultural History
of Horror. Sankt-Peterburg: Amfora. К ВОПРОСУ ХОРРОР КАК К ЖАНРУ
СОВРЕМЕННОГО ЭКРАННОГО ПРОСТРАНСТВА Анна Чмиль1a, Марина Войцещук2a Анна Чмиль1a, Марина Войцещук2a 1 доктор философских наук, доцент; e-mail: gannachmil@gmail.com; ORCID: 0000-0001-6569-1066
2 ассистент кафедры кино-, телеискусства; e-mail: dekanat25kino@ukr.net; ORCID: 0000-0001-8164-012X
a Киевский национальный университет культуры и искусств, Киев, Украина 171 171 REFERENCES Artemeva, O.E., 2010. Evoliutciia esteticheskoi modeli zhanra ″khorror″ v amerikanskom kino [The
evolution of the aesthetic model of the horror genre in American cinema]. Moscow. Delez, Zh., 2004. Kino [Cinema]. Moscow: Ad Marginem. Аннотация ц
Цель исследования заключается в том, чтобы раскрыть особенности жанра и констатировать его значение
для решения художественных задач, как отдельной отрасли аудиовизуального искусства. Методология
исследования. В предлагаемом исследовании применяются следующие методы: аналитический – для
выделения отдельных частей общей проблемы, их исследование для полного понимания предмета;
комплексный подход – для целостного и разностороннего освещения темы, в частности в ходе рассмотрения
теоретического раздела наряду с исследовательскими проблемами; искусствоведческий – применяется
во время анализа фильма в художественном дискурсе; сравнительный – в процессе изучения жанров
и поджанров разных годов; описательный – во время исследования особенностей жанровых категорий
и отдельных фильмов; диалектический – во время анализа взглядов и мнений различных кинокритиков
и ученых касаемо данного жанра и поджанров. Научная новизна полученных результатов состоит в том,
что благодаря анализу и синтезу исследовано влияние жанра ужасов в осознаваемое миропонимание
человека, а также характер построения такого фильма, принципы обращения авторов к внутреннему миру
аудитории через соотношение различных уровней развития – режиссера и аудитории. Выводы. В работе
исследован хоррор как жанр современного экранного пространства. Доказано, что сегодня фильм
ужасов – один из самых известных и стабильных жанров голливудского кинематографа. Его приемами
активно пользуются и другие создатели, а некоторые фильмы вызывают серьезный культурный резонанс. Конкретная категория искусства играет определяющую роль в формировании нового кинематографического
будущего. Также в работе приведены примеры традиционных границ жанра и его кризисного положения. Ключевые слова: искусство; ужасы; кинематограф; фильм; хоррор; страх Chmil, H., Voitseshchuk, M. To the question of horror
as a genre of modern screen space
ISSN 2617-2674 (Print),
ISSN 2617-4049 (Online) Chmil, H., Voitseshchuk, M. To the question of horror
as a genre of modern screen space ISSN 2617-2674 (Print),
ISSN 2617-4049 (Online) РЕЖИСУРА ТА ЗВУКОРЕЖИСУРА КІНО І ТЕЛЕБАЧЕННЯ
РЕЖИСУРА ТА ЗВУКОРЕЖИСУРА КІНО І ТЕЛЕБАЧЕННЯ 2019 · 2(2) · с.165-172
Вісник Київського національного університету культури і мистецтв. Серія: Аудіовізуальне мистецтво і виробництво 2019 · 2(2) · с.165-172
Вісник Київського національного університету культури і мистецтв. Серія: Аудіовізуальне мистецтво і виробництво TO THE QUESTION OF HORROR
AS A GENRE OF MODERN SCREEN SPACE Hanna Chmil1a, Maryna Voitseshchuk2a 1 Doctor of Philosophy, Associate Professor, Cinema and Television Arts Department;
e-mail: gannachmil@gmail.com; ORCID: 0000-0001-6569-1066
2 Assistant, Cinema and Television Arts Department;
e-mail: dekanat25kino@ukr.net; ORCID: 0000-0001-8164-012X
a Kyiv National University of Culture and Arts, Kyiv, Ukraine 1 Doctor of Philosophy, Associate Professor, Cinema and Television Arts Departm
e-mail: gannachmil@gmail.com; ORCID: 0000-0001-6569-1066
2 Assistant, Cinema and Television Arts Department;
e-mail: dekanat25kino@ukr.net; ORCID: 0000-0001-8164-012X
a Kyiv National University of Culture and Arts, Kyiv, Ukraine Abstract The purpose of the study is to reveal the genre’s peculiarities and ascertain its significance for solving artistic
tasks as a separate branch of audiovisual art. Research methodology. In our study, the following methods
were applied: analytical is to isolate certain parts of the general problem and study for a full understanding
of the subject of research; comprehensive approach is for holistic and versatile coverage of the theme, in
particular during the examination of the theoretical section along with research problems; art is applied
during the analysis of film works in the Art discourse; comparative is in the research process of genres and
subgenres of different years; descriptive is during the study features of genre categories and individual
films; dialectical is during the analysis of views, opinions of various film critics and scholars regarding the
genre and subgenres. Scientific novelty of the obtained results is that due to analysis and synthesis and
the nature of the influence of the horror genre on the perceived person’s worldview, as well as the nature of
constructing such a film, the principles of applying authors to the internal The different levels of development
of the Worldview Director and spectator. Conclusions. Horror paper as a genre of modern screen space has
been investigated. It has been proved that today horror film is one of the most famous and stable genres of
Hollywood cinema. His tricks are actively used by other creators, and some films cause a serious cultural
resonance. A specific art category plays a decisive role in shaping the new cinematic future. Also there are
examples of traditional boundaries of the genre and its crisis situation in work. Keywords: art; horror; cinema; film; fear This is an open access journal and all published articles are licensed under a Creative Commons «Attribution» 4.0. ISSN 2617-2674 (Print),
ISSN 2617-4049 (Online)
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Theological Functions for Human Life in the Perspective of Asian Theologies in the Era of Society 5.0
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Millah
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cc-by-sa
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Article History: Received: June 7, 2022
Revised: December 23, 2022
Accepted: December 26, 2022
Published: December 28, 2022 Millah: Journal of Religious Studies
ISSN: 2527-922X (e); 1412-0992 (p)
Vol. 21, No. 3 (2022), pp. 973-1002
DOI: 10.20885/millah.vol21.iss3.art13 Millah: Journal of Religious Studies
ISSN: 2527-922X (e); 1412-0992 (p)
Vol. 21, No. 3 (2022), pp. 973-1002
DOI: 10.20885/millah.vol21.iss3.art13 Millah: Journal of Religious Studies
ISSN: 2527-922X (e); 1412-0992 (p) Vol. 21, No. 3 (2022), pp. 973-1002
DOI: 10.20885/millah.vol21.iss3.art13 Theological Functions for Human Life in
the Perspective of Asian Theologies in the
Era of Society 5.0
GP Harianto
Department of Theology (Bachelor), Sekolah Tinggi Teologi Excelsius,
Surabaya, Indonesia
hariantogp@sttexcelsius.co.id
Article History:
Received: June 7, 2022
Revised: December 23, 2022
Accepted: December 26, 2022
Published: December 28, 2022 Keywords: Asian Theologies; Human Life; Society 5.0; Theological Functions Theological Functions for Human Life in
the Perspective of Asian Theologies in the
Era of Society 5.0 GP Harianto
Department of Theology (Bachelor), Sekolah Tinggi Teologi Excelsius,
Surabaya, Indonesia
hariantogp@sttexcelsius.co.id GP Harianto
Department of Theology (Bachelor), Sekolah Tinggi Teologi Excelsius,
Surabaya, Indonesia
hariantogp@sttexcelsius.co.id Article History:
Received: June 7, 2022
Revised: December 23, 2022
Accepted: December 26, 2022
Published: December 28, 2022 Abstract Human civilization has changed so quickly since humans entered the era of the
industrial revolution. In this context, the role of theology seems to be dead, i.e. it
has not yet functioned for the benefit of humanity in the world. This study
examines the development of the meaning of theology, theology for Asian
theologians, and the function of theology towards human suffering in the midst of
world crises. This study used literature studies. The study's findings include the
functions of Muangthai's Waterbuffalo Theology, Catholicism's Polulorum
Progression, Choan-Seng Song, Indonesian Pluralism Theology, Secularism
Theology, Liberation Theology, Indian Dalif Theology, Korean Minjung Theology,
and Philippine Struggle Theology. Basic conclusion from the process of human
understanding of God's will for the present and future of humans based on the
Bible Humans use the formulation of theology to free themselves from the problems
of life, including poverty, education, the economy, security, and politics. Man
invites God to intervene in human life to depends on the goodness of God. Sound
theology is rooted in God as a way of life that has a closer relationship with God,
the Creator. This hope positions theology as a solution to human problems in the
world, specifically giving humans life in holiness, giving humans life in
eschatological victory, giving humans life as the Salt and Light of the World, and
theology of safeguarding humanity's hope against suffering calamity. words: Asian Theologies; Human Life; Society 5.0; Theological Functions Asian Theologies; Human Life; Society 5.0; Theological Functions Millah Vol. 21, No. 3 August 2022 | 973 Harianto INTRODUCTION Theological positions were deemed "doubtful" during the
"Society 5.0" era, which saw the birth of modern technology such as
artificial intelligence (Deguchi et al., 2020; Fukuda, 2020; Fukuyama,
2018). Theology is considered unable to solve the most basic
problems for humans. The findings of this era tend to use modern
technological developments as a basis for answering human needs
(clothing, security, happiness, and so on). It's as if life's problems can
only be solved with modern technological sophistication, even
though human power remains the main driving force behind this
development. Theology is being tested for its usefulness in humanitarian
issues: does theology have a role for the good of mankind, or does it
only play a role in monotonous rituals? The development of modern
technological sophistication has covered the role of theology in
human life. In this regard, Fuller (2021) said that this condition
shows the theological unconscious of artificial intelligence, which
gives life to two contrasting interpretations of the logos with basic
human needs. With various changes in the technological era,
theology became separated. Theology was always understood by ancient writers through its
etymology, which denotes the compound words, i.e. opinion,
conjecture, or reasoning. In early Christian life, "theology" referred to
the Word of God, the eternal Logos, namely Jesus Christ. According
to Aristotle, "theologizing" refers to someone who talks about gods
or about things related to gods, whereas in Plutarch's work, it
denotes research on "divine topics" (Dodson, 1913; Guthrie, 1933;
Györkösy et al., 2010, p. 496). Benedict Pictet notes that in the works
of ancient pagan scholars, the term "theology" is used in three
contexts: (1) "theologia fabulosa" (mythica) denotes the theology of
the poet; (2) "theologia naturalis" denotes the theology of 974 | Millah Vol. 21, No. 3 August 2022 Theological Functions for Human Life philosophers; and (3) "theologia politica et civilis" means the
theology of priests (Pictet, 1711). From time to time, the understanding of theology continues to
develop. Alister McGrath gives a very clear picture, and for ancient
witnesses he refers to Tertullian, according to whom theology is a
reflection of the God that Christians worship and revere (McGrath,
2016, p. 158). Augustine stated that theology is reasoning about or
talking about God (Augustinus, 2014). INTRODUCTION Church fathers such as Peter
Abelard and Gilbert of Poitiers explained that the Latin word
theologia means "the discipline of sacred learning, embracing the
totality of Christian doctrine and not merely the doctrine of God”
(McGrath, 2016, p. 121). In the 20th century, neo-orthodox theologians began to redefine
the essence of theology using classical definitions. Karl Barth said
theology as a science is a measure taken by the church, whose goal is
self-examination (McGrath, 2016, pp. 158–159). The development of
the meaning of theology cannot end, especially now that the world is
in crisis and theology cannot answer humanity's problems. As
Aquinas explained, theology is like the mind of God, God's
teachings, and what leads to God (Ferguson et al., 2008, pp. 680–681). Meanwhile, the editor of the Encyclopedia of Philosophy places
theology as part of the philosophy of religion (Edwards, 1972, p. 18). Vern defines theology as the study of problems related to God
and the world of reality (Stewart, 2017, pp. 64–67). Brandon is
sharper, saying that theology is a discourse conversation about God
that includes His systematics and ways of thinking such as dogmatic
theology, biblical theology, moral theology, and religious theology. ascetic,
mystical,
symbolic,
sacramental,
apologist,
pastoral,
philosophical, liturgical, and natural theology (Brandon, 1970, p. 610). Steenbrink discusses fundamental issues in theology: God's Millah Vol. 21, No. 3 August 2022| 975 Harianto relationship with His creation, human beings, and ethical values in
the context of practical life (Steenbrink, 2005). Circumstances have changed due to the sophistication of
modern technology. Uncontrolled change raises various questions
and statements about the role of theology. Haught said, "God is the
essence of moral values and is not cruel to the suffering that happens
to humans in the world" (Haught, 2008, pp. 40–51). More sharply,
Aarde said that postmodern spirituality currently does not need to
involve religion, and institutional religion is "dying" (Van Aarde,
2008). Thus, spirituality is not a religious ritual, but it must have a
role in overcoming changes in world civilization. The purpose of
writing this article is to answer the question, "How does theology
function for human life in the Asian context in the midst of the world
crisis in the Era of Society 5.0?" METHOD This research can be categorized as a literature review. A
literature review examines academic books, articles, and other
sources that are pertinent to a particular problem, field of
study, or theory, and by doing so offers a description, a
synopsis, and a critical assessment of these works. A literature
review entails much more than merely summarizing the
sources that were cited. While it may be a list of sources on a
particular topic, it usually adopts a critical, analytical stance,
demonstrating the connections between the different writings
and how they relate to the research (Anderson, 2022). The collection technique is through literature study
methods with the following steps: First, gather research
materials with the goal of identifying research issues: how
does theology function for human life during the World Crisis
Era and Society 5.0? Second, collecting literature on the 976 | Millah Vol. 21, No. 3 August 2022 Theological Functions for Human Life meaning of theology and theological formulations from
various theologians to look for the functions of these
theologies. Third, analyzing the findings of the literature in the
context of how theology functions for human life in the World
Crisis Era Society 5.0. Reviewing the literature can be considered a goal in and
of itself, either to guide practice or to give a thorough grasp of
what is known about a subject. The procedure takes a lot
longer when a literature review is done to give a thorough
grasp of what is known about a subject (Mertens, 2019). This
research get primary data about the development of
theological meanings from the thoughts of theologians
through literature research. RESULTS AND DISCUSSION The findings of literature studies from Kosuke Koyama's
(Muangthai) Theology, Waterbuffalo Theology, Catholic Polulorum
Progression, Asian Theologian, Choan Seng Song, Indonesian
Pluralism Theology (Eka Darmaputera), Religionum Theology (Th. Sumartana), Secularism Theology (Banchoff), Liberation Theology,
Dalif India's Liberation Theology of Asia (Ramoswaney Periyan;
Bhimarao Ambedkan), Korean Minjung Theology, and Philippine
Theology of Struggle (Fr. Louei Hechanova & Edicio de La Torre) are
found in the following table: Millah Vol. 21, No. 3 August 2022| 977 Harianto Harianto Table 1
Names of and formation function of theological movem
Name of theology
Formation function of the
movement
Water Buffalo
Theology
Muangthai
1. Responding to human fo
2. Communicate
Jesus
appropriate but critical l
of one's culture is consi
formation of theology. 3. Become interpersonal ra
through ongoing conv
enrichment. 4. Apply universal values
environmental justice. Polulorum
Progression
Catholic
1. Theology makes man t
heart
2. Theology changes a pe
attitude of solidarity
Choan Seng Song
Jesus is the answer to hum
including suffering and th
Indonesian
Pluralism
Theology
1. Theology pays attentio
human life. 2. Theology must be able
attention to loving and
another. Secularist
Theology
Theology determines the
life. Liberation
Theology
Theology is the basis for p
and regulates human life
Dalif Theology of
Asian Liberation
India
Theology to fight hum
suffering in the world. Korean Minjung
Theology
Theology to liberate the o
people, in all aspects of li
economic, social or politic
Philippine
Theology of
Struggle
Theology to free oneself f
oppression (Catholic and
politics). Source: Primary data. Table 1
Names of and formation function of theological movement
Name of theology
Formation function of theological
movement
Water Buffalo
Theology
Muangthai
1. Responding to human food needs
2. Communicate
Jesus
Christ
in
culturally
appropriate but critical language. Improvement
of one's culture is considered important in the
formation of theology. 3. Become interpersonal rather than interdoctrinal
through ongoing conversation and mutual
enrichment. 4. Apply universal values because of health and
environmental justice. Polulorum
Progression
Catholic
1. Theology makes man to have a kind
heart
2. Theology changes a person to have an
attitude of solidarity
Choan Seng Song
Jesus is the answer to human problems
including suffering and the rich-poor gap
Indonesian
Pluralism
Theology
1. Theology pays attention to the needs of
human life. 2. Theology must be able to pay serious
attention to loving and supporting one
another. Secularist
Theology
Theology determines the purpose of human
life. RESULTS AND DISCUSSION Liberation
Theology
Theology is the basis for political doctrine
and regulates human life in society. Dalif Theology of
Asian Liberation
India
Theology to fight humans who experience
suffering in the world. Korean Minjung
Theology
Theology to liberate the oppressed class of
people, in all aspects of life: educational,
economic, social or political. Philippine
Theology of
Struggle
Theology to free oneself from Western
oppression (Catholic and authoritarian
politics). Source: Primary data. Names of and formation function of theological movement 2. Communicate
Jesus
Christ
in
culturally
appropriate but critical language. Improvement
of one's culture is considered important in the
formation of theology. 3. Become interpersonal rather than interdoctrinal
through ongoing conversation and mutual
enrichment. 4. Apply universal values because of health and
environmental justice. 1. Theology makes man to have a kind
heart 2. Theology changes a person to have an
attitude of solidarity Jesus is the answer to human problems
including suffering and the rich-poor gap 1. Theology pays attention to the needs of
human life. 2. Theology must be able to pay serious
attention to loving and supporting one
another. Theology determines the purpose of human
life. Theology determines the purpose of human
life. Theology is the basis for political doctrine
and regulates human life in society. Theology is the basis for political doctrine
and regulates human life in society. g
y
Theology to fight humans who experience
suffering in the world. Theology to fight humans who experience
suffering in the world. Theology to liberate the oppressed class of
people, in all aspects of life: educational,
economic, social or political. Theology to free oneself from Western
oppression (Catholic and authoritarian
politics). 978 | Millah Vol. 21, No. 3 August 2022 Theological Functions for Human Life Waterbuffalo
Theology
(Muangthai):
Theology
for
Answering Food Solutions Muangthai has a theology represented by Kosuke
Koyama, the theologian who wrote the book "neighborology,"
who says theology should be able to answer food issues such
as giving them food, water, clothing, and care. The pagan issue
is a basic human need (Koyama, 1993; Meyers, 2005; Thaut, 2009;
Ager & Ager, 2015). Ager & Ager, 2015). The Pontifical Council provides a model of spiritual
formation for migrants and refugees, emphasizing that a
peaceful life is "closely linked to the growth of a friendly
mentality" (Meyers, 2005). "Hospitality" is more than concern
for or closeness to people. Jesus demonstrated this hospitality
by sharing food with lepers and exiles (Matthew 25:35) (Paul
VI, 1967). Jesus also fed 5,000 men and 1,000 women and
children to their fill, healed people, performed miracles, and
defended those who were despicable. Furthermore, Koyama, who formulated "Water Buffalo
Theology," said that theology must be able to become a reality
for humans. Theology must address aspects of society's daily
lives.In this context, Koyama examines the life of farmers in
Thailand; there is the reality of rice fields, farmers, and
buffaloes. Departing from this situation, he continued the
theological model by introducing God into it. Theology must
be able to address the life of a community and participate in
responding to the real needs of local people because theology
contains the light of God's Word. Theology is always able to
answer the problems of human life. Buffalo theology focuses
on the roots of Thai culture, religion (Buddhism and
Christianity), and history. Christian theology solves problems: Millah Vol. 21, No. 3 August 2022| 979 Harianto rich and poor, disease, tribal religion, spirituality, and the
problems facing humanity (Koyama, 1974, 1999). The
aims
of
Waterbuffalo
theology
are
to:
(1)
communicate Jesus Christ in culturally appropriate but critical
language. Improvement of one's culture is considered
important
in
the
formation
of
theology;
(2)
become
interpersonal rather than interdoctrinal through ongoing
conversation and mutual enrichment; and (3) applying
universal values because of environmental health and justice
(England & Prior, 2003). Catholic Polulorum Progression: Theology for Kindness and
Solidarity Catholic Polulorum Progression: Theology for Kindness and
Solidarity Polulorum
Progression
Catholicism
illustrates
that
theology makes humans have kindness and changes a person
to have an attitude of solidarity. Not only humans, but even
bigger states, must be able to do this with the following three:
(1) Reciprocal solidarity in the form of cross-government gifts
(2) Social justice in the form of increasing relations in the field
of trade (3) Struggle through universal charity to develop a
society that respects human dignity and where people can help
each other (Etchegaray & Cheli, 1981). Basically, no one is lacking
in a community of believers because members share with one
another (Acts 4:34). Populorum Progressio said that true
Christian humanism always focuses on God, not human
standards (Paul VI, 1967). However, human life has not reached
its proper level if his actions do not encourage others to seek
God. 980 | Millah Vol. 21, No. 3 August 2022 Theological Functions for Human Life Choan-Seng Song: Jesus as the Mission Center for the Relief
of Suffering in the World Choan-Seng Song: Jesus as the Mission Center for the Relief
of Suffering in the World Jesus is believed as the answer to human problems,
including suffering and the rich-poor gap, thus making human
existence come alive (Song, 1999, 2002a). The beginning of
theology is rooted in God's relationship with humans. In the
beginning, human problems were perceived by God and
involved Him. Human suffering makes God decide to
overcome it. God gave Jesus Christ to suffer so that Jesus could
lead human existence out of the problems of the world. Jesus
Christ is the solution to the world's problems (Song, 2002b;
Chambers, 2014; Balc, 2016). Jesus became the focal point of the mission of putting the
concrete
experience
of
God's
worship
into
practice. Meanwhile, the church building is a social reality. So Jesus is at
the center of preaching the good news throughout the ages
(Song, 1977, p. 66). People can be liberated based on their
suffering in this world. The fact is, people are too stressed in
their lives to be sure. The Christian understands redemption to
be the atonement for the guilt of something that does not exist
(Song, 1977, p. 68). Song shared an understanding of Christian beliefs and an
appreciation for community activities in politics. So, theology
evaluates all sides of human life. Catholic Polulorum Progression: Theology for Kindness and
Solidarity This thought is known as the
"theology of the cross" and talks about what is experienced by
humans in all conditions. This theology makes people free
from
the
snares
of
suffering,
poverty,
and
various
environmental pressures (Song, 1977, p. 279). Popular theology
presents people as historical subjects, not objects. His task is to
tell the story as a "socio-political biography" of the city
(Harianto GP, 2019). Millah Vol. 21, No. 3 August 2022| 981 981 Harianto Indonesian Pluralism Theology: Theology is concerned with
the needs of human life. Indonesia has a theology of pluralism. Theology must be
able to pay attention to the needs of human life. A pluralist in
Indonesia, Tanja, emphasized that the need to solve
humanitarian problems requires a way of togetherness,
wholeness, to achieve ethical togetherness (Harianto GP, 2001). At least three obligations are carried out by fellow religious
communities: (1) creating animosity and envy interferes with
good relations between religious adherents; (2) religious
people need not be afraid to reveal religious abuse; and (3) the
search for an ethic of togetherness must be commented on
(Tanja, 1994, p. 17). According to Darmaputera (1988), theology
could be called theology if theology answered universal
human needs. For Sumartana (Sumartana, 1999a, 1999b),
theology must be able to pay serious attention to loving and
supporting fellow human beings. Theology
of
Secularism
(Banchoff):
Theology
for
"Determining the Purpose of Human Life" Banchoff (2007), a figure in secularization theology who
masters technology and knowledge, proposed that secular
theology determines human goals (Volf, 2007). Furthermore,
Lerner said that secular theology must be applied to all aspects
of human life (Karim, 1997). The purpose of secularization is to
renew ways of thinking and acting. In general, this means that
people reject beliefs and teachings that rule their lives. People
are careful to put their trust where it belongs. While religion is
part of the human individual, it does not become a rule of
human life, and humans must follow the rules that exist in
society. Secularism is a rule that must be obeyed by humans. 982 | Millah Vol. 21, No. 3 August 2022 Theological Functions for Human Life Secularization means separating religious issues from the
government and society. Religion performs its task of
enlivening the beliefs and character of its individual followers. Society lives according to the assumptions and rules that are in
place. According to Turner (2010), in a time of global crisis,
religion is needed and has an impact on human life. Secularization means separating religious issues from the
government and society. Religion performs its task of
enlivening the beliefs and character of its individual followers. Society lives according to the assumptions and rules that are in
place. According to Turner (2010), in a time of global crisis,
religion is needed and has an impact on human life. Dalit India Liberation Theology of Asia: Theology to fight
against suffering people in the world India has a Dalit theology, meaning "broken" (oppressed),
which is a theological understanding of the caste system
(Amaladoss, 2014, p. 40). Followers of this theology are called
"Dalif." Their activities are based on Western Christian
theology and are oriented towards the life of Hindu society. Dalit itself consists of Christians, Hindu low castes, and others. Dalif figures: Ramoswaney Periyan and Bhimarao Ambedkar. They show that theology and religious mechanisms that
tolerate social subordinates use their new beliefs. Caste is a
belief and a god for society. This idea is supported by
Mahatma Gandhi. Dalif's theological basis, "The Ashram
Movement," is characterized by "pathos" and "doxology."
Pathos is a symbol of the shocking power and struggle for
liberation that engulfed the Dalai Lama. Doxology represents
the power that expelled the Dalai Lama from the Hindu caste
and converted them to Christianity (Amaladoss, 2014, p. 55). Thus, Dal's theological struggle is against humans who suffer
in the world. Korean Minjung Theology: Theology to liberate the
oppressed class of people in all matters of life: educational,
economic, social, or political Liberation Theology: Theology is the basis for political
doctrine and regulates human life in society. Liberation theology (theology of liberacism, contextual
theology, humanist theology, transformative theology, and
rational theology), known as third world theology, serves as
the basis for political doctrine and regulates human life in
society. The theology of freedom was born and developed in
Africa, Latin America, and Asia. The term "liberal" in
theological thought was originally put forward by Gustavo
Gutierrez from Latin America as "theology of Lebacion"
(Steenbrink, 2005, p. 138). The concept of sustainable liberation evolved from
previous ideas that had dimensions of freedom over injustice,
such as between 1930 and 1962. Dussel described it as a
prophetic theology (colonization, new Christian theology) that
focused on the struggle for equality in political and social life
(Wahono, 2000, p. 18). The idea of liberation first focused on
social personality and forms of orthodox theology before
shifting to speculative rationalistic theology through various
social movements. By taking into account educational,
political, social, cultural, and security factors, these theological
characteristics provide humans with an interpretation that the
quality of theology is rooted in God's will. Thus, the theology
of liberation requires fellowship in the form of the Millah Vol. 21, No. 3 August 2022| 983 983 Harianto involvement of the followers as a whole in the struggle for
independence and life in the Third World (Wahono, 2000, p. 23). Korean Minjung Theology: Theology to liberate the
oppressed class of people in all matters of life: educational,
economic, social, or political Korea has Minjung theology. The word "minjung" is a
combination of two Chinese words: "min" (community) and
"jung" (general). The term minjung first appeared between
1392-1960. That year was during the "Yi" dynasty, which 984 | Millah Vol. 21, No. 3 August 2022 Theological Functions for Human Life regulated the distinction between the aristocratic class and the
common people (minjung). Minjung is the theology of the
oppressed class of the people in all aspects of life: educational,
economic, social, or political. They were treated unfairly for a
long time, then divided into the North and the South
(Commission on Theological Concerns of the Christian Conferences
of Asia, 1983, p. 17). Hyun Yong Hak and Suk Nan Dong are Minjung
characters. The main ideas based on social and political
hermeneutics are: (1) "Han" liberators, who felt anger,
hardship, pressure, powerlessness, and grudges against the
government at that time. (2) The "Han" struggle uses the "Dan"
(consciousness) method. There are 2 levels of "Y," namely
direct and social. "Y" is denial in its direct meaning, based on
cyberspace. "And" at the social level, through four things:
understanding, maintaining, practicing, and overcoming
various problems regarding the truth of God's faith (Smith,
2001, p. 260). (3) The Trinity Idea: God-Jesus-Holy Spirit is
motivated by a desire to escape the pressures of political and
social life. The idea has its roots in patheism, which believes
God can use universal power, as well as spiritual shamanism
(Sugirtharajah, 1994, p. 262). Filipino Theology of Struggle: Theology for Liberation from
Western Oppression (Catholic and Authoritarian Politics) Filipino Theology of Struggle: Theology for Liberation from
Western Oppression (Catholic and Authoritarian Politics) The Philippines has a theology of struggle, which is
understood by and through resistance and is a reflection of
Filipino Christians' desire for the liberation of economically
weak people. This struggle is also known as "theology of the
people" (ummah), which is filled with "choreos" to free oneself Millah Vol. 21, No. 3 August 2022| 985 985 Harianto from
Western
oppression
(Catholicism
and
political
authoritarianism) (Amaladoss, 2014, p. 26). from
Western
oppression
(Catholicism
and
authoritarianism) (Amaladoss, 2014, p. 26). Fr. Louei Hechanova and Edicio de La Torre are figures of
the theology of struggle. The idea is rooted in Latin American
theology. At that time, in the 1970s, they participated in the
Economic Association of Third World Theologians. The aims
of the theology of struggle are as follows: (1) Progressive
spirituality places liberation theology in the effort to liberate
human beings between two poles, namely, suffering and living
in a new world for Christians, which is not just a dream but a
promise from God. (2) Ekklesia, who rejects church politics. The church is a community based on myriad bands of people
(ummas) that are run as committed "people churches." (3) A
new social analysis from Marxist theory, emphasizing the
economic
and
political
dimensions
that
differentiate
landowners on the basis of poverty. (4) The new social analysis
is reinforced by "Practice Zen," a collective liberation
movement using the possibility of a commitment to social
change (Amaladoss, 2014, p. 38). The Function of Theology in Human Life in a World Crisis
Era Society5.0 The Function of Theology in Human Life in a World Crisis
Era Society5.0 Theology is a life expectancy that has a close relationship
with God, the Creator. Benz (2001) explained that hope is not a
scientific term but comes from experience at the level of
personal perception and religion that requires participation. Anticipating the future, science and theology of creation meet,
and the tension between practical knowledge and visionary
hope enters a constructive dialogue. This hope places the
function of theology as a solution to human problems in the
world. Horton (2011) described theology as the grammar of the 986 | Millah Vol. 21, No. 3 August 2022 Theological Functions for Human Life Christian faith, which is expressed, discussed, taught, and
used as a lens for Christians to practice their lives. So theology
is the practice of Christian life. Life is inseparable from the
problems of poverty, natural disasters, illness, and even death. Furthermore, Appasamy (1942) formulated theology for
indigenous peoples in India. This formulation is an elaboration
of Christian beliefs that are linked by Hindu beliefs. Christianity and Hinduism have something in common when
it comes to spiritual matters, so Hindus are younger to enter
the Christian realm (Wellem, 2003, pp. 15–17). Appasamy (2010)
maintained theology to solve real human problems on earth. Theology is a way of life that has a close relationship with
God, the Creator. Benz said that "hope" is not a scientific term
but comes from experience at the level of personal perception
and religion that requires participation. Anticipating the
future, science and theology of creation meet, and the tension
between practical knowledge and visionary hope enters into a
constructive dialogue. This hope positions theology as a
solution to the world's human problems (Benz, 2001). Bonding
can be seen in Figure 1 about the plot of the Bible as the basis
for theology. Millah Vol. 21, No. 3 August 2022| 987 Harianto Harianto Figure 1
The plot of the Bible as the basis for theology The plot of the Bible as the basis for theology Theology
Bible
Theology Gives Human Hope to
Live in Holiness
Theology gives hope to humanity in
eschatological victory
Theology gives hope to humans as
salt and light in the world
Theology of Safeguarding
Humanity's Hope against Suffering Theology Gives Humans Hope to Live in Holiness The Christian faith must be strengthened by Christ's
painful suffering on the cross in anticipation of His glorious
resurrection. Christian faith must be consistent with the hope
of God's promise that "human suffering brings holiness", that
with God all things are possible for those who believe in Him. In
facing
life's
challenges,
love
and
compassion
are
essential.As a Christian, his pain and suffering are likened to
the painful sufferings of Christ on the Cross, which brought
him a glorious resurrection, happiness, and joy (Isidiho &
Obioma, 2020; Pearson, 2020; Pityana, 2020; Cordero, 2021). There is
hope in human suffering. Moltmann said, hope gives
humanity the possibility of a new life and a return to the love
of God and society (Moltmann & Kohl, 1995, p. 40). The theology of being a suffering human is that he reflects
on himself based on his relationship with God, his Creator, 988 | Millah Vol. 21, No. 3 August 2022 Theological Functions for Human Life and finds the essence of everything he experiences. Man
changes his dark life circumstances towards the light of Christ,
and he discovers a new world with Christ: to live in the
holiness of Christ. Theology Gives Humans Hope to Live in Eschatological
Victory Theology Gives Humans Hope to Live in Eschatological
Victory Beker (1994) showed that suffering and hope are in the
balance of human life. Suffering without hope (leads to
hopelessness, cynicism, unhealthy submission, and passivity)
or hope without recognizing the reality of suffering (leads to
the illusion of hope). In a state of suffering, humans must
maintain hope, namely, for eschatological victory over death. That hope is based on God's victory over death on the cross
and the resurrection of Christ. Hope is a practice found in worshiping God, not a theory. The word "worship" in Greek (the New Testament) appears
with the terms: ainein (epainein), epicaleisthai, eusebein,
eucharistein, phobein, proskynein, leitourgein, latreuein, and
sebein. These words generally refer to his actions being
directed by God, with fewer cases where Jesus is the recipient,
Jesus is not only spoken of as the agent or embodiment of
God's glory but as a sharer of that glory (Hooker, 2008; Dunn,
2010). The eschatological hope of Christ fills the void humans
live in, thereby creating a strong representation of Christ. Theology gives hope for the future of human life, both on
earth and in heaven (Hayes, 1990; O’Callaghan, 2011; Middleton,
2014). The Apostle Paul also spoke of "eternity." In 1
Corinthians 15:51, the phrase "incorruptible" refers to (KJV
"incorruptible", NAS, VIB, NJB, NRS "imperishable") using the Millah Vol. 21, No. 3 August 2022| 989 989 Harianto Harianto Greek ἄφθαρτοι (aphtartoi) is an adjective, nominative,
masculine, plural, and of no degree, meaning eternal, eternal,
or indestructible. Although aphtartoi is an adjective, it has a
nominative function, namely to express the subject in a clause. So aphtartoi (eternal) becomes the most important part in
verse 51. Eternal in the New Testament, which appears 66
times out. The first 65 verses talk about a period of time that
never stops (ends), does not change, does not shift, and is
forever eternal. Furthermore, in 1 Timothy 6:16, the word "eternal" is
rendered in Greek (aionion), whereas the KJV, NJB, NAS, RSV,
and NIV translate it as "forever."The word "aionion" is an
adjective, nominative, neuter, singular, and no degree means
"eternity." Although the Greek language is different from 1
Corinthians 15:52 (aphtartoi), aionion also refers to "enternal,"
which is an adjective that has a nominative function (subject
form in a sentence). The word aionion becomes significant in
verse 16 which explains the word "everlasting power". Theology Gives Humans Hope to Live in Eschatological
Victory So,
there is authority over something that shows the government
is eternal (everlasting forever). Eternal reign is intended to be
gender neutral and spiritual in nature, rather than masculine
or feminine (Muel-Dreyfus, 2001; Fudge, 2012). The words "aphartartoi" and "aionion" describe God and
His life as being untouched by death. God's life cannot be
measured in terms of time, the life of the Spirit, or even
dominion over eternity itself. In this regard, several
theologians have used and defended the term "immortality of
the soul," according to the Bible. According to Calvin (2008),
Adam had a living soul, did not have immortality, but God
gave him immortality (Prins, 1972; Kister, 2009). 990 | Millah Vol. 21, No. 3 August 2022 Theological Functions for Human Life Theological Functions for Human Life Hodge (1957) presented a number of arguments in his
writings, in defense of words and teachings about the
immortality of the soul. Shedd uses the word immortality in
his book to show that the soul is immortal apart from the
death of the body. This truth was discovered in ancient times. Thus, theology offers human hope from an eschatological
standpoint. Theology Offers Hope: Humans Can Serve as the Salt and
Light of the World Theology has a role, function, and impact on human life
in the world. Theology makes a person live as "salt" and "light"
in the world. Such a life can be carried out by someone who
has a sound theology. A healthy person can manage his time
between the spiritual and his daily life, which both come from
Jesus Christ (Andrian et al., 2021). He lives in the world in the same way that Christ did: he
seeks the kingdom of God (Matthew 6:33-34). The text
emphasizes the phrase "seek first the kingdom of God,"
pointing to a focus on Christ's faith pointing to eternity. Faith
brings eternal life. God's eternity is an attribute that indicates
that God does not change His nature, will, or promises (Num. 23:19, 1 Sam. 15:29, Ps. 102:26, Mal. 3:6, 2 Tim. 2:13, Heb. 6:17–
18, and Jas. 1:17). In this regard, Bloesch (1981) wrote that faith
justifies acts of mutual love between believers. Personal faith
comes alive because of the acts of love it performs (Paul VI,
1967). As a result of adopting the lifestyle of "seeking first the
kingdom of God,” humans can position themselves as salt and
light in the world. Millah Vol. 21, No. 3 August 2022| 991 Harianto Theology
of
Safeguarding
Humanity's
Hope
against
Suffering Calamity Theology is the desire to improve oneself. Theology is
obligated to give a person hope for humanity in the face of his
time's suffering disasters (Bonino, 1975; Johnson, 2007). These
hopes are: (1) helping people understand the presence of God,
who strengthens their lives in the midst of disaster problems;
(2) believers become "light" to greet others, make friends, and
join the nation in solving various crises that the world is
experiencing. This hope contains togetherness and unity
because the essence of theology is partiality towards one
religion. Togetherness and unity are carried out to find the
expression of all religious teachings. According to Baum (1977,
pp. 46–49), the true nature of religion can be understood by the
promise of togetherness to live in mutual respect on this earth. Going deeper, Armstrong (2001) stated that the Holy
Scriptures have been accepted by various religions with the
goal of converting humans into followers of their Creator. Thus, religion is a practice of life separate from the
supernatural world. Religion can realize what is needed by its
adherents. So if religion cannot answer human needs, it must
be abandoned by its adherents. According to Habermas (2014) it was time for religion to
side with humanity and fight injustice, so that religion would
become a charm of peace for mankind (Bahri, 2020). Furthermore, Erikson said that Christian theology is the
majesty of God in terms of power, knowledge, his nature, his
superiority, and the beauty of his moral nature. This can
happen because God is the beginning and the end, the alpha
and omega, so it is very suitable if theology is structured 992 | Millah Vol. 21, No. 3 August 2022 Theological Functions for Human Life Theological Functions for Human Life within the framework of greatness and goodness (Erikson, 2012,
pp. 533–560; Wellum, 2020). CONCLUSION Muangthai Waterbuffalo Theology serves the following
purposes: Catholic Polulorum Progression, Asian Theologian,
Choan Seng Song, Indonesian Pluralism Theology, Secularism
Theology, Liberation Theology, Asian Liberation Indian Dalit
theology, Korean Minjung theology, and Filipino theology of
struggle are as follows: First, based on a conclusion from the
process of human understanding of God's will for the present
and future of humans based on the Bible. Second, humans use
theological formulations to free themselves from the problems
of life, including poverty, education, economy, security, and
politics. Third, humans invite God to intervene in human life. So humans depend on God's goodness. A healthy theology can solve problems of human
suffering in the midst of world crises. A healthy theology is
rooted in God, and if it is not, then it will be difficult or even
impossible to properly solve the problem of the humanitarian
crisis. Theology is a way of life that has a closer relationship
with God, the Creator. This hope places the function of
theology as a solution to human problems in the world,
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The ammonite faunas of the upper Hypselocyclum to Divisum zones (Lower Kimmeridgian, Upper Jurassic) at Małogoszcz, Holy Cross Mts., central Poland: their stratigraphical interpretation and evolutionary development
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University of Warsaw, Faculty of Geology University of Warsaw, Faculty of Geology The ammonite faunas of the upper Hypselocyclum
to Divisum zones (Lower Kimmeridgian, Upper
Jurassic) at Małogoszcz, Holy Cross Mts., central
Poland: their stratigraphical interpretation and
evolutionary development Andrzej Wierzbowski Andrzej Wierzbowski Research Article DOI: https://doi.org/10.21203/rs.3.rs-4008521/v1 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 Journal of Iberian Geology on September
26th, 2024. See the published version at https://doi.org/10.1007/s41513-024-00260-y. Page 1/32 Abstract The transgressive succession of deposits of the late Early Kimmeridgian at Małogoszcz in the south-
western margin of the Holy Cross Mts. in central Poland yielded diversified faunas of ammonites. The
commonly represented Submediterranean ataxioceratid ammonites include the last members of
Ataxioceras in the Hypselocyclum Zone and the assemblage of various Crussoliceras, Garnierisphinctes
and Progeronia, mostly developed in the Divisum Zone, and associated upwards (Uhlandi Subzone) with
aspidoceratids (Pseudhimalayites). A few typical Mediterranean ammonites (Nebrodites, Presimoceras,
Idoceras, Taramelliceras) are indicative of the Herbichi Zone. The Subboreal ammonites include mostly
Eurasenia, Involuticeras in addition to some Rasenia and Rasenioides of the uppermost Cymodoce Zone,
corresponding to the Askepta Subzone. The changes in composition of ammonite faunas and
comparison with the coeval faunas of other areas of Europe give indications on the evolutionary
development of some Ataxiocertidae and Aulacostephanidae at the end of Early Kimmeridgian. The
development of the Crussoliceras, Garnierisphinctes and Progeronia, having possibly their roots in the
Mediterranean areas, was strictly correlated with the overall transgression and oscillations of sea-level
controlled possibly by climatic eccentricity cycles in northern Tethyan shelf. This resulted also in decline
of older Ataxioceras and its nearby allies. The indigenous lineage of Aulacostephanidae includes the
transition from Eurasenia to Pararasenia. The development from Rasenioides to Aulacostephanoides
occurred mostly in the Subboreal areas – although some late representatives of Rasenioides like R. moeschi reached the area of study. The existence of an independent lineage leading from Rasenia
involuta to heavily-ribbed Aulacostephanoides/Aulacostephanus is also suggested. Introduction The most valuable specimens
of the collection are housed in the Museum of the Geological Faculty of the University of Warsaw
(collection number ZI/100). Materials and methodology. The ammonites occur fairly commonly. The collection of ammonites
consisting of over 200 specimens determined to the genus level (and additionally in a large part to the
species level) is composed of common representatives of three families: Ataxioceratidae, The dimorphism of ammonites, if strongly marked in shell morphology, is expressed
morphotaxonomically both at the generic and species levels. This refers especially to the most
commonly recognized forms of the families Ataxioceratidae and Aulacostephanidae whose taxonomical
interpretation accepted herein is especially close to those presented by Atrops (1982) in relation to
Ataxioceratidae, and by Wierzbowski (2022) in relation to Aulacostephanidae. Such an approach,
although purely descriptive is “fully conscious”, because it makes easier study of evolutionary
transformations in the lineages, especially those constraint by heterochrony, closely related to changes
in environmental conditions. The whole succession studied can be subdivided into five units differing in lithology which along with
stated differences in composition and preservation of faunal assemblages make possible recognition of
the changes in the environmental conditions. Beside the chronostratigraphical interpretation of the
succession, the composition and changes in ammonites faunas offer new data on their evolutionary
development in relation to changing environment. Introduction Geological setting. The Upper Jurassic deposits occurring around the Paleozoic core of the Holy Cross
Mountains (Polish: Świętokrzyskie Mountains) in central Poland, in the north-eastern, the north-western
and the south-western margins, represent remnants of the primary cover developed originally over the
whole area. The lateral extent of the Upper Jurassic deposits was modified by Neo-Cimmerian (pre-
Albian) tectonic movements and markedly changed by Laramian (at the end of Maastrichtian and during
Palaeocene) uplift of the mountains which became the south-eastern fragment of the elevated zone of
the Middle Polish Anticlinorium (Kutek & Głazek, 1972). The Upper Jurassic deposits are accessible for detailed study in several large quarries, including that o
the Małogoszcz cement works in the south-western margin of the mountains (Fig. 1), where the detaile
Kimmeridgian succession of carbonates has been described from (Matyja et al., 2006). The
stratigraphical succession studied here, attaining about 25 meters in thickness, corresponds to the
Skorków Lumachelle of Kutek (1968). It consists of oyster lumachelles, micritic limestones and marls
rich in various fossils and oncoids representing the transgressive part of sedimentary sequence,
covering the older shallow-water marls and sublithographic limestones deposited in restricted
environment with a very poor fauna. The latter represent the final regressive fragment of an older
sequence developed at the decline of the shallow-water carbonate platform (e.g., Kutek, 1994). A Page 2/32 Page 2/32 substantial fossil assemblage listed by Radwańska & Radwański (in: Matyja et al., 2006) comprises
numerous bivalves (both deep infaunal to epifaunal oysters – such as Actinostreon and Nanogyra –
forming mass accumulations), ammonites, brachiopods, lobsters, regular and irregular echinoids,
starfish, crinoids and various fish remains, some of them bored by bryozoans and bivalves and/or
encrusted by diverse epizoans such as serpulids and bryozoans. Materials and methodology. The ammonites occur fairly commonly. The collection of ammonites
consisting of over 200 specimens determined to the genus level (and additionally in a large part to the
species level) is composed of common representatives of three families: Ataxioceratidae,
Aulacostephanidae and Aspidoceratidae, and rare ones of two others: Perisphinctidae and Oppeliidae. These ammonites have been picked during several years personally and donated by other persons
during occasional visit in the quarry, being usually referred to the particular lithostratigraphical units (also
after study of the matrix), but commonly without precise location therein. Description of the lithological succession For the purpose of this study the whole succession of the Skorków Lumachelle at Małogoszcz is
subdivided into five units A-E (see Fig. 2). The general faunal and lithological characters of the
succession were given in several older studies (Kutek, 1968; Kaźmierczak & Pszczółkowski, 1968;
Seilacher et al., 1985; Machalski, 1996; Matyja et al., 2006; Matyja, 2011), all of them have been taken
into account along with some new observations when prepared the following description. Page 3/32
Unit A consists of thick-bedded, soft bioclastic limestones with numerous internal moulds of various
infaunal bivalves, including those of Pholadomya, often preserved in life-position. There occur also
commonly epifaunal bivalves like Actinostreon, Trichites, Nanogyra and others, as well as various Page 3/32 Page 3/32 brachiopods and echinoids. The deposits attain 6.5 m in thickness. Their base is marked by the well-
developed omission surface having character of hard-ground, and representing the successive stages of
its development from soft- and firm-ground stages with numerous burrows (Thalassinoides and
Rhizocorallium ) to bivalve and polychaete borings, and encrustation by serpulids of the hard-ground
stage. This level marks the lower boundary of the Skorków Lumachelle (or lumachelle/coquina
formation) with the underlying “sublithographic” limestones of the Buczyna Limestone Mbr. of the
Spinkowa Góra Fm. (Wierzbowski, 2020). Ammonite occur rather commonly, especially of the family
Ataxioceratidae: Ataxioceras (Parataxioceras) to “Orthosphinctes(Ardescia)”, Crussoliceras,
Garnierisphinctes and Progeronia, and of the family Aulacostephanidae: Eurasenia, Involuticeras,
Prorasenia, Rasenia (Pachypictonia) (see Kutek, 1968, 1994; Matyja, Wierzbowski, 2000; Wierzbowski,
2022; see also herein). Unit B consists of oyster lumachelles about 2.5 m in thickness. The lumachelles are composed of
densely packed shells of Actinostreon (“Lopha”, “Alectryonia”) with marked admixture of other bivalve
shells, and those of diverse other groups – brachiopods, gastropods, echinoids; additionally are found
remains of starfish, crinoids, fish and others. The fossils are commonly overgrown by epizoans (sponges,
serpulids, bryozoans) and bored by lithophags. The deposit reveals the features of non-deposition and
winnowing of finer particles, as well as temporary reworking. The latter is shown e.g., by the occurrence
of multiphase growth of oysters from the elongated forms attached to seminfaunal bivalve shells to the
cup-shaped forms all of them preserved in deposit, but commonly not in life position; it is also the case
of occurrence of disoriented internal moulds of deep burrowing bivalves, and clasts of coral colonies. All
these features indicate the residual lag type character of the discussed deposit. Description of the lithological succession The ammonites are
fairly common – but usually fragmentarily preserved. These are especially represented by
Ataxioceratidae: mostly Crussoliceras, Garnierisphinctes, less commonly Progeronia, but to the virtual
absence of Ataxioceras and “Orthosphinctes”, and somewhat less abundantly by Aulacostephanidae:
Eurasenia, Rasenia, Prorasenia (see Kutek, 1968, 1994; Matyja, Wierzbowski, 2000; Wierzbowski, 2022,
see also herein). Unit C having only 1.75 m in thickness is developed as oncolitic limestone. Oncolites differ markedly in
final size from less than millimeter to 3–4 cm, but the most commonly around 0.5-1 cm in diameter. The
associated fauna is represented mostly by various bivalves, both infaunal to seminfaunal (often
preserved in life position, such as Pleuromya) and epifaunal types (especially Nanogyra), associated with
brachiopods, gastropods and echinoids. The ammonites occur fairly commonly. There is observed a
marked domination of Ataxioceratidae: Crussoliceras, Garnierisphinctes, and Progeronia, and less
common occurrence of Aulacostephanidae: mostly Prorasenia, subordinately Involuticeras and
Eurasenia, but also of Aspidoceratidae: Pseudhimalayites and Psedowaagenia (see detailed comments
below). Unit D, attaining 8.25 m in thickness, consists of lumachelles with Nanogyra: these are composed of
Nanogyra shells and their hash occurring in micritic limestone or marly matrix; the deposits are well-
bedded with intervening beds of micritic limestones and marls, especially common in the middle part of Page 4/32 Page 4/32 the unit. The faunal assemblage is rather monotonous, dominated by epifaunal oysters Nanogyra, less
commonly serpulids, crinoids, echinoids, starfish. The overlying deposits of unit E, 5.50 m in thickness,
are thick-bedded bioclastic limestones with numerous internal moulds of various infaunal bivalves,
including those of Pholadomya, intervening in the middle by a prominent marly bed without macrofana. A
highest bed of limestones of unit E yielded numerous specimens of aspidoceratid ammonite
Pseudhimalayites. The collection of ammonites coming from units D and E is, however, treated jointly,
because of difficulties in proper location of some collected ammonites. It is composed of occurring
mostly in nearly equal proportions Ataxioceratidae (Crussoliceras, Garnierisphinctes, Tolvericeras,
Progeronia) and Aspidoceras (Pseudhimalayites, very commonly). Some specimens of
Aulacostephanidae represent a very diversified assemblage ( Prorasenia, Rasenia, Rasenioides, and
possibly Pararasenia) (Kutek, 1968; Matyja, Wierzbowski, 2000; Wierzbowski, 2022; see also comments
below). A few large specimens of phylloceratids attaining about 0.5 m in diameter discovered in an
upper part of unit E represent possibly allochtonous post-mortem drifted shells. Stratigraphical and palaeobiogeographical interpretation of
the ammonite faunas The studied ammonite faunas contain besides the dominating Submediterranean-Mediterranean forms,
also fairly commonly the Subboreal ones, as well as some of the strictly Mediterranean origin. Such a
spectrum of ammonites enables not only the recognition of the various biostratigraphical zonations, but
also makes possible correlation between the particular zonal schemes of different bioprovinces. The ammonites of the family Ataxioceratidae are the typical Submediterranean (partly also
Mediterranean) forms. Although “the family is still one of the most difficult to classify” ( Enay & Howarth,
2019, p. 112; see also discussion in chapter on evolution), its differentiation has became the basis for
foundation of the Submediterranean zonations of the Lower Kimmeridgian, especially that established in
south-eastern France, in the Jura Mts. in Switzerland, and in Swabian Alb and Franconian Alb in southern
Germany (see e.g., Geyer, 1961; Atrops, 1982; Gygi, 2003; Enay et al., 2014), which is evidently
recognizable in the area of central Poland (e.g., Kutek, 1968), including the section studied. The
ammonites studied herein can be attributed to three different groups. The first of them recognized only
in unit A in the Małogoszcz section is closely related to the genus Ataxioceras. It is represented by
Ataxioceras (Parataxioceras) cf. planulatum (Quenstedt) (Fig. 3a), A. (P.) oppeli parvum Atrops (Fig. 3b),
and “Orthosphinctes (Ardescia)” perayensis Atrops (Fig. 3c). The representatives of Ataxioceras are
indicative of the semistriatum horizon, possibly beginning from its base as suggests occurrence of a
form similar to A. planulatum characteristic of still older hypselocyclum horizon, of the upper part of the
Lothari Subzone of the Hypselocyclum Zone in the accepted herein subdivision of Atrops (1982). The
occurrence of “O”. perayensis, being a form of the Ataxioceras (Parataxioceras) oppeli group, possibly
reduced in ontogenetic development due to the heterochrony, is indicative moreover of the topmost part
of the Hypselocyclum Zone (Atrops, 1982; see also comments on evolution below). The stratigraphical
interpretation of all these ammonites is consistent with the occurrence of Crussoliceras and Page 5/32 Page 5/32 Garnierisphinctes of the second group of Ataxioceratidae in the discussed ammonite fauna of unit A. Both these genera appear for the first time in the Submediterranean succession in the semistriatum
horizon of the Hypselocyclum Zone (Atrops, 1982). Of the recognized here three species of that group –
G. melliconense (Geyer) (Fig. 3d), Crussoliceras sayni (Camus et Thieuloy) (Fig. Stratigraphical and palaeobiogeographical interpretation of
the ammonite faunas Although the
appearance of Progeronia in the upper part of the Hypselocyclum Zone has been commonly recognized
(e.g., Atrops, 1982), the reported new findings for the first time firmly prove the occurrence of the
discussed species at that stratigraphical level. The ammonites coming from unit B do not reveal the representatives of the first group related to the
genus Ataxioceras. Instead there are commonly represented forms of two other groups of
Ataxioceratidae: Crussoliceras and Garnierisphinctes ones, as well as that of the genus Progeronia. Unfortunately because of the taphonomic reasons (see above) the specimens are mostly incomplete
and difficult for closer determination. The only recognized specifically forms are: Garnieriphinctes cf. garnieri (Fontannes) (Fig. 3e) and Crussoliceras cf. aceroides Geyer (Fig. 4c-d). Nevertheless, the
assemblage is diagnostic of the lower part of the Divisum Zone – and may be treated as corresponding
to the Crusoliensis or Tenuicostatum Subzone according to stratigraphical interpretation accepted herein
(see e.g., Atrops, 1982; Enay et al., 2014). It should be remembered, however, that in stratigraphical scheme of similar Submediterranean
succession of southern Germany, the lower boundary of the Divisum Zone has been originally correlated
with the level of the first occurrence of Crussoliceras and Garnierisphinctes (e.g., Geyer, 1961). According to that stratigrapical subdivision, the whole interval corresponding to unit A in the Małogoszcz
section (compared with the upper part of the Lothari Subzone herein) would be correlated already with
the lowest part of the Divisum Zone. It should be remembered, however, that in stratigraphical scheme of similar Submediterranean
succession of southern Germany, the lower boundary of the Divisum Zone has been originally correlated
with the level of the first occurrence of Crussoliceras and Garnierisphinctes (e.g., Geyer, 1961). According to that stratigrapical subdivision, the whole interval corresponding to unit A in the Małogoszcz It should be remembered, however, that in stratigraphical scheme of similar Submediterranean
succession of southern Germany, the lower boundary of the Divisum Zone has been originally correlated
with the level of the first occurrence of Crussoliceras and Garnierisphinctes (e.g., Geyer, 1961). According to that stratigrapical subdivision, the whole interval corresponding to unit A in the Małogoszcz
section (compared with the upper part of the Lothari Subzone herein) would be correlated already with
the lowest part of the Divisum Zone. Stratigraphical and palaeobiogeographical interpretation of
the ammonite faunas 4a), and Garnierisphinctes of the second group of Ataxioceratidae in the discussed ammonite fauna of unit A. Both these genera appear for the first time in the Submediterranean succession in the semistriatum
horizon of the Hypselocyclum Zone (Atrops, 1982). Of the recognized here three species of that group –
G. melliconense (Geyer) (Fig. 3d), Crussoliceras sayni (Camus et Thieuloy) (Fig. 4a), and
Garnierisphinctes garnieri (Fontannes) (Fig. 4b)– the middle has been reported from the upper part of
the Lothari Subzone (Gygi, 2003), whereas the level of appearance of the latter two has not been so far
precisely recognized. The genus Progeronia of the third group of Ataxioceratidae is represented in unit A Garnierisphinctes of the second group of Ataxioceratidae in the discussed ammonite fauna of unit A. Both these genera appear for the first time in the Submediterranean succession in the semistriatum
horizon of the Hypselocyclum Zone (Atrops, 1982). Of the recognized here three species of that group –
G. melliconense (Geyer) (Fig. 3d), Crussoliceras sayni (Camus et Thieuloy) (Fig. 4a), and
Garnierisphinctes garnieri (Fontannes) (Fig. 4b)– the middle has been reported from the upper part of
the Lothari Subzone (Gygi, 2003), whereas the level of appearance of the latter two has not been so far
precisely recognized. The genus Progeronia of the third group of Ataxioceratidae is represented in unit A
both by macroconchs: Progeronia (Progeronia) progeron (von Ammon) (Fig. 5a) and P. (P.) cf. eggeri (von
Ammon), and microconch Progeronia (Hugueninsphinctes) breviceps (Quenstedt) (Fig. 5b). Although the
appearance of Progeronia in the upper part of the Hypselocyclum Zone has been commonly recognized
(e.g., Atrops, 1982), the reported new findings for the first time firmly prove the occurrence of the
discussed species at that stratigraphical level. Garnierisphinctes garnieri (Fontannes) (Fig. 4b)– the middle has been reported from the upper part of
the Lothari Subzone (Gygi, 2003), whereas the level of appearance of the latter two has not been so far
precisely recognized. The genus Progeronia of the third group of Ataxioceratidae is represented in unit A
both by macroconchs: Progeronia (Progeronia) progeron (von Ammon) (Fig. 5a) and P. (P.) cf. eggeri (von
Ammon), and microconch Progeronia (Hugueninsphinctes) breviceps (Quenstedt) (Fig. 5b). Stratigraphical and palaeobiogeographical interpretation of
the ammonite faunas According to that stratigrapical subdivision, the whole interval corresponding to unit A in the Małogoszcz
section (compared with the upper part of the Lothari Subzone herein) would be correlated already with
the lowest part of the Divisum Zone. On the other hand, a markedly different ammonite succession is recognized in the coeval deposits of the
Spanish sections of the Iberian Range, which can treated as representative of the western part of
Submediterranean Province. It includes the occurrence there of a special ataxiocertid group
distinguished as corresponding to the new genus Geyericeras, not known in other European areas. These
ammonites define the aragoniense stratigraphical horizon occurring directly below the appearance of
Crussoliceras indicating already the base of the Divisum Zone. The relevant correlation with SE France
and eastern parts of the Submediterranean Province, including central Poland, strongly suggests the
position of the aragoniense horizon directly below the upper part of the semistriatum horizon and the
perayensis horizon (see Moliner & Olóriz, 2009, 2010; see also Moliner, 2009). Page 6/32 The ataxioceratid ammonites from unit C of the Małogoszcz section are still markedly dominated by
Garnierishinctes and Crussoliceras, although the species are mostly different when compared with older
units: Garnierisphinctes semigarnieri (Geyer) (Fig. 6a), G. championneti (Fontannes) (Fig. 6b), G. plebejus
(Neumayr) (Fig. 6c), as well as Crussoliceras cf. lacertosum (Fontannes), C. cf. atavum (Schneid) –
aceroides (Geyer). It is worth noting also the first occurrence of a form which possibly can be referred to
the genus Tolvericeras; the corresponding specimens (Fig. 6d) are very similar to that described as
“Tolvericeras (Tolvericeras) n. sp.” by Gygi (2003, Figs. 165–166) showing the polygyrate subdivision of
ribs placed low in the whorl-side; this form was treated subsequently as taking the intermediate position
between Garnierisphinctes and Tolvericeras (Enay et al., 2014, p. 327). Some representatives of
Progeronia with well preserved specimen of Progeronia (Hugueninsphinctes) breviceps (Quenstedt)
(Fig. 5c) are also recognized. The occurrence of several specimens of Aspidoceratidae represented
mostly by Pseudohimalayites uhlandi (Oppel) unequivocally indicates that the whole ammonite
assemblage from unit C corresponds already to the Uhlandi Subzone of the upper part of the Divisum
Zone (see e.g., Geyer, 1961). The last ataxioceratid ammonites coming from units D and E are dominated by representatives of
Crussoliceras: C. cf. crusoliense (Fontannes); Garnierisphinctes: G. championneti (Fontannes); and
Tolvericeras group; the representatives of the Progeronia group: Progeronia (Progeronia) cf. eggeri (von
Ammon) (Fig. Stratigraphical and palaeobiogeographical interpretation of
the ammonite faunas Similarly, the co-occurrence of P. uhlandi with
Nebrodites sp. and Taramelliceras (Taramelliceras) compsum (Oppel) (Fig. 7l) as recognized in still
higher units units D and E indicates the presence of some upper parts of the Uhlandi Subzone (see
Caracuel et al., 1998). Some problem arises with interpretation in term of the Mediterranean zonation of the lowest part of the
studied succession – unit A of the Małogoszcz section, corresponding in the Submediterranean
subdivision to the uppermost part of the Hypselocyclum Zone and the upper part of the Lothari Subzone. The only ammonite of a strictly Mediterranean character coming from that stratigraphical interval is
Nebrodites maccerimus (Quenstedt) (Fig. 7e), according to interpretation of the species by Ziegler
(1959). Just that species was reported, however, from the boundary beds of the two Mediterranean
zones – the Strombecki Zone and the Herbichi Zone or corresponding beds of the Divisum Zone
(Caracuel et al., 1998; see also Olóriz, 1978; Sapunov, 1979). This, together with observations given
above, suggests the discussed unit A corresponds most probably to the lowermost part of the Herbichi
Zone. The ammonite faunas of the family Aulacostephanidae commonly represented in the succession studied
are indicative of the Subboreal Province. These ammonites numerously occurring in units A to B, less
commonly in unit C, are mostly belonging to the genera Eurasenia and Involuticeras of the north-eastern
European branch of the family, and rarely to the genus Rasenia (and its local ally Pachypictonia) related
to its north-western European branch (Wierzbowski, 2022); both these groups of macroconchs occur
together with their microconch counterparts of the genus Prorasenia. Some of these ammonites were
presented and discussed previously: Rasenia (Pachypictonia) aff. indicatoria (Schneid), which seems to
be related to Rasenia evoluta Spath (Wierzbowski, 2022, pl. 7), Eurasenia rolandi (Oppel) (Wierzbowski,
2022, pl. 10:4), and E. trimera (Oppel) (Wierzbowski, 2022, pl. 11: 4) as well as Involuticeras involutum
(Quenstedt) (Wierzbowski, 2022, pl. 14: 2) – all of them from unit A; Eurasenia pendula (Schneid)
(Wierzbowski, 2022, pl. 11: 2–3) – from unit B; and Eurasenia trimera (Oppel) (Wierzbowski, 2022, pl. 11:
5), possibly from unit C. In addition, several specimens of E. trimera (Oppel), E. trifurcata (Reinecke), E. pendula (Schneid), associated with Involuticeras, including I. crassicostatum (Geyer), along with
common Prorasenia – especially P. quenstedti Schindewolf and P. witteana (Oppel), are reported from
units A to C. Stratigraphical and palaeobiogeographical interpretation of
the ammonite faunas 5d) are less common. On the other hand, very numerously occur Aspidoceratidae
represented almost exclusively by Psudhimalayites uhlandi (Oppel). These ammonites represent the
upper parts of the Uhlandi Subzone of the Divisum Zone. Several ammonites of the families Ataxioceratidae (such as Crussoliceras, Garnierisphinctes and
Progeronia), as well as of Aspidoceratidae (mostly Pseudhimalayites) occurring in the studied
succession of the Holy Cross Mts., central Poland, are in common with areas of the Mediterranean
Province such as the Venetian Alps (see e.g., Pavia et al., 1987; Sarti, 1993; Caracuel et al., 1998), and the
Central Appenines (Cecca et al., 1985; Cecca & Santantonio, 1986) in Italy, the Betic Cordillera in
southern Spain (Olóriz, 1978), the Gerecse-Pils Moutains and the Bakony Mountains in Hungary (Főzy &
Scherzinger, 2013; Főzy et al., 2022), the Romanian Carpathians (e.g., Grigore, 2000), and the Balkan Mts. in Bulgaria (Sapunov, 1979). This along with occurrence in the succession studied of some strictly
Tethyan ammonites, such as Presimoceras, Idoceras and Nebrodites allows for recognition of the
Mediterranean zonation (see Sarti, 1993; Caracuel et al., 1998, and correlations with older subdivisions). The ammonites of the genus Presimoceras occur rarely in unit B of the Małogoszcz section. They are
fragmentarily preserved, nevertheless they can be attributed to the P. herbichi group (Fig. 7g-h-i), being
possibly close to P. nodulatum (Quenstedt). Such taxonomical interpretation indicates the correlation
with the Mediterranean Herbichi Zone, additionally because of common occurrence of Crussoliceras -
with its middle part – the Divisum Subzone (see Sarti, 1993). The occurrence of a single specimen of
Idoceras balderum (Oppel) (Fig. 7k) in the discussed unit B suggests also similar correlation. Although
the typical forms of I. balderum are commonly referred to the upper part of the Divisum Zone in
Submediterranean Province (SE France, southern Germany, see e.g., Geyer, 1961; Atrops, 1982; see also Page 7/32 Page 7/32 Schweigert & Kuschel, 2017), some specimens coming from a lower part of the Divisum Zone in other
areas (Swiss Jura Mts., Venetian Alps, Italy) are compared with that species as well (see e.g., Gygi &
Persoz, 1986; Pavia et al., 1987; Sarti, 1993). The overlying deposits of unit C yielded a few specimens of
Nebrodites including N. favaraenenis (Gemmellaro) (Fig. 7f) and N. cf. agrigentinus (Gemmellaro)
(Fig. 7j), which together with appearance of Pseudhimalayites uhlandi (Oppel) indicate the upper part of
the Herbichi Zone – the Uhlandi Subzone (see Sarti, 1993). Stratigraphical and palaeobiogeographical interpretation of
the ammonite faunas On the other hand, some specimens of the genus Rasenia, including form similar to R. involuta Spath (Fig. 7a-b), have been recognized in units B and D-E (near the top of the section). Summarizing, the ammonites of the genera Eurasenia and Involuticeras (associated with Prorasenia
microconchs) occur in units A-C in the Małogoszcz section, indicating a stronger NE Subboreal
influences, whereas NW Subboreal Rasenia are sporadically encountered nearly throughout the whole Page 8/32 Page 8/32 succession up to its top. Additionally, a few fragmentarily preserved specimens from unit E (and possibly
its upper part), showing the ribbing similar to that of Eurasenia, reveal some weakening of ornamentation
on the ventral side of whorls resembling thus younger genus Pararasenia (see Ziegler, 1962). They are
especially similar to P. quenstedti Durand, differing in more elongated primary ribs, and in a weaker
development of the ventral smooth band: thus, they are referred herein to as ? Pararasenia aff. quenstedti Durand (Fig. 7c-d). Moreover, in the upper part of the succession there appear rarely
ammonites of the genus Rasenioides, representing a different branch of Aulacostephanidae, such as R. moeschi (Oppel), coming from the lowermost part of unit D, and illustrated by Matyja & Wierzbowski
(2000, Fig. 4c) and Wierzbowski (2022, pl. 15:2A-B), and some other poorly preserved and difficult for
closer determination specimens of that genus currently recognized in units D-E. The stratigraphical interpretation of the discussed Subboreal ammonites in term of the Subboreal
zonation indicates the correlation of the whole discussed succession with the NW European Cymodoce
Zone. The occurrence of ammonites similar to Rasenia evoluta and R. involuta, strongly suggests the
presence of the higher levels of that zone (see Birkelund et al., 1978, 1983). It should be remembered,
however, that the highest part of the Cymodoce Zone was often treated in the past as an interval
characterized by common occurrence of the fine-ribbed Rasenioides. In the studied succession at
Małogoszcz, dominated by Submediterranean ammonites, the Subboreal Rasenioides ammonites are
very rare, which precludes the precise differentiation of stratigraphical intervals corresponding to the
Askepta Subzone, and/or Chatelaillonensis Subzone – both defined by occurrence of the Rasenioides
faunas in northern European areas (see Birkelund et al., 1983; Hantzpergue, 1989, 1995). Stratigraphical and palaeobiogeographical interpretation of
the ammonite faunas The former can
be possibly correlated with the bulk of the succession studied from the Submediterranean horizon
perayensis at its base, whereas the base of the latter subzone runs possibly somewhat lower, near the
base of the semistriatum horizon, as resulted from wider biostratigraphical correlations of the Subboreal
and Submediterranean sections (see Matyja, Wierzbowski, 2000; Comment et al., 2015). Changes in environment versus evolutionary development of ammonites Changes in environment versus evolutionary development of ammonites The stratigraphical interval corresponding to the upper part of the Hypselocyclum Zone and to the
Divisum Zone (or in a broader sense to the whole Divisum Zone) of the uppermost Lower Kimmeridgian
represents one of the most prominent faunal turnover in the whole Upper Jurassic. It is markedly
correlated with changes in the depositional environment as based on sedimentological data. The main
reasons of changes were of climatic and tectonic nature resulting from sea-level oscillations, and/or
opening of new sea-routs. It should be remembered that the deepest faunal changes generally occurred
during the transgressions, whereas endemism was rather related to dominance of the shallow-water
environments (Atrops & Ferry, 1989; Hanzpergue, 1995; Wierzbowski, 2022). The stratigraphical interval discussed herein corresponded in the Submediterranean Province to decline
of the shallow-water carbonate platforms due to progress of a large transgression. This resulted from
the tectonic subsidence of the wide shallow-water carbonate areas including that of the Holy Cross
Mountains in central Poland: at the boundary between the COK and the LUK tectono-stratigraphic Page 9/32 Page 9/32 sequences (Kutek, 1994) which corresponds to the boundary between the Buczyna Mbr. of the Spinkowa
Góra Fm., and the Skorków Lumachelle of the Coquina Fm. in the Małogoszcz section (Wierzbowski,
2020). The progress of the transgression is well shown in the section studied by several
sedimentological observation summarized by Matyja et al. (2006), and by analysis of the geochemical
data, mostly oxygen and carbon composition of shells of various oysters by Wierzbowski (2019). A
similar tectonic phenomenon is observed in the development of the Banné Member of the Lower
Reuchenette Fm. in the Jura Mountains of northern Switzerland which marks the change from an older
flat carbonate platform topography into that of a “prominent basin and swell morphology” (Jank et al.,
2006). In addition, the climatic oscillations had also a marked influence on sea-level changes, especially
those of longer duration related to 100-kyr and 405-kyr eccentricity cycles. Such a study to attempt to
recognize the climatic cycles was presented on the basis of detailed geochemical analysis of the Early
Kimmeridgian pelagic deposits of the south-eastern France having a good ammonite stratigraphy
(Boulila et al., 2008; see also Atrops, 1982). Changes in environment versus evolutionary development of ammonites It resulted in recognition of three main eccentricity cycles
within deposits strictly coeval to those studied herein and showing similar assemblage of ammonites:
beginning with the important transgressive level corresponding to minimum of 405-kyr MS cycle
developed at the base of the whole succession (Min. 4 in Fig. 2 of Boulila et al., 2008), and the following
two minima of 100-kyr cycles. The duration of the whole stratigraphical interval corresponding to the
broadly treated Divisum Zone can be estimated as 260 to 300 kyr (Boulila et al., 2008). The overall transgression at the decline of the Early Kimmeridgian attained its very high level in central
Poland (and south-eastern France) at the end of the Hypselocyclum Chron – during the semistriatum
and perayenis horizons, when the transgressive deposits of a basal part of the Skorków Lumachelle
(denoted herein as unit A, see Fig. 2) completely covered the earlier shallow-water carbonate platform
deposits. This level additionally corresponds to the minimum (Min. 4 after Boulila et al., 2008) of the 405-
kyr eccentricity cycle: it marks the minimum of the magnetic susceptibility (MS), and corresponds to
enhanced carbonate production, showing the maximum insolation, which appears to have induced a very
high sea-level according to the model proposed by Boulila et al. (2008, 2010). The transgression in the Submediterranean Province controlled both by climatic and synsedimentary
tectonics resulted in a marked exchange of the ammonite faunas (Fig. 8). The extinction of the older
lineage of typical Submediterranean ammonites Ataxioceratinae, corresponding to decline of the genus
Ataxioceras, was associated with the appearance of new members of the family Ataxioceratidae, such
as genera Crussoliceras and Garnierisphinctes, beginning a new evolutionary stage of their development
(see Atrops & Ferry, 1989; Enay & Howarth, 2019). The former lineage is represented in the studied
interval of the uppermost Hypselocyclum Zone of the Submediterranean Province, from south-eastern
France to central Poland, by the last smaller-sized forms of the genus Ataxioceras, both micro- and
macroconchs (Atrops, 1982). The final, somewhat isolated microconch member of that lineage is
“Orthosphinctes (Ardescia)” perayensis Atrops. This form has been sometimes treated as the end-
member of the separate Ardescia lineage (or even Lithacosphinctes lineage; see Moliner & Olóriz, 2010),
but it can be also considered as the final member of the small-sized Ataxioceras (Parataxioceras) oppeli
group, originated due to the heterochrony process. Changes in environment versus evolutionary development of ammonites It may be concluded thus, it is possibly a small-sized Page 10/32 Page 10/32 form developed due to progenesis as shown by its ornamentation very similar to that of the inner whorls
of its direct forerunner – the subspecies A. (P.) oppeli parvum Atrops (see also Atrops, 1982, p. 228,
Fig. 53). When discussing the phylogenetical position of the genus Crussoliceras the most commonly
interpretation given (Hantzpergue, 1989; see also Enay et al., 2014) suggested its origin from the Early
Kimmeridgian Lithacosphinctes due to growth heterochrony – mostly progenesis and neoteny. The
genus Lithacosphinctes as interpreted recently (e.g., Moliner & Olóriz, 2010) includes both micro- and
macroconchs corresponding to “the most conservative lineage” among the Early Kimmeridgian
Submediterranean Ataxioceratinae: according to such interpretation the genus comprises also some
evolute, smaller-sized microconch species occurring in the lower part of the Lothari Subzone, at the end
of the lineage, and attributed previously to the Orthosphinctes (Ardescia) inconditus group (see Atrops,
1982), but excluding “O. (A.)” perayensis as shown herein (see above). However, in accordance to that,
there does not exist any link in the Submediterranean successions, both stratigraphical and
morphological nature, between the older Lithacosphinctes group, and the younger well-developed
Crussoliceras group. It is the reason that the proposed lineage of evolutionary development from the
indigenous Submediterranean genus Lithacosphinctes to the suddenly appearing genus Crussoliceras is
not accepted herein. It should be remembered that according to Moliner (2010), the genus
Garnierisphinctes has been treated as descendent of a special group of the genus Ardescia (recognized
as the separate genus, composed both of micro-and macroconchs), and moreover it was suggested that
the genus Progeronia evolved from Garnierisphinctes during the late Divisum Chron. Also that
interpretation is not accepted because of the stratigraphical reasons – the occurrence of the typical
representatives of the genus Progeronia in much older deposits of the Early Kimmeridgian (see
assemblage of unit A herein, see also e.g., Sarti, 1993). The new ammonite genera – Crussoliceras, Garnierisphinctes and Progeronia which appeared during
discussed transgression at the end of the Hypselocyclum Chron have had possibly their roots in the
Mediterranean areas. Such an opinion was expressed already by Pavia et al. (1987) who suggested
affinity of Crussoliceras to the Mediterranean Passendorferiinae, which interpretation has been, however,
partly questioned by Enay et al. (2014). Changes in environment versus evolutionary development of ammonites The Mediterranean origin of the discussed genera can be,
however, additionally supported by the occurrence of the genera Progeronia and possibly Crussoliceras
deep in the Mediterranean Strombecki Zone – correlated with some lower parts of the Submediterranean
Hypselocyclum Zone, markedly below the Lothari Subzone (see e.g., Sarti, 1993). The principal reason
for suggesting that representatives of the genera Crussoliceras and Garnierisphinctes have been
originated from Mediterranean migrants is, however, that there are not known their earlier
Submediterranean forerunners. Somewhat different situation is with the genus Progeronia only, because
there are known some forms occurring in older deposits of the Submediterranean succession which
seem similar to typical representatives of the genus – e.g., such as “Orthoshinctes (Ardescia)” enayi
Atrops occurring at the narrow interval between the lower and middle parts of the Platynota Zone from
SE France to central Poland (Atrops, 1982; see also Wierzbowski, 2017). The open problem is, however, if
they represent the fragmentarily recognized members of a single (?Mediterranean) lineage, or the local Page 11/32 Page 11/32 offshoots of Ataxioceratidae developed during the transgressive episodes. A generally poor knowledge
on details of the succession (and their precise dating) yielding ammonites referred to as Orthosphinctes,
Crussoliceras and Progeronia (e.g., Pavia et al., 1987; Sarti, 1993) in the crucial intervals of the Lower
Kimmeridgian (mostly the Strombecki Zone) in the Mediterranean sections prevents the detailed
recognition of the evolutionary development of the discussed lineages. The evolutionary development of ammonites of the family Aulacostephanidae proceeded a different way. In central Poland, in the area of Burzenin, north-west of the Holy Cross Mountains, in the deposits of
open-marine environment of the middle part of the Hypselocyclum Zone (i.e. older than studied herein),
was noted a marked increase in number, and in morphological development of representatives of the
family (Wierzbowski, 2017). These ammonites belonged here to two different groups: that showing a
more heavily-ribbed shells and representing possibly a more shallow-water environment such as
Eurasenia and Involuticeras, and another one composed of Vineta to Balticeras and the first Rasenioides
showing more subdued ribbing, exploring possibly more-open marine, nektonic environment
(Wierzbowski, 2022). The overall transgression at the end of the Hypselocyclum Chron brought the
heavily-ribbed Aulacostephanidae into flooded area of the shallow-water carbonate platform of the Holy
Cross Mountains. Their share within the whole ammonite fauna of unit A in the Małogoszcz section
ranges even up to about 35%. Changes in environment versus evolutionary development of ammonites 2), suggests also
a local higher upward range of that species. Anyway, the sudden invasion of the the Subboreal
Rasenioides to the north, coincided possibly in time with a sudden spread across the Submediterranean
Province of ammonites of the Mesogean affinity – such as Crussoliceras, Garnierisphinctes and
Progeronia, as discussed above. Unit B in the Małogoszcz section has clearly the regressive character as indicated by its lithological
characteristics (see above). The assemblage of ammonites consists mostly of Ataxioceratidae
(Crussoliceras, Garnierisphinctes and Progeronia) and to lesser degree of the heavily-ribbed
Aulacostephanidae (Eurasenia, Prorasenia, rare Rasenia), showing thus a marked similarity to that of unit
A, but without Ataxioceras. The younger ammonite assemblage of unit C reveals, however, a markedly
different character. The most important new faunal elements in ammonite assemblage of unit C are the suddenly appearing
fairly numerous Aspidoceratidae, especially those of the genus Pseudhimalayites with the species P. uhlandi (Oppel). The roots of the genus were possibly in the Western Tethyan areas, or even far outside,
thus a wide distribution of this genus in many European sections was an effect of migration (see e.g.,
Schweigert, 1997). The species P. uhlandi associated with many other Tethyan ammonites, such as
various species of Nebrodites, occurs commonly in the Western Tethyan areas, and their foreland – in
the Submediterranean Province, defining everywhere the upper part of the Divisum Zone or of the
Herbichi Zone – the Uhlandi Subzone (e.g., Geyer, 1961, Sarti, 1993; Caracuel et al., 1998). Even in the
studied Submediterranean succession at Małogoszcz – some representatives of various species of
Nebrodites have been encountered along with P. uhlandi in unit C. Additionally, the not numerous
Aulacostephanidae are markedly impoverished here, both in number and in presence of heavily-ribbed
forms. All these data strongly suggests a transgressive character of unit C. It should be remembered
that the deposits of the Uhlandi Subzone are treated as transgressive in character in many areas of
Europe (e.g., Marques & Olóriz, 1992). This high sea-level can be correlated possibly with the minimum of
100-kyr eccentricity cycle (see Boulila et al., 2008, 2010). Units D and E in the Małogoszcz section yielded numerous specimens of Pseudhimalayites uhlandi
(Oppel). Unfortunately the distribution of these specimens in the succession cannot be traced in details
mostly because of lack of precise location of the particular finds. Changes in environment versus evolutionary development of ammonites On the other hand, the aulacostephanids of the second group were totally
absent in this area. Similar features of distribution of ammonites of the family Aulacostephanidae have been observed also
in other shallow-water areas of the Western European Shelf, corresponding to the so-called “Western
European Swell” (Hantzpergue, 1989; Hantzpergue et al., 1997; see also Enay et al., 2014). A sudden
appearance of ammonites of the genus Eurasenia, along and above the occurrence of Ataxioceras of A. lothari group, “coincided with a maximum sea-level rise” (Hantzpergue, 1995, p. 247). The horizons
aulsnisa and manicata as defined on the basis of Eurasenia in the Western European Shelf can be just
correlated with the semistriatum horizon of the upper part of the Hypselocyclum Zone of the
Submediterranean zonation (Hantzpergue, 1989; Matyja & Wierzbowski, 2000). On the other hand, a sudden development of the genus Rasenioides representing the second group of
Aulacostephanidae took place in the areas of northern and north-western Europe corresponding to the
Subboreal Province, or representing the transitional areas between Submediterranean and Subboreal
provinces. The dominance of these ammonites is observed e.g., in England (Birkelund et al., 1983),
where their appearance defines the base of the Askepta Subzone correlated with the upper part of the
Subboreal Cymodoce Zone, but also in a similar stratigraphical position in the Peri-Baltic Syneclise from
the north-eastern Poland to Lithuania (e.g., Wierzbowski et al., 2015). In Aquitaine (Hantzpergue, 1989;
Hantzpergue et al., 1997), the appearance of Rasenioides defines the base of the askeptus horizon
within the Chatelaillonensis Subzone – which is located directly above the aulsnisa-manicata horizons
with Eurasenia. The crucial for correlation of the discussed aulacostephanid (Subboreal) and the
ataxioceratid (Submediterranean) zonations appeared the data from boreholes in the Zalesie Anticline in
northern Poland studied by the present author. The recognized here (Matyja & Wierzbowski, 2000, Page 12/32 Page 12/32 Figs. 3–4) nearly coeval occurrence of early Rasenioides and “Orthosphinctes (Ardescia)” perayensis
Atrops indicates, the correlation of the lowermost part of the Askepta Subzone or askeptus horizon of
the Chatelaillonensis Subzone in Subboreal or transitional areas of the northern Europe with the
perayensis horizon of the uppermost part of the Hypselocylum Zone of the Submediterranean Province. It should be remembered, however, that the occurrence of “O.(A.)” perayensis along with late
representatives of the genus Rasenioides transitional to Aulacostephanoides, as reported in archival
materials from core Kcynia IG-IV in northern Poland (Matyja & Wierzbowski, 2000, Fig. Changes in environment versus evolutionary development of ammonites It is only the topmost part of unit E
recognized which shows a marked concentration of the Pseudhimalayites shells, some of them attaining
large sizes. In a similar stratigraphical position has been found also a few specimens of Phylloceratidae
of giant sizes (about 0.5 m in diameter), interpreted herein as the allochtonous elements which Page 13/32 Page 13/32 appearance has been possibly related to the post-mortem drift of shells from the Tethyan areas (thus not
taken into account in the diagram of distribution of the ammonite genera in the succession – see Fig. 2). It seems highly probable that the concentration of all these shells at the top of unit E resulted from a very
high sea-level. This may correspond to the minimum of the next 100-kyr eccentricity cycle well
documented in the late Divisum Subchron (see Boulila et al., 2008, 2010). This level corresponds
possibly also to the balderum horizon (or subzone) as marked by common occurrence of the Tethyan
form Idoceras balderum (Oppel) well documented at the top of the Uhlandi Subzone of the Divisum Zone
in the Submediterranean areas of southern Germany to south-eastern France (see e.g., Hantzpergue et
al., 1997). Such stratigraphical interpretation can be also confirm by finding in the Małogoszcz section, at
the top of unit E, of a single specimen of Taramelliceras (Taramelliceras) compsum (Oppel) of the
Tethyan origin. Some changes in ammonites of the family Ataxioceratidae seen already in unit C, recognized also in
units D-E include the emerging of the new genus Tolvericeras. The genus as interpreted by Enay et al. (2014) derived possibly from Garnierisphinctes and Crussoliceras. The rare specimens discovered in the
Małogoszcz succession show a marked similarity to “ Tolvericeras n. sp.” in Gygi (2003, p. 144,
Figs. 165–166) coming from the Divisum Zone of northern Switzerland, showing independently some
similarity to Garnierisphinctes. On the other hand, the units C-D-E yielded also numerous specimens of
Crussoliceras [C. cf. crusoliense (Font.)], Garnierisphinctes [G. plebejus (Neumayr and G. championneti
(Neum., G. semigarnieri (Geyer] and less common Progeronia [ P. (P.) eggeri (von Ammon), P. (H.)
breviceps (Quenstedt)]. The recognized changes within family Aulacostephanidae in units D-E of the Małogoszcz section include
especially the rare occurrences of the advanced morphologically representatives of the genus
Rasenioides, such as R. moeschi (Oppel). Changes in environment versus evolutionary development of ammonites Their appearance, already at the base of unit D, can be treated
as a manifestation of “southern drift” of the genus which developed earlier in the Subboreal Province
(see Wierzbowski, 2022; see also comments above): this phenomenon resulted possibly from
appearance in the succession studied of marly facies resembling the “Virgulian Facies” of north-western
Europe suitable for development of the Subboreal fauna (cf. Hantzpergue, 1995). On the other hand, the
local occurrence of a special group of aulacostephanids intermediate between some Eurasenia and
Pararasenia has been recognized in upper parts of unit E. They show heavy ornamentation on the whorl
side characteristic of both Eurasenia and Pararasenia, but reveal an incipient obliteration of ribbing in the
ventral side of whorls typical of the genus Pararasenia. The specimen (Fig. 7c-d) studied resembles the
heavily ribbed species Pararasenia quenstedti Durand (cf. Ziegler, 1962) reported in younger beds of the
earliest Late Kimmeridgian in the Małogoszcz section (Kutek, 1968). Two specimens coming from units B and D-E (Fig. 7a-b) are representatives of NW European Subboreal
genus Rasenia. They show a weakly involute to weakly evolute coiling, rather distant primary ribs, and a
high number of secondary ribs (the secondary/primary rib ratio equals 5.0 at about 50 mm diameter). The specimens belong to Rasenia involuta Spath, a very characteristic species commonly encountered in
southern England where the specimens coming from (e.g., Birkelund et al., 1983, Fig. 3A-D) are very Page 14/32 Page 14/32 similar to the specimens studied. It is worth noting that all these specimens resemble also the heavily-
ribbed form referred to as Rasenioides ecolisnus (Hantzpergue, 1989, pl. 35: c-f). It should be
remembered that the latter is treated as a form which begins a side branch of the Rasenioides lineage,
leading in its evolutionary development to origin of a more heavily-ribbed forms of the genus
Aulacostephanoides (Hantzpergue, 1989, Figs. 130,133), or marking the transition to the genus
Aulacostephanus (Borreli, 2014, Fig. 2). The possible phylogenetical link between late Rasenia involuta,
and some early forms of Aulacostephanoides/Aulacostephanus (as suggested herein) can be thus
considered as an alternative proposal for the lineage (assuming the phylogenetical affinity between R. involuta and R. ecolisnus), which developed independently of that leading from the densely-ribbed
Rasenioides to the typical Aulacostephanoides (cf. Borreli, 2014). General comments The late Early Kimmeridgian corresponding to the late Hypselocyclum and Divisum chrons (or to the late
Cymodoce Chron) was a fairly short time (about 260 to 300 kyr) when the diversity in development of
many ammonites lineages changed markedly. This phylogenetical turnover resulted in origination and
termination of several lineages, and has been strictly controlled by changes in the environment, generally
in progress of the overall transgression, which occurred especially along the northern Tethyan Shelf in
the wide areas of Submediterranean to Subboreal provinces of Europe. The transgression was strictly
controlled by syn-sedimentary tectonic activity, and additionally superimposed transgressive pulses
corresponding to climate changes, related mostly with orbitally-controlled cyclicity. The studied
Submediterranean succession at Małogoszcz in the south-western margin of the Holy Cross Mountains,
central Poland was formed in changing water depth – from initial transgression flooding the shallow-
water carbonate platform to a fairly deep water environment, which showed similarity to that of the
“Virgulian Facies” of north-western Europe. The development of the coeval deeper-water sedimentation
is recognized widely in Submediterranean areas from central Poland to south-eastern France and Spain,
being everywhere strictly correlated to the marked evolutionary transformations of the ammonite faunas:
their migrations, decline or radiation. The beginning of the marine transgression already at the end of the Hypselocyclum Chron brought into
the whole discussed areas of Submediterranean Europe the new ataxioceratid ammonites including
closely related genera Crussoliceras, Garnierisphinctes and Progeronia having possibly their roots in the
Mediterranean Tethys. The development of these ammonites was strictly related to changes of the
environmental conditions into those compatible with their physiological tolerance, which also affected
the group of older indigenous Ataxioceratinae, such as Ataxioceras, Orthosphinctes and Ardescia, and
resulted in its total decline. On the other hand, the changes in environment strongly disturbed also the
development of other groups of ammonites, mostly Aulacostephanidae: some of them, especially the
heavily- ribbed forms (Eurasenia, Involuticeras) fluorished on the flooded areas of the carbonate platform
like that of the Holy Cross Mts., some other like the more open-marine Rasenioides migrated toward the
north into a deeper-water environment of Subboreal Province. The progress of the transgression brought Page 15/32 Page 15/32 onto the Tethyan Shelf (but also into the Mediterranean Tethys) during the late Divisum Chron also other
ammonites – mostly Aspidoceratidae, especially of the genus Pseudhimalayites. The taxonomic diversity within Crussoliceras-Garnierisphinctes-Progeronia faunas increases upwards in
the succession studied at Małogoszcz. General comments This is shown by the appearance of larger number of species, but
also by the evolutionary development of the new genus Tolvericeras showing affinity to
Garnierisphinctes. It is in general accordance with observation of Enay et al. (2014) who suggested the
evolutionary path from Garnierisphinctes to restricted group of species placed around the type species
of Tolvericeras already during the late Divisum Chron, at the end of Early Kimmeridgian, in the
Submediterranean Province in SE France and Switzerland. Later on, Crussoliceras and Tolvericeras were
rather not commonly encountered here, occurring mostly during the Late Kimmeridgian in the Western
European Swell – the transitional area between Submediterranean and Subboreal provinces, where also
some forms close to Crussoliceras evolved at the end of the Kimmeridgian into Pseudogravesia to
Gravesia group (Enay et al., 2014; cf. also Hantzpergue et al., 1989). On the other hand, a more common
occurrence of Crussoliceras and Tolvericeras during the Late Kimmeridgian has been recognized in the
Subboreal Province in England: the appearance there of Subdichotomites, which possibly evolved from
Crussoliceras at the end of the Kimmeridgian, gave subsequently rise to the Boreal Dorsoplanitinae
(Enay et al., 2014). The evolutionary development of Aulacostephanidae at the end of the Early Kimmeridgian – beginning of
the Late Kimmeridgian in Europe has been the subject of fairly diversified interpretations. This is mostly
because of the incompleteness of the fossil records, at least partly resulting from marked changes of
palaeobiogeographical ranges of these ammonites in time. The only indigenous lineage traced in the
Holy Cross Mts. area is that leading from the heavily-ribbed Eurasenia, like E. pendula and E. trifurcata
recorded in the succession studied, to Pararasenia which shows the smooth band in the ventral side of
whorls (Ziegler, 1962; see also Wierzbowski, 2022). Another lineage, already discussed above, originated
from the weakly-ornamented Vineta to Rasenioides, but its further development included migration of
younger forms of the lineage towards the north where they gave rise to early Aulacostephanoides. Even
more nebulous is the development of the third lineage of aulacostephanids. This possibly included at its
base some moderately involute and heavily-ribbed Rasenia (like R. involuta and possibly similarly looking
“Rasenioides” ecolisnus, as discussed above). They seem to intergrade even with the “transitional”
genus Zonovia showing some weakening of the ribbing on the ventral side of outer whorls (see Birkelund
et al., 1978). The author declares no conflicts of interest. The author declares no conflicts of interest. Declarations Achnowledgements: The study was financed by the Polish National Science Center (project no
2020/39/B/ST10/01489). Special thanks are to Błażej Błażejowski for his help in preparation of
photographs of ammonites, and to Hubert Wierzbowski for fruitful discussion. General comments The lineage may continue into some more heavily-ribbed early Late Kimmeridgian
aulacostephanids referred in older classifications to as Aulacostephanoides, and more recently
correlated with Aulacostephanus (Borreli, 2014). The changes in distribution of ammonites were accompanied by important development in their
evolution. Such a drastic example of overlapping of ranges of distribution of the ammonite faunas
belonging to different lineages and representing the various stages of their evolutionary development
has been reported in the successions of central and southern parts of the Russian Platform, especially
Tatarstan, central European Russia (see Rogov et al., 2017). The recognized there a narrow Page 16/32 Page 16/32 stratigraphical horizon with Submediterranean Crussoliceras atavum (Schneid) - C. lacertosum
(Fonatnnes), possibly corresponds only to some upper parts of the Divisum Zone, and it is underlain by
beds with Subboreal Rasenia and Zonovia, and overlain by beds with Rasenioides, all of them belonging
to the upper part of the Cymodoce Zone – the Askepta Subzone. Additionally, the occasional occurrence
of the Boreal ammonites of the genus Amoebites, transitional already to younger Euprionoceras,
indicates the evolutionary changes in the cardioceratid lineage during the late Kitchini Chron. The problems of general classification of the Late Kimmeridgian ammonites attributed to the genus
Aulacostephanus is generally outside the scope of the present study. It is only the genus Zenostephanus
(formerly Xenostephanus) which evolutionary development is partly related to those of some discussed
here aulacostephanids, and which represents a similar stratigraphical range. The genus evolved probably
from the Subboreal Rasenia evoluta, as evidenced by occurrence of the intermediate forms already at the
end of the Cymodoce Chron in southern England (Birkelund et al., 1983). These migrated successively
into the Arctic areas of the Boreal Province at the boundary between the Early and Late Kimmeridgian as
a consequence of a large transgression giving then the onset to the new lineage (e.g., Wierzbowski &
Smelror, 2020), but appeared soon thereafter as representatives of the fully developed genus
Zenostephanus in north-eastern Europe in areas of the Russian Sea (Rogov et al., 2017). The genus can
be considered as a possible forerunner of a group of the Late Kimmeridgian Aulacostephanus, widely
distributed in NW, central, and NE European areas, as already suggested by Birkelund & Callomon (1985),
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Badenerschichten) im sȕddeutschen Jura. Palaeontographica, 117A(1–4), 1–157. 19. Gygi, R. (2003). Perisphinctacean ammonites of the late Jurassic in northern Switzerland: a versatile
tool to investigate the sedimentary geology of an epicontinental sea. Schweizerische
Paläontologische Abhandlungen, 123, 1–232. 19. Gygi, R. (2003). Perisphinctacean ammonites of the late Jurassic in northern Switzerland: a versatile
tool to investigate the sedimentary geology of an epicontinental sea. Schweizerische
Paläontologische Abhandlungen, 123, 1–232. 20. Gygi, R., & Persoz, F. (1986). Mineralogy, litho- and biostratigraphy combined in correlation of the
Oxfordian (late Jurassic) formations of the Swiss Jura range. Eclogae Geologicae Helvetiae, 79(2),
385–454. 20. Page 17/32 Gygi, R., & Persoz, F. (1986). Mineralogy, litho- and biostratigraphy combined in correlation of the
Oxfordian (late Jurassic) formations of the Swiss Jura range. Eclogae Geologicae Helvetiae, 79(2),
385–454. 21. Hantzpergue, P. (1989). Les ammonites kimmeridgiénnes du haut-fond d’Europe occidentale:
biochronologie, systématique, evolution, paléobiogéographie (pp. 1–425). Centre National de la
Recherche Scientifique. Paris. 21. Hantzpergue, P. (1989). Les ammonites kimmeridgiénnes du haut-fond d’Europe occidentale:
biochronologie, systématique, evolution, paléobiogéographie (pp. 1–425). Centre National de la
Recherche Scientifique. Paris. 22. Hantzpergue, P. (1995). Faunal trends and sea-level changes: biostratigraphic patterns of
Kimmeridgian ammonites on the Western European Shelf. Geologische Rundschau, 84, 245–254. 23. Hantzpergue, P., Atrops, F., & Enay, R. (1997). Kimméridgien. In E.Cariou & P. Hantzpergue (Coord.):
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of NW Switzerland (Swiss Jura): stratigraphical and palaeogeographical implications in the
transition area between the Paris Basin and the Tethys. Sedimentary Geology, 186, 237–263. 25. Kaźmierczak, J., & Pszczółkowski, A. (1968). Sedimentary discontinuities in the Lower Kimmeridgian
of the Holy Cross Mts. Acta Geologica Polonica, 18(3), 587–612. 25. Kaźmierczak, J., & Pszczółkowski, A. (1968). Sedimentary discontinuities in the Lower Kimmeridgian
of the Holy Cross Mts. Acta Geologica Polonica, 18(3), 587–612. 26. Książkiewicz, M., & Samsonowicz, J. (1953). Zarys geologii Polski. Państwowe Wydawnictwa
Naukowe. Warszawa. 26. Książkiewicz, M., & Samsonowicz, J. (1953). Zarys geologii Polski. Państwowe Wydawnictwa
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Polonica, 44(3–4), 167–221. 29. Kutek, J., & Głazek, J. (1972). The Holy Cross area, central Poland in the Alpine cycle. Acta Geologica
Polonica, 22(4), 603–652. 29. Kutek, J., & Głazek, J. (1972). The Holy Cross area, central Poland in the Alpine cycle. Acta Geologica
Polonica, 22(4), 603–652. Page 19/32 30. Machalski, M. (1993). Ławice ostrygowe kimerydu obrzeżenia Gór Świętokrzyskich. Unpublished
Ph.D Thesis (pp. 1-215), Institute of Paleobiology, Polish Academy of Sciences. 31. Marques, B., & Olóriz, F. (1992). Page 17/32 The Orthaspidoceras uhlandi (Oppel) record and the maximum
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Kimmeridgian: new data from northern Poland. GeoResearch Forum, 6, 129–136. 34. Matyja, B. A., Wierzbowski, A., Radwańska, U., & Radwański, A. (2006). Małogoszcz, large quarry of
cement works (Lower and lowermost Upper Kimmeridgian). In A. Wierzbowski, et al. (Eds.), Jurassic
of Poland and adjacent Slovakian Carpathians, Field trip guidebook of 7th International Congress on
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containing the ammonite Ataxioceras from the eastern Iberian Range, northeastern Spain. GFF, 131,
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(Zona Subbetica). Paleontologia. Biostratigrafia. Tesis Doctorales de la Universidad de Granada,
184, tomo I (pp 1-758), tomo 2 (atlas). 39. Pavia, G., Benetti, A., & Minetti, C. (1987). Il Rosso Ammonitico dei Monti Lessini Veronesi (Italia NE). Fauna ad ammoniti e discontinuità stratigrafische nel Kimmeridgiano inferiore. Bolletino della
Società Paleontologica Italiana, 26(1–2), 63–92. 39. Pavia, G., Benetti, A., & Minetti, C. (1987). Il Rosso Ammonitico dei Monti Lessini Veronesi (Italia NE)
Fauna ad ammoniti e discontinuità stratigrafische nel Kimmeridgiano inferiore. Bolletino della
Società Paleontologica Italiana, 26(1–2), 63–92. 40. Rogov, M. A., Wierzbowski, A., & Shchepetova, E. (2017). Ammonite assemblages in the Lower to
Upper Kimmeridgian boundary interval (Cymodoce to Mutabilis zones) of Tatarstan (central
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Museo di Storia Naturale di Verona (II serie), Sezione Scienze della Terra, 5, 1-145. 41. Sarti, C. (1993). Il Kimmeridgiano delle Prealpi Veneto-Trentine: fauna e biostratigrafia. Memoire del
Museo di Storia Naturale di Verona (II serie), Sezione Scienze della Terra, 5, 1-145. Page 20/32 Page 20/32 42. Sapunov, I. G. (1979). Les Fossiles de Bulgarie. III.3. Jurassique supérieur. Ammonoidea (pp. 1-237). Academie Bulgare des Sciences. Sofia, 1979. 43. Schweigert, G. (1997). Die Ammonitengattungen Simocosmoceras Spath and Pseudhimalayites
Spath (Aspidoceratidae) im sȕddeutschen Oberjura. Stuttgarter Beiträge zur Naturkunde Serie B
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palaeogeography and climate-controlled cycles. Volumina Jurassica, 15, 41–120. 10.5604/01.3001.0010.5659). 47. Wierzbowski, A. (2020). The Kimmeridgian of the south-western margin of the Holy Cross Mts.,
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and palaeogeographical significance. Volumina Jurassica, 18(1), 1–22. 10.7306/VJ.18.1). 50. Wierzbowski, A., Smoleń, J., & Iwańczuk, J. (2015). The Oxfordian and Lower Kimmeridgian of the
Peri-Baltic Syneclise (north-eastern Poland: stratigraphy, ammonites, microfossils (foraminifers,
radiolarians), facies and palaeogeographical implications. Neues Jahrbuch fȕr Geologie und
Paläontologie, 277(1), 63–104. 10.1127/njgpa/2015/0496). 51. Wierzbowski, H. (2019). Palaeoenvironmental changes recorded in oxygen and carbon isotope
composition of Kimmeridgian (Upper Jurassic) carbonates from central Poland. Geological
Quarterly, 63(2), 259–274. http://dx.doi.org/10.7306/gq.1471). 51. Wierzbowski, H. (2019). Palaeoenvironmental changes recorded in oxygen and carbon isotope
composition of Kimmeridgian (Upper Jurassic) carbonates from central Poland. Geological
Quarterly, 63(2), 259–274. http://dx.doi.org/10.7306/gq.1471). 52. Ziegler, B. (1979). Page 17/32 Idoceras und verwandte Ammoniten_Gattungen im Oberjura Schwabens. Eclogae
Geologicae Helvetiae, 52(1), 19–56. 52. Ziegler, B. (1979). Idoceras und verwandte Ammoniten_Gattungen im Oberjura Schwabens. Eclogae
Geologicae Helvetiae, 52(1), 19–56. 53. Ziegler, B. (1962). Die Ammoniten Gattung Aulacostephanus im Oberjura (Taxonomie, Stratigraphie,
Biologie). Palaeontographica, 119A, 1–172. 53. Ziegler, B. (1962). Die Ammoniten Gattung Aulacostephanus im Oberjura (Taxonomie, Stratigraphie,
Biologie). Palaeontographica, 119A, 1–172. Figures Figures Page 21/32 Figure 1 Geological map of the Holy Cross Mountains (after Samsonowicz in Książkiewicz & Samsonowicz, 1953,
somewhat modified) showing the position of the Małogoszcz cement-work quarry section. Geological map of the Holy Cross Mountains (after Samsonowicz in Książkiewicz & Samsonowicz, 1953,
somewhat modified) showing the position of the Małogoszcz cement-work quarry section. Geological map of the Holy Cross Mountains (after Samsonowicz in Książkiewicz & Samsonowicz, 1953,
somewhat modified) showing the position of the Małogoszcz cement-work quarry section. Page 22/32 igure 2
Geological section of the discussed deposits of the uppermost Lower Kimmer
ection (after Matyja et al., 2006) showing position of the distinguished litholo
hronostratigraphical interpretation, and changing patterns of ammonite distri
he succession. Figure 2 Figure 2 Geological section of the discussed deposits of the uppermost Lower Kimmeridgian in the Małogoszcz
section (after Matyja et al., 2006) showing position of the distinguished lithological units A-E, their
chronostratigraphical interpretation, and changing patterns of ammonite distribution at the family level in
the succession. Geological section of the discussed deposits of the uppermost Lower Kimmeridgian in the Małogoszcz
section (after Matyja et al., 2006) showing position of the distinguished lithological units A-E, their
chronostratigraphical interpretation, and changing patterns of ammonite distribution at the family level in
the succession. Page 23/32 Page 23/32 Figure 3
Ammonites Ataxioceras and Garnierisphinctes from the upper Hypselocyclum Zone (
to lower Divisum Zone (Crusoliensis Subzone). Figure 3 Ammonites Ataxioceras and Garnierisphinctes from the upper Hypselocyclum Zone (Lothari Subzone)
to lower Divisum Zone (Crusoliensis Subzone). Ammonites Ataxioceras and Garnierisphinctes from the upper Hypselocyclum Zone (Lothari Subzone)
to lower Divisum Zone (Crusoliensis Subzone). Ammonites Ataxioceras and Garnierisphinctes from the upper Hypselocyclum Zone (Lothari Subzone)
to lower Divisum Zone (Crusoliensis Subzone). a – Ataxioceras (Parataxioceras) oppeli parvum Atrops, microconch; ZI/100/41; unit A. a – Ataxioceras (Parataxioceras) oppeli parvum Atrops, microconch; ZI/100/41; unit A. Page 24/32 b – Ataxioceras (Parataxioceras) cf. planulatum (Quenstedt); microconch; ZI/100/42; un
c – “Orthosphinctes (Ardescia)” perayensis Atrops, microconch; ZI/100/44; unit A. d – Garnierisphinctes melliconense (Geyer), ? microconch; ZI/100/45; unit A. e – Garnierisphinctes cf. garnieri (Fontannes), ? microconch; ZI/100/85; unit B. Phragmocone/body chamber boundary is arrowed. Page 25/32
Phragmocone/body chamber boundary is arrowed. Page 25/32 a – Rasenia involuta Spath; macroconch; ZI/100/74; unit B. Figure 4 Ammonites Crussoliceras and Garnierisphinctes from the upper Hypselocyclum Zone (Lothari Subzone)
to lower Divisum Zone (Crusoliensis Subzone). Ammonites Crussoliceras and Garnierisphinctes from the upper Hypselocyclum Zone (Lothari Subzone)
to lower Divisum Zone (Crusoliensis Subzone). a – Crussoliceras sayni (Camus et Thieuloy), phragmocone, ? macroconch; ZI/10046; unit A. y
(
y), p
g
,
;
;
b – Garnierisphinctes garnieri (Fontannes), microconch; ZI/100/56 (imprint from original specimen); unit
A. c-d – Crussoliceras cf. aceroides Geyer; fragments of body-chamber with a part of phragmocone (B);
ZI/100/87,88; unit B. Page 26/32 Figure 5 Figure 5
Ammonites Progeronia from the upper part of the Hypselocyclum Zone (Lothari Subzone) and the upper
part of the Divisum Zone (Uhlandi Subzone). a – Progeronia (Progeronia) progeron (von Ammon), macroconch; ZI/100/52; unit A. Figure 5 Ammonites Progeronia from the upper part of the Hypselocyclum Zone (Lothari Subzone) and the upper
part of the Divisum Zone (Uhlandi Subzone). Ammonites Progeronia from the upper part of the Hypselocyclum Zone (Lothari Subzone) and the upper
part of the Divisum Zone (Uhlandi Subzone). a – Progeronia (Progeronia) progeron (von Ammon), macroconch; ZI/100/52; unit A. a – Progeronia (Progeronia) progeron (von Ammon), macroconch; ZI/100/52; unit A. Page 27/32 b – Progeronia (Hugueninsphinctes) breviceps (Quenstedt), microconch; ZI/100/53; unit A. c - Progeronia (Hugueninsphinctes) breviceps (Quenstedt), microconch; ZI/100/100; unit C. d – Progeronia (Progeronia) cf. eggeri (von Ammon); ZI/100/120; units D-E. Page 28/32
Phragmocone/body chamber boundary is arrowed. Figure 6 Figure 6 Page 28/32 a – Garnierisphinctes semigarnieri (Geyer), phragmocone with a part of the body-chamber; ZI/100/101;
unit C. b - Garnierisphinctes championneti (Fontannes), microconch; ZI/100/10; unit C. c – Garnierisphinctes plebejus (Neumayr), phragmocone, imprints of fragmentary preserved ribs of the
outer whorl belong possibly to the body-chamber; ZI/100/103; unit C. d - ? Tolvericeras sp.; ZI/100/106; unit C. Phragmocone/body chamber boundary is arrowed. Phragmocone/body chamber boundary is arrowed. Page 29/32 Figure 7
Ammonites of the families Aulacostephanidae, Perisphinctidae and Oppeliidae from
the Hypselocyclum Zone (Lothari Subzone) and the Divisum Zone Figure 7 Figure 7 Ammonites of the families Aulacostephanidae, Perisphinctidae and Oppeliidae from the upper part of
the Hypselocyclum Zone (Lothari Subzone) and the Divisum Zone a – Rasenia involuta Spath; macroconch; ZI/100/74; unit B. Page 30/32 Page 30/32 b - Rasenia involuta Spath; macroconch; ZI/100/116; unit D-E. b - Rasenia involuta Spath; macroconch; ZI/100/116; unit D-E. c-d - ? Pararasenia aff. quenstedti (Durand), macroconch, phragmocone, lateral (A) and ventral (B) views;
ZI/100/118, units D-E. c-d - ? Pararasenia aff. quenstedti (Durand), macroconch, phragmocone, lateral (A) and ventral (B) views;
ZI/100/118, units D-E. e – Nebrodites maccerimus (Quenstedt), microconch; ZI/100/51; unit A. e – Nebrodites maccerimus (Quenstedt), microconch; ZI/100/51; unit A. e – Nebrodites maccerimus (Quenstedt), microconch; ZI/100/51; unit A. f – Nebrodites favaraensis (Gemmellaro), body chamber; ZI/100/98; unit C. f – Nebrodites favaraensis (Gemmellaro), body chamber; ZI/100/98; unit C. g-h-i – Presimoceras ex gr. P. herbichi (? P.nodulatum (Quenstedt); body chamber, lateral (A and B) and
ventral view (C); ZI/100/82,83; unit B. g-h-i – Presimoceras ex gr. P. herbichi (? P.nodulatum (Quenstedt); body chamber, lateral (A and B) and
ventral view (C); ZI/100/82,83; unit B. g-h-i – Presimoceras ex gr. P. herbichi (? P.nodulatum (Quenstedt); body chamber, lateral (A and B) and
ventral view (C); ZI/100/82,83; unit B. j – Nebrodites cf. agrigentinus (Gemmellaro), phragmocone with a part of the body-chamber; ZI/100/99,
unit C. j – Nebrodites cf. agrigentinus (Gemmellaro), phragmocone with a part of the body-chamber; ZI/100/99,
unit C. k – Idoceras balderum (Oppel); outer whorl is a body chamber; ZI/100/81 (imprint from original
specimen); unit B. k – Idoceras balderum (Oppel); outer whorl is a body chamber; ZI/100/81 (imprint from original
specimen); unit B. l – Taramelliceras (Taramelliceras) compsum (Oppel), phragmocone; ZI/100/111; units D-E (?E). Phragmocone/body chamber boundary is arrowed. l – Taramelliceras (Taramelliceras) compsum (Oppel), phragmocone; ZI/100/111; units D-E (?E). l – Taramelliceras (Taramelliceras) compsum (Oppel), phragmocone; ZI/100/111; units D-E (?E). Phragmocone/body chamber boundary is arrowed. l – Taramelliceras (Taramelliceras) compsum (Oppel), phragmocone; ZI/100/111; units D-E (?E). Phragmocone/body chamber boundary is arrowed. Page 31/32 Figure 8
Phylogenetical development and palaeobiogeographical affinity of ammonites from the uppermo
Lower Kimmeridgian of the Małogoszcz section. Figure 8 Figure 8 Phylogenetical development and palaeobiogeographical affinity of ammonites from the uppermost
Lower Kimmeridgian of the Małogoszcz section. Page 32/32
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Olaf Scholz/Ulrich Becker (Hrsg.) Die Auswirkungen der Rechtsprechung
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Unterstützung durch einen intensiven Diskurs mit Wissenschaftlern, Politikern und
Verbänden. Unser herzlicher Dank gilt allen, die an der Diskussion über "Die Auswirkungen der
Rechtsprechung des Europäischen Gerichtshofes auf das Arbeitsrecht der Mitgliedstaa-
ten" mitgewirkt haben – auf dem Symposium ebenso wie in diesem Buch. Der Ta-
gungsband soll einen Beitrag zur weiteren Diskussion über die Zukunft des sozialen
Europa im Wechselspiel zwischen Politik und Rechtsprechung leisten, die wir unter
Einbeziehung aller Betroffenen, der gesellschaftlichen Interessengruppen und vor allem
der Sozialpartner, führen müssen. Olaf Scholz
Ulrich Becker im Mai 2009 5 5 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb 9783845219165, am 24.10.2024, 06:46:57
- https://www.nomos-elibrary.de/agb Inhaltsverzeichnis Inhaltsverzeichnis
I. Analyse der aktuellen Rechtsprechung des EuGH
1. Aus wissenschaftlicher Sicht
Europäisches Marktrecht vs. soziales Europa
Antoine Lyon-Caen
9
Die aktuelle Rechtsprechung des EuGH vor dem Hintergrund des
Verhältnisses von Grundfreiheiten und Gemeinschaftsgrundrechten
Robert Rebhahn
17
Die Auslegung der Entsenderichtlinie durch den EuGH in den Ent-
scheidungen Laval und Rüffert
Juliane Kokott
25
Diskussionsbericht
33
2. Aus richterlicher Sicht
Das Europäische Sozialrecht und die Rechtsprechung des EuGH
Egils Levits
37
Das soziale Europa aus der Perspektive der deutschen Gerichtsbar-
keit
Ulrich Koch
45
Diskussionsbericht
57
II. Spielräume für die künftige Entwicklung
1. Auf europäischer Ebene
Scope of Action at the European Level
Brian Bercusson
61
Scope of Action at the European and National Level from the
Perspective of the European Commission
Armindo Silva
91
Diskussionsbericht
97
7
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htt
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b 1 EuGH vom 11. Dezember 2007, Viking C-488/05, Slg. 2007, I-10779; vom 18. Dezember 2007,
Laval, C-341/05, Slg. 2007, I-11767; vom 3. April 2008, Rüffert, C-346/06. Europäisches Marktrecht vs. soziales Europa Antoine Lyon-Caen 9
9
10
11
11
12
13
14
14
15 I. Einleitung
1. Zwischen Überraschung und vorhersehbarer Entscheidung
2. Ausblick
II. Die Trennung von Marktrecht und Arbeitsrecht
1. Die Gleichstellung des Arbeitsrechts
2. Die Trennung als Bestandteil der demokratischen Verfassung der
Europäischen Staaten
3. Die demokratische Verfassung der Mitgliedsstaaten als Säule des
Europäischen Einigungswerkes
III. Der Beginn einer gefährlichen Beziehung
1. Die Tendenz in Richtung einer neuen Unterordnung
2. Die Gefahren der Abhängigkeit Einleitung
1. Zwischen Überraschung und vorhersehbarer Entscheidung 1. Zwischen Überraschung und vorhersehbarer Entscheidung
2. Ausblick II. Die Trennung von Marktrecht und Arbeitsrecht g
1. Die Gleichstellung des Arbeitsrechts 2. Die Trennung als Bestandteil der demokratischen Verfassung der
Europäischen Staaten 3. Die demokratische Verfassung der Mitgliedsstaaten als Säule des
Europäischen Einigungswerkes III. Der Beginn einer gefährlichen Beziehung 1. Die Tendenz in Richtung einer neuen Unterordnung 2. Die Gefahren der Abhängigkeit 1. Aus wissenschaftlicher Sicht 33 37 57 61 2. Auf nationaler Ebene 101 105 III. Podiums- und Plenumsdiskussion
127
Autorenverzeichnis
141
Verzeichnis der Moderatoren und Diskussionsteilnehmer
143
Anhänge:
Entschließung des Europäischen Parlaments vom 22. Oktober 2008 zu den
Herausforderungen für Tarifverträge in der EU (2008/2085(INI))
Jan Andersson
145
The recent jurisprudence of the ECJ viewed against the background of the
relation between Fundamental Freedoms and Community Fundamental
Rights
Robert Rebhahn
157
The ECJ’s Interpretation of the Posting Directive in the Laval and Rüffert
Judgements
Juliane Kokott
165 157 165 8 8 1. Zwischen Überraschung und vorhersehbarer Entscheidung Es ist nicht sicher, dass jeder in ihnen das Gleiche sucht oder findet. Aber eines ist
sicher: die drei Urteile des Europäischen Gerichtshofes, die unter den Namen Viking,
Laval und Rüffert berühmt geworden sind1, lösen auch über die Fachkreise der Juristen
hinaus ein reges Interesse aus. Diese Urteile behandeln die schwierigen Beziehungen
zwischen der Niederlassungs- und Dienstleistungsfreiheit einerseits und dem Arbeits-
recht andererseits. Aus praktischen Erwägungen und insbesondere, um sich der Fachsprache zu bedie-
nen, wird vorgeschlagen vom Marktrecht zu sprechen. Dessen Komponenten sind, ne-
ben anderen Grundfreiheiten, die Niederlassungsfreiheit und die Dienstleistungsfreiheit
sowie das Wettbewerbsrecht; zusammen gefasst schaffen diese so bezeichneten Rege- 9 /10.5771/
– Antoine Lyon-Caen lungen einen Markt mit dem Ziel, einen funktionierenden Wettbewerb zu gewährleisten. Das Kernstück dieser Urteile bildet die Kollision des Marktrechtes, als Teil des europäi-
schen Einigungswerkes, mit dem Arbeitsrecht, das nationale Wurzeln hat. lungen einen Markt mit dem Ziel, einen funktionierenden Wettbewerb zu gewährleisten. Das Kernstück dieser Urteile bildet die Kollision des Marktrechtes, als Teil des europäi-
schen Einigungswerkes, mit dem Arbeitsrecht, das nationale Wurzeln hat. Schon früher war der Gerichtshof in Luxemburg Schauplatz einer solchen Kollisi-
on.2 Er legte sie jedoch nicht offen dar. Er hat den analytischen Rahmen festgelegt und
die Denkweisen aufgezeigt, um die Modalitäten für eine solche Kollision festzulegen. Mit einem Geschick, um das andere ihn beneiden würden, scheint er in den Urteilen
Viking, Laval und Rüffert das vorher zum Ausdruck gebrachte, wieder aufgenommen
und fortgesetzt zu haben. Kurz gesagt, die Kontinuität ist gewahrt, jede Überraschung
ausgeschlossen, sofern sie nicht gespielt ist. Dieser erste Eindruck ist jedoch nicht gerechtfertigt, denn die Kollision, die er in den
jüngsten Fällen zu beurteilen hat, weist neuartige Züge auf. Was der Gerichtshof glaubt,
dem europäischen Marktrecht – mit seinen Komponenten Niederlassungs- und Dienst-
leistungsfreiheit – gegenüberstellen zu können, ist das Rechtssystem, d.h. das nationale
Rechtssystem der industriellen Beziehungen. Es wird vorgeschlagen, dieses als „Recht
der industriellen Beziehungen“ zu bezeichnen. Der Begriff industrielle Beziehungen
wird verwendet, um die gesamten Beziehungen zu bezeichnen, d.h. Konfrontation, Ver-
handlungen oder Zusammenarbeit, die sich zwischen Arbeitnehmern und ihren Organi-
sationen sowie den Arbeitgebern und deren zuständigen Organisationen und teilweise
auch mit den staatlichen Behörden entwickeln. 2 S. insbesondere EuGH vom 5. Juni 1997, SETTG, C-398/95, Slg. 1997, I-3091, worin der Gerichts-
hof unvorsichtig erklärte, dass “die Wahrung des sozialen Friedens als Mittel, um einen Tarifkon-
flikt zu beenden keinen Grund im Allgemeininteresse darstelle, der eine Einschränkung eines
Grundrechts rechtfertige, das durch den Vertrag garantiert sei" (in diesem Falle handelte es sich um
die Dienstleistungsfreiheit). 1. Zwischen Überraschung und vorhersehbarer Entscheidung Hinsichtlich der Regelungen zur Über-
wachung und Gestaltung dieser Beziehungen braucht wohl kaum daran erinnert zu wer-
den, dass sie unterschiedlichsten Ursprungs sind: es handelt sich weder zwangsläufig
um Heteronomie noch unbedingt um Autonomie, die Kombinationen sind je nach den
nationalen, sektoriellen und lokalen Erfahrungen vielfältig und unterschiedlich. Die Situation bei dieser Kollision ist ganz neu, denn was der Gerichtshof in seinen
jüngsten Urteilen anspricht, ist das Rechtssystem der industriellen Beziehungen, auf das
er bisher nicht offen eingegangen ist. 2. Ausblick Durch die neuartigen Züge der Kollision entstehen bestimmte Anforderungen. Die
erste betrifft die Art und Weise wie diese Urteile zu lesen und auszulegen sind. Es ist
sicherlich erforderlich den Aufbau, den Zusammenhang und das rechtliche Regelwerk
zu verstehen. Aber man muss sich auch bemühen zu verstehen, was der Gerichtshof tut, 10 /10.5771/
– Europäisches Marktrecht vs. Soziales Europa wenn er zum Verhältnis zwischen dem Recht der industriellen Beziehungen und dem
Marktrecht Stellung bezieht. Die hier vorgebrachte These hebt an erster Stelle hervor, was der Gerichtshof nicht
für wesentlich erachtet, auf jeden Fall nicht (durch ihn) für unantastbar hält: die Tren-
nung zwischen dem Recht der industriellen Beziehungen und dem Marktrecht. Wir ha-
ben den Eindruck, dass die Tragweite und Bedeutung dieser Urteile klarer zum Aus-
druck kommen, wenn man die Bedeutung dieser Trennung unterstreicht. 1. Die Gleichstellung des Arbeitsrechts In den westeuropäischen Gesellschaften hat sich das Arbeitsrecht durch die Gleich-
stellung gegenüber dem Marktrecht herausgebildet, das mit unterschiedlichen Begriffen
bezeichnet wurde, die nicht in Vergessenheit geraten dürfen: hier Handelsfreiheit woan-
ders freedom of trade. Es ist nicht so, als hätte es vor dieser Gleichstellung keine Rege-
lungen im Bereich der Arbeit gegeben, aber sie fielen eher in den Zuständigkeitsbereich
einer Arbeitspolizei, ein mehr oder weniger unabhängiger Teil der Polizei mit Zustän-
digkeiten für die Märkte. Wie hat sich diese Gleichstellung in großen Zügen abgespielt? Sie erfolgte aufgrund der institutionellen Neuerungen, die einen Bruch mit den Prinzi-
pien und der dem Wettbewerbsmarkt eigenen Rationalität bedeuten; aus diesem Grund
aber auch wegen der beispielhaften Rolle, die sie im Rahmen der Gleichstellung ge-
spielt haben, bilden sie immer noch die historischen Säulen des Arbeitsrechts. Zwei Neuerungen gilt es auf jeden Fall zu erwähnen. Die Erste ist die Schaffung oder
Anerkennung eines Freiraums für kollektive Maßnahmen der Arbeitnehmer. Die Wege,
über die diese Anerkennung erreicht wurde, sind vielfältig. Dazu zählt zweifellos auch
der Rückgang von Strafverfahren im Zusammenhang mit Bündnissen und Zusammen-
schlüssen von Arbeitnehmern. Jedoch wurde in einigen europäischen Ländern Straffrei-
heit bei kollektiven Maßnahmen im Zusammenhang mit Schadensersatzklagen einge-
führt. Im Allgemeinen fand an anderer Stelle etwas später, die Proklamation der Grund-
rechte statt, insbesondere des Streikrechts, als verfassungsmäßiges Recht mit allen Ga-
rantien, die dieses Recht begleiten; was offensichtlich der Ausdruck diese Gleichstel-
lung ist. Eine zweite institutionelle Neuerung muss ebenfalls angesprochen werden und zwar:
die Einführung von Verfahren oder Mechanismen zur Festlegung eines üblichen Tarifs
oder Lohns, der tendenziell für alle Arbeitnehmer Anwendung finden sollte. Diese
grundlegende Regelung erfolgte in einigen Fällen als Fortsetzung der vorausgegangenen
institutionellen Neuerung. Dies trifft dort zu, wo der Tarifvertrag der Weg zur Einfüh-
rung einer üblichen Entlohnung ist. Aber die Tarifverhandlung ist ganz und gar nicht 11 11 Antoine Lyon-Caen der alleinige Weg dorthin; insbesondere dann nicht, wenn für bestimmte Vorhaben oder
gewisse Gruppen, die von jeglichen Tarifverhandlungen ausgeschlossen sind, eine übli-
che Entlohnung vereinbart werden muss. Aus dieser Situation heraus entstand Ende des
19. Jahrhunderts in Frankreich das Verfahren, das der Gerichtshof in der Rechtssache
Rüffert kritisiert hat. 1. Die Gleichstellung des Arbeitsrechts Der Zwiespalt, der sich bei der Einhaltung einer üblichen Entloh-
nung für die Zuschlagsempfänger eines öffentlichen Auftrags ergibt, wurde damals
schon erkannt und betont: es geht darum, einen fairen Wettbewerb durchzusetzen, des-
sen Anwendungsbereich eingegrenzt ist und bei dem vermieden wird, dass sich die Ar-
beits- und Entlohnungsbedingungen verschlechtern; aus rechtsgrundsätzlichen Erwä-
gungen geht es auch darum, die Würde der Arbeitnehmer zu wahren. g
g
g
Es ist nicht nötig, hier noch weiter auszuholen: die Gleichstellung gegenüber dem
Marktrecht ist erfolgt, zu Gunsten des Arbeitsrechts, der Zulassung von Kollektivmaß-
nahmen und der Einführung einer üblichen Entlohnung – die häufig zum Mindestlohn
geworden ist –, das Eine oder Andere entspricht nicht dem Prinzip der individualisti-
schen Optimierung oder dem optimierenden Individualismus. 3 EuGeI vom 17. Juni 1998, CGPME, T-135/96, Slg. 1998, II-2335 Rdnr. 89. 2. Die Trennung als Bestandteil der demokratischen Verfassung der Europäischen
Staaten In den westeuropäischen Gesellschaften hat die Trennung von Arbeitsrecht und
Marktrecht, die bedingt ist durch Gleichstellung des Arbeitsrechts, eine tief greifende
Bedeutung. Man kann sich logischer Weise vorstellen, dass der Aufschwung des Markt-
rechtes, das eine besondere Rationalität aufweist, durch einen Differenzierungseffekt
möglich war. Aber der eigentliche Sinn dieser Trennung soll hier erläutert werden. Sie
ist tatsächlich der Grundpfeiler der demokratischen Ordnung der westeuropäischen Ge-
sellschaften. Um dies nachzuvollziehen, muss man sich das besondere Statut der Akteu-
re, der Maßnahmen und Tätigkeiten, die unter das Arbeitsrecht fallen genauer ansehen. Der Arbeitnehmer als Person genießt einen Schutz, der keinem anderen Vertragspartner
zuteil wird. Und dieser Schutz ist Bestandteil der demokratischen Ansprüche der euro-
päischen Staaten. In den meisten Staaten ist die gewerkschaftliche Handlungsfreiheit,
das Recht auf Tarifverhandlungen durch die Verfassung fest garantiert. Und seither sind
die Tarifverhandlungen und die Kollektivmaßnahmen fest im Grundrecht der Vereini-
gungsfreiheit verankert, so wie es in der Europäischen Konvention zum Schutz der
Menschenrechte in Artikel 11 proklamiert und garantiert wird. All diese Aussagen wür-
den an Banalität grenzen, wenn man nicht auf den daraus resultierenden Schutz für die
Arbeitnehmer, für ihre Organisationen und Aktionen vor der Übermacht der Marktrege-
lungen hinweisen würde. Anders gesagt: der verfassungsmäßige Schutz des Arbeitneh-
mers als Person und das fundamentale Recht auf Organisationsfreiheit und auf Kollek-
tivmaßnahmen haben logischerweise zur Folge – oder finden ihre notwendige Fortset- 12 Europäisches Marktrecht vs. Soziales Europa zung dadurch –, dass die Handlungen und Aktionen der Arbeitnehmer und ihrer Organi-
sationen gegenüber dem Einfluss der Marktregelungen abgeschirmt sind. Diese Äußerung kann durch ihre Bestimmtheit eine skeptische Reaktion auslösen. Jedoch hat die Trennung im verfassungsmäßigen Sinne einen grundlegenden Charakter. Dadurch wird nicht vermieden, dass die Maßnahmen und Aktionen der Arbeitnehmer
und ihrer Organisationen beobachtet und begrenzt werden, aber die Bedingungen und
Beschränkungen erfolgen durch speziell für diesen Zweck ausgearbeitete Regelungen. Dadurch wird auch nicht verhindert, dass die Tarifverträge und Lohnvereinbarungen in
unterschiedlichem Umfang die Wettbewerbsbedingungen auf dem Markt berücksichti-
gen. Aber die Tatsache, dass die sozialen Protagonisten das Wettbewerbsumfeld be-
rücksichtigen, bedeutet keinesfalls, dass sie sich den Regeln des Marktes unterwerfen. 4 EuGH vom 21. September 1999, Albany, C67/96, Slg. 1999, I-5751; vom 21. September 2000, Van
der Wonde, C-222/98, Slg. 2000, I-7111. 3. Die demokratische Verfassung der Mitgliedsstaaten als Säule des Europäischen
Einigungswerkes 3. Die demokratische Verfassung der Mitgliedsstaaten als Säule des Europäischen
Einigungswerkes Es soll hier nicht darum gehen, erneut die Beziehungen zwischen der Verfassung der
Mitgliedsstaaten und der Verfassung der Union, man könnte sogar sagen - seiner politi-
schen Verfassung - zu erörtern. Es soll nur daran erinnert werden, dass die Trennung
von Arbeitsrecht und Marktrecht als Bestandteil der demokratischen Verfassung der
Staaten ein Teilstück des europäischen Einigungswerkes ist; davon sollte das Gericht
nicht abweichen können. Im Übrigen würdigt das Gemeinschaftsrecht bezeichnender-
weise im Namen seines demokratischen Anspruchs, die normative Tätigkeit der Sozial-
partner – oder das Recht der industriellen Beziehungen – so wie es von den Mitglieds-
staaten gestaltet wurde. Diese Wertschätzung wird nicht nachdrücklich betont, und es
bedarf einer konstruktiven Interpretation, um aus der Achtung der Grundrechte gemäß
den gemeinsamen verfassungsmäßigen Traditionen (Art. 6 EU) zu schließen, dass die
im Verfassungsrecht der Mitgliedsstaaten verankerte Trennung von Arbeitsrecht und
Marktrecht für die Institutionen der Gemeinschaft verbindlich ist. Aber das erstinstanz-
liche Gericht hat den Weg aufgezeigt; es hat nicht gezögert aufgrund des demokrati-
schen Prinzips, auf dem die Europäische Union beruht, zu folgern, dass wenn keine par-
lamentarische Intervention erfolgt, die Beteiligung des Volkes an der Ausarbeitung ei-
ner Rechtsakte über die Sozialpartner stattfindet.3 Vom Artikel 137 des Vertrages geht
eine doppelte Botschaft aus. Eine eindeutige Aussage, wenn er die Entlohnung und ins-
besondere die Vereinigungsfreiheit und das Recht auf Kollektivmaßnahmen außerhalb
des Aktionsradius der Gemeinschaft ansiedelt. Diese erste Aussage könnte ausreichen,
um einer Institution der Gemeinschaft jegliche Befugnis abzusprechen, das Recht und
die nationalen Praktiken auf diesen Gebieten zu ändern. Aber der Text hat noch einen
anderen Sinn. Denn die Entlohnungen und die Art ihrer Festlegung, sowie die Regelun- 13 /10.5771/9
– Antoine Lyon-Caen gen für die Organisation und für die Kollektivmaßnahmen sind die wesentlichen Be-
standteile, welche entsprechend der demokratischen Verfassung der Mitgliedsstaaten,
vom Marktrecht ausgenommen sind. Der Artikel 137 ist sozusagen das Markenzeichen
einer Verpflichtung der Gemeinschaft, die darin besteht, die Gleichstellung des Arbeits-
rechts zu respektieren. Und dennoch… 1. Die Tendenz in Richtung einer neuen Unterordnung Durch die drei jüngsten Urteile zeichnet sich eine neue Beziehung zwischen Markt-
recht und Arbeitsrecht ab und, was noch viel besorgniserregender ist, dies betrifft be-
sonders die industriellen Beziehungen. Die Trennung bewegt sich in Richtung Unter-
ordnung. Wie manifestiert sich diese Unterordnung? Die Regelungen, die den Markt bestimmen, d. h. die Niederlassungsfreiheit und die
Dienstleistungsfreiheit haben, gemäß dem Diskurs des Gerichtshofes, den Status eines
Prinzips. Der Begriff entfaltet nun seine ihm inhärente dreifache Bedeutung. Das heißt
seine Stärke. Es handelt sich in erster Linie um Regelungen, die mit dem umfangreichs-
ten Geltungsbereich ausgestattet sind. Nichts oder fast nichts entgeht ihnen. Vorbei ist
es nun mit der ermutigenden Vorsicht, die der Gerichtshof bei den Verbotsregelungen
für Absprachen walten ließ. Er hatte nämlich beschlossen, dass die Sozialpartner diesen
nicht unterliegen, da anderenfalls die Ziele der Sozialpolitik, die ihre Absprachen
rechtmäßig verfolgen, gefährdet werden.4 Folgt man den Urteilen Laval, Viking und
Rüffert, so trifft dieser venünftige Vorbehalt nicht auf die Niederlassungs- und Dienst-
leistungsfreiheit zu, die jedoch auch auf die Organisation und das Funktionieren eines
Wettbewerbsmarktes ausgerichtet sind. Dieser Bereich ist nach dem Prinzip der Allge-
meingültigkeit gestaltet. Hinzu kommt noch der besondere Einfluss, den diese haben. Zur zweiten Bedeutung ist zu sagen, dass der Gerichtshof keine Neuerungen einführt
und seine früheren Vorschläge wieder aufgreift. Er bereichert sie jedoch, indem er die-
sen Prinzipien umfassende Effizienz verleiht und die Möglichkeit hinzufügt, dass sie
von allen geltend gemacht werden können. Im Gegenzug werden die nationalen Regelungen, die Tarifverhandlungen und Kol-
lektivmaßnahmen zulassen und garantieren sowie die Regelungen, die Lohnstandards
festlegen, in ihren Beziehungen zu den gemeinschaftlichen Marktregelungen zu Aus-
nahmen. Was nicht heißt, dass sie ihre Anwendungsbestimmung verlieren. Aber im Fal-
le eines angeblichen Widerspruchs zu den Marktregeln ist zu ihrer Aufrechterhaltung 14 https://doi.org/1
Open Access – Europäisches Marktrecht vs. Soziales Europa der Nachweis zu erbringen, dass sie gerechtfertigt sind. Diese Nachweisforderung kann
mehr oder weniger streng ausfallen; sie kann schonungslos sein oder als ein mehr oder
weniger wesentlicher Bestandteil der Verhältnismäßigkeitsprüfung berücksichtigt wer-
den. Das Wesentliche ist dennoch die neue Aufsicht, die das Markrecht gemeinschaftli-
chen Ursprungs über das Recht der industriellen Beziehungen ausübt. 2. Die Gefahren der Abhängigkeit Um die europäische Integration beim Namen zu nennen, die im Wesentlichen durch
die Grundfreiheiten erfolgt, zieht man die Idee der negativen Integration heran. Was die
negative Integration auslöst, so wie sie durch die Urteile Laval, Viking und Rüffert dar-
gestellt wurde, ist ein Prozess der Zersetzung der Fundamente des Arbeitsrechts. Und
wenn man weiß, mit welcher Kraft diese Zersetzung auf anderen Gebieten stattfand, so
ist vorherzusehen, dass der Zersetzungsvorgang beträchtlich sein könnte. Gleichzeitig
könnte dadurch das Gemeinschaftsrecht und auch das Marktrecht an Ansehen verlieren. Sein Eindringen in die industriellen Beziehungen, die in ihrer Gestaltung und ihren
Auswirkungen auf Arbeit und Entlohnung dadurch beeinträchtigt werden, lösen soziale
Proteste aus. Dies umso mehr, als seine Dynamik nur eine Einbahnstrasse ist: sie trägt
niemals dazu bei, das Ungleichgewicht zwischen den Unternehmen, deren Macht durch
die Erweiterung des Marktes gewachsen ist, und den geschwächten Arbeitnehmen zu
verringern. Aber die neue Bevormundung, die im Namen des Marktrechtes ausgeübt werden
soll, hat eine noch tiefere Bedeutung. Wenn die Trennung des Arbeitsrechts, vor allem
des Rechts der industriellen Beziehungen und des Marktrechts ein Bestandteil der de-
mokratischen Verfassung der Mitgliedsstaaten der Union ist, wenn diese demokratische
Verfassung eine Säule des europäischen Einigungswerkes ist, bzw. der politischen Ver-
fassung Europas – wie das Suggestivwort einiger Autoren lautet –, dann bedeutet das
Infragestellen dieser Trennung von Arbeitsrecht und Marktrecht, die demokratischen
Ansprüche Europas noch weiter zurückzuschrauben. 15 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb 783845219165, am 24.10.2024, 06:46:57
- https://www.nomos-elibrary.de/agb https://doi.org/10.5771/97838
Open Access –
- 1 Vgl zum Urteil Viking zB Rebhahn, Grundfreiheit vor Arbeitskampf – der Fall Viking, ZESAR
2008, 109 – 117. Die aktuelle Rechtsprechung des EuGH vor dem Hintergrund
des Verhältnisses von Grundfreiheiten
und Gemeinschaftsgrundrechten Robert Rebhahn 2 EuGH vom 12.6.2003, Schmidberger, Rs. C-112/00, Slg. 2003, I-5659. I. Einleitung Viele Mitgliedstaaten schützen Tarifautonomie und Arbeitskampf durch Grundrech-
te. Der EuGH hat Gemeinschaftsrecht bislang jedoch nie hinter ein nationales Grund-
recht zurücktreten lassen, weil dies die Einheit gefährdet. Im Gemeinschaftsrecht kön-
nen nationale Grundrechte als solche nur relevant sein, soweit sie eine dem Mitglied-
staat eröffnete Möglichkeit zur Beschränkung ausfüllen. Als Gegengewicht zu Grundfreiheiten kommen somit nur Grundrechte des Gemein-
schaftsrechts in Betracht. Die Gemeinschaft hat bislang keinen verbindlichen Grund-
rechtskatalog zustande gebracht. Der Katalog wäre längst erforderlich, weil ohne ihn als
fundamentale Werte im geschriebenen Primärrecht und in der Judikatur noch immer nur
die Grundfreiheiten im Vordergrund stehen. Dies ist in Anbetracht des enormen Zu-
wachses an Kompetenzen und Regelungen nicht mehr angemessen. Der EuGH aner-
kennt aber zumindest ungeschriebene Grundrechte als allgemeine Grundsätze. Zum
Grundrecht auf Tarifautonomie hat das Urteil Albany jedoch noch 1999 beredt nicht
Stellung genommen. Die Urteile Viking und Laval anerkennen nun ausdrücklich „das
Recht auf Durchführung einer kollektiven Maßnahme einschließlich des Streikrechts als
Grundrecht“.1 Dies ist an sich ein großer Schritt, mag auch die konkrete Ableitung 17 /10.5771/97
– Robert Rebhahn durch den EuGH rechtsdogmatisch nicht recht überzeugen. Bejaht man dieses Gemein-
schaftsgrundrecht, so wird man umso mehr jenes auf Tarifautonomie bejahen können. Allerdings spielt das Grundrecht auf kollektive Maßnahmen in der Argumentation der
Urteile und für deren Ergebnis keine erkennbare Rolle. Auch zu diesem Grundrecht gibt
es noch keine Grundrechtsdogmatik des EuGH. Der geringe Stellenwert liegt auf der
bisherigen Linie der Judikatur. III. Neue Urteile und Grundrechte Aus Viking und Laval folgt, dass jedenfalls kollektive Maßnahmen, die sich gegen
eine Standortverlagerung in einen anderen Mitgliedsstaat oder eine Entsendung richten,
an Niederlassungs- oder Dienstleistungsfreiheit zu messen sind. Die Ausübung des Ge-
meinschaftsgrundrechts kann die Gewerkschaften nicht vor der Unterwerfung unter die
Grundfreiheiten bewahren. Dann müsste die Grundrechtsausübung aber in der Folge bei
den Überlegungen berücksichtigt werden, was wie gesagt ausbleibt. Auch wenn man
das Grundrecht beiseite lässt, wird die Unterwerfung der Arbeitnehmer und Gewerk-
schaften unter die Grundfreiheit wohl nicht schlüssig begründet. Einmal wird gesagt, es
reiche aus, dass die Aktion auf eine normative Regelung – den Tarifvertrag – abzielt,
dann wird gesagt, dass es darauf nicht ankomme. Schlüssig wäre nur die These, dass
jede faktisch erhebliche Beeinträchtigung einer Grundfreiheit auch durch Private diese
beschränkt und daher nur bei Rechtfertigung zulässig ist. Es mag sein, dass diese weite
These den Urteilen zugrunde liegt – ausgesprochen wird sie aber nicht. Die Beschränkung einer Grundfreiheit ist nur rechtmäßig, wenn sie gerechtfertigt
werden kann. Sie unterliegt dem Gebot der Verhältnismäßigkeit. Bei Streiks bedeutet
dieser Maßstab für viele Mitgliedsstaaten im Süden und Norden eine grundlegende Än-
derung, im Süden auch eine Einschränkung der nationalen Grundrechte. Bei Verhält-
nismäßigkeitsprüfungen weiß man leider meist erst nach dem Urteil, wie sie ausgeht. Die Unsicherheit belastet den, der agieren will, hier die Grundrechtsträger. Eine Recht-
fertigung ist laut EuGH nicht nur für Streiks jener Arbeitnehmer denkbar, um deren
Arbeitsbedingungen es geht, sondern auch für Unterstützungsboykotte und damit für
Sympathiemaßnahmen. Auch dies ist für manche Staaten neu. Als Rechtfertigungsgrund nennen die Urteile den Schutz der Arbeitnehmer und kon-
kretisieren dies durch die Ziele der EG-Sozialpolitik. Die Grundfreiheit ist mit diesen
Zielen abzuwägen. Ein Streik kann nach Viking aber wohl nur gerechtfertigt sein, wenn
Arbeitsplätze oder Arbeitsbedingungen gefährdet oder ernstlich bedroht sind, was die
Grenze enger zieht als manch nationale Grundrechte, u.zw. wohl nicht nur beim Streik
um sozialpolitische Gesetze. Gemeinschaftsrechtlich ist es wohl ein Novum, dass der
EuGH hier primär ökonomische Interessen zur Rechtfertigung akzeptiert. Dies ersetzt
aber nicht das jedenfalls verbale Ausblenden des Grundrechts. Auch wenn Gefährdung oder Bedrohung vorliegt ist zu prüfen, ob die Maßnahme zur
Erreichung des Zieles geeignet und erforderlich ist. Die Erforderlichkeit soll fehlen,
wenn das nationale Recht andere, die Niederlassungsfreiheit weniger beschränkende
Mittel bereitstellt, um zu einem Tarifabschluss zu kommen. Daraus könnte folgen, dass
zuerst eine freiwillige Schlichtung zu beschreiten sei, was die Grenze wieder enger zöge
als nationale Grundrechte. II. Grundrechte und Grundfreiheiten im Allgemeinen Das Verhältnis von Grundfreiheiten und Gemeinschaftsgrundrechten bei Kollision ist
eher dunkel. Es lässt sich weniger durch Thesen als durch die Praxis des EuGH bestim-
men. Ein Urteil kann den Eingriff in eine Grundfreiheit oder in ein Grundrecht prüfen. Die bisherigen, wenigen Urteile zu Kollisionen betrafen die Beschränkung einer Grund-
freiheit. Das Grundrecht kam nur als deren potentielle Einschränkung in den Blick. Kaum kam der EuGH in die Lage zu prüfen, ob der Eingriff in ein Gemeinschaftsgrund-
recht seitens der Gemeinschaft oder eines Staates im Interesse einer Grundfreiheit ge-
rechtfertigt werden kann. Auch in Viking und Laval wurde nicht verhandelt, ob das
Recht zu streiken wegen der Grundfreiheit eingeschränkt werden kann, sondern das
Gegenteil. Die Fragstellung beeinflusst aber stets die Sicht, die wiederum die Aussage
zum Recht beeinflussen kann. Kommt eine Kollision vor allem in den Blick, wenn es
um die Beschränkung einer Grundfreiheit durch Grundrechtsbetätigung geht, so kann
dies leicht zur Grundrechtsausübung „nach Maßgabe“ der Grundfreiheit führen. Gleich geordnet wären Grundfreiheit und Grundrecht nur, wenn man von einer Kol-
lision primärrechtlich geschützter Interessen ausgeht, die dann entweder schon auf der
Ebene des Schutzbereiches oder auf der Ebene der Eingriffsschranken miteinander
„auszugleichen“ sind. Dies müsste das Gericht aber ausdrücklich artikulieren. Die Urtei-
le des EuGH prüfen und berücksichtigen beide Positionen bislang jedoch nicht erkenn-
bar auf derselben Ebene, sondern setzen die Grundfreiheit an den ersten Platz. Allein
das Urteil Schmidberger schien die beiden Rechte zumindest sprachlich auf gleicher
Ebene anzusiedeln.2 In Viking und Laval ist von dieser Annäherung im Rang nichts zu
bemerken. Denn das eben anerkannte Grundrecht auf kollektive Maßnahmen wird zur
Rechtfertigung der Beschränkung der Grundfreiheit nicht ins Spiel gebracht. Wohl steht
am Anfang der Überlegungen dazu, dass die „Ausübung des Rechts zu kollektiven
Maßnahmen“ ein zwingendes Allgemeininteresses sein kann. Das Urteil spricht hier
aber nicht von Grundrecht. Und es konkretisiert das Allgemeininteresse nur durch den
Schutz der Arbeitsbedingungen und die Sozialpolitik der EG. Ausgeklammert bleibt
hingegen der spezifische Gehalt von Tarifautonomie und Arbeitskampf – die kollektive
Vertretung von Interessen – , falls erforderlich durch Druck. 18 10.577
– Verhältnis von Grundfreiheiten und Gemeinschaftsgrundrechten in der aktuellen EuGH Rechtsprechung Verhältnis von Grundfreiheiten und Gemeinschaftsgrundrechten in der aktuellen EuGH Rechtsprechung Robert Rebhahn Robert Rebhahn chen Staaten wird dies erstmals wirksame Sanktionen gegen Streiks erfordern. Dass die
Akteure Grundrechtsträger sind, kommt im Urteil diesbezüglich kaum zum Ausdruck. chen Staaten wird dies erstmals wirksame Sanktionen gegen Streiks erfordern. Dass die
Akteure Grundrechtsträger sind, kommt im Urteil diesbezüglich kaum zum Ausdruck. Die Urteile Laval und Rüffert betreffen Entsendungen. Im Vordergrund steht die
Entsenderichtlinie. Dazu wird im Urteil Laval erstmals klar vertreten, dass die Richtli-
nie die Möglichkeiten des Arbeitsstaates, sein Arbeitsrecht für Entsendungen vorzu-
schreiben, abschließend regelt. Ich hielt und halte diese Auslegung wegen der in An-
spruch genommenen Binnenmarktkompetenz an sich für gut vertretbar, sie ist aber nicht
zwingend und hätte daher wohl mehr an Begründung erfordert. Fraglich ist aber jeden-
falls das Verhältnis zu den Grundrechten der Arbeitnehmer. In Laval ging es nicht um einen Streik der Entsendeten, sondern um eine Blockade
der schwedischen Arbeitnehmer gegen die Entsendung. Die Schweden sahen ihre Ar-
beitsbedingungen gefährdet. Das Urteil lässt grundsätzlich auch diesen Boykott, der
kein Sympathiearbeitskampf mehr ist, in den Schutzbereich des Grundrechts fallen. Dies ist für viele Mitgliedsstaaten ein Novum. Spätestens hier fällt auf, dass das Urteil
andere Interessen der Unternehmer als die Grundfreiheiten, die gegen einen Boykott
sprechen und die in manchen Staaten grundrechtlich geschützt sind, (ebenfalls) aus-
blendet. Auch der Interessenkonflikt der Boykottierenden mit den entsendeten Arbeit-
nehmern wird nicht ausdrücklich thematisiert. Der EuGH lässt die Zulässigkeit dieses Boykotts jedenfalls an der Entsenderichtlinie
enden. Schweden hat allerdings nicht alle Möglichkeiten dieser Richtlinie genutzt, weil
es für Entsender nicht auch jene Tarifverträge vorgeschrieben hat, die zwar nicht allge-
mein verbindlich, aber doch allgemein wirksam sind. Jedenfalls unzulässig ist es nach
dem Urteil, wenn eine nationale Friedenspflicht nur durch nationale Tarifverträge aus-
gelöst wird, weil dann der ausländische Tarifvertrag nicht gleichbehandelt wird. Ebenso
ist es jedenfalls unzulässig, wenn der Entsender die Mindestlöhne erst mit der Gewerk-
schaft verhandeln soll. Das Urteil erscheint in sich geschlossen. Näheres Hinsehen erweckt Zweifel. So be-
ruht schon die Entsenderichtlinie auf der Prämisse, dass ausländische Tarifverträge den
inländischen nicht stets gleichgestellt werden. Ferner geht das Urteil davon aus, dass
Tarifautonomie und Arbeitskampf nur nach Maßgabe der Entsenderichtlinie gewährleis-
tet sind. Man hätte aber fragen können und wohl auch müssen, ob die Entsenderichtlinie
die Gemeinschaftsgrundrechte auf kollektive Maßnahmen und Tarifautonomie ausrei-
chend beachtet, insbesondere wenn man bedenkt, dass die Gemeinschaft auch das Tarif-
system nach verbreiteter Auffassung nicht regeln darf. III. Neue Urteile und Grundrechte Viking hat überdies gesagt, dass das in seiner Grundfreiheit
verletzte Unternehmen daraus Rechte gegen die Gewerkschaft ableiten kann. In man- 19 19 Robert Rebhahn Robert Rebhahn Das Urteil erwähnt zur Rechtfer-
tigung der Beschränkung den Schutz des nationalen Tarifsystems aber nicht besonders,
sondern fährt darüber hinweg. Das Urteil Rüffert betrifft ein Tariftreuegesetz. Das in diesem Gesetz Verlangte geht
über die Entsenderichtlinie hinaus. Laut EuGH widerspricht es auch der Dienstleis-
tungsfreiheit, insbesondere weil nicht erkennbar sei, warum Entsendete bei öffentlichem
Auftraggeber eines stärkeren Schutzes bedürften. Damit wird wohl gesagt, dass diese
Unterscheidung keine sachliche Rechtfertigung sei. Mit dieser „Erwägung“ kann man 20 Verhältnis von Grundfreiheiten und Gemeinschaftsgrundrechten in der aktuellen EuGH Rechtsprechung wohl alle arbeitsrechtlichen Regelungen in Frage stellen, die nur bestimmten Personen –
etwa dem Staat – stärkere Pflichten als Arbeitgeber oder Auftraggeber auferlegen, etwa
im Antidiskriminierungsrecht. Überdies erörtert auch Rüffert nicht, ob das Tariftreuege-
setz der Tarifautonomie und damit einem Gemeinschaftsgrundrecht dient. Das BVerfG
hat hingegen 2006 zur Rechtfertigung des Tariftreuegesetzes vor der Erwerbsfreiheit
ausgeführt, das Gesetz unterstütze die Ordnungsfunktion der Tarifverträge und die
grundrechtlich geschützte Tarifautonomie. Der EuGH geht darauf nicht ein, obwohl der
Dialog mit nationalen Höchstgerichten häufig beschworen wird. Das Eingehen erübrigt
sich auch nicht, weil es dem BVerfG um den Schutz der nationalen Tarifautonomie
ging. Ein Gemeinschaftsgrundrecht auf Tarifautonomie könnte ja auch den Schutz der
jeweils national bestehenden Möglichkeiten umfassen. IV. Stellungnahme und Ausblick Aus Sicht des Arbeitsrechts fällt auf, dass die Urteile kollektive Maßnahmen und Ta-
rifvertrag nicht als Regelungsproblem und Institutionen des Arbeitslebens abhandeln,
sondern als Randerscheinungen des Binnenmarktes. Man überlegt nicht erkennbar, in-
wieweit kollektive Regelungen und Aktionen wegen eines behaupteten Ungleichge-
wichtes am Arbeitsmarkt abstrakt und konkret sinnvoll oder erforderlich sind. Man sieht
die Phänomene nur als Störfaktoren des Leistungsaustausches. Diese Sicht erscheint
verkürzt. Die Verkürzung mag erklären, dass Blockaden dem Streik um eigene Arbeits-
bedingungen einfach gleichgestellt werden, sodass andere Interessen der Arbeitgeber
neben den Grundfreiheiten ausgeblendet werden. Ausgeblendet wird auch folgender Widerspruch. Das Gemeinschaftsrecht beschränkt
nun Aktionen der Arbeitnehmer in Lagen, die erst durch den Binnenmarkt geschaffen
wurden. Die Aktionen sind oft Surrogat dafür, dass es keinen gesicherten Rechtsrahmen
für Tarifvertrag und Streik über Binnengrenzen hinweg gibt, auch weil die Gemein-
schaft Streik und vielleicht Tarifvertrag nicht regeln darf. Solange die Gemeinschaft
diese Regelung nicht leistet, sollte sie sich jedoch bei Beschränkung der nationalen
Möglichkeiten zurückhalten. Gemessen an traditionellen Maßstäben nicht nur der Rechtsdogmatik, sondern all-
gemeiner Anforderungen an Begründung, erscheinen die Ausführungen zum Verhältnis
von Grundfreiheit und Grundrecht in Viking und Laval wenig überzeugend. Aufmerk-
samkeit und Sorge des EuGH gelten nur der Grundfreiheit. Andere Aspekte werden an
den Rand gedrängt. Zugunsten der Grundfreiheit wird alles als Argument verwertet. Das
weite Verständnis der Grundfreiheit entspricht zwar dem Satz vom effet utile. Bei Ge-
meinschaftsgrundrechten fehlt hingegen ein ähnlicher Eifer zum effet utile, außer man
sieht bereits in der Anerkennung ungeschriebener Gemeinschaftsgrundrechte einen
Ausdruck dieses Auslegungstopos. Grundfreiheiten haben, jedenfalls im Effekt, bislang 21 21 Robert Rebhahn einen höheren Rang als Grundrechte. Als Hüter der Grundrechte kann man den EuGH
daher schwerlich sehen. Das ist zwar im bestehenden positiven Recht angelegt, aber
dennoch wenig erfreulich. Die Unterordnung des Grundrechts unter die Grundfreiheit zeigt sich auch, wenn
man das Problem in nationales Verfassungsrecht transponiert. Niederlassungs- und
Dienstleistungsfreiheit sind materiell Ausprägungen der Unternehmerfreiheit. Kein
normaler Staat würde bestimmte Unternehmerrechte, nur weil sie ein Binnengrenzen
überschreitendes Handeln betreffen, stets höher stellen als andere Grundrechte. Warum
sollte eine Organisation, die nicht einmal Staat sein will, dies tun? Es stehe hier dahin, ob man oder der EuGH zu den Ergebnissen der Urteile auch
kommen würde, wenn man alle hier vermissten Aspekte einbezöge. Eine umfassende
Argumentation hätte aber den Vorzug deutlich zu machen, dass der EuGH Fragen, die
vielen Menschen und Mitgliedstaaten wichtig sind, ernst nimmt. IV. Stellungnahme und Ausblick Überdies würde sie es
jenen leichter machen die Ergebnisse zu akzeptieren, welche sie nicht schon vorher
rechtspolitisch wünschten. Justice should not only be done, but should manifestly be
seen to be done. Experten des Europarechts sagen allerdings, Urteile des EuGH dürften nicht an tradi-
tionellen Maßstäben gemessen werden. Man dürfe nicht bloße Rechtsdogmatik oder gar
Orientierung an Absichten des Gesetzgebers erwarten, sondern müsse neben der juristi-
schen Oberfläche die Tiefenstruktur bedenken und das Recht im politischen Kontext
sehen. Die neuen Entscheidungen begünstigen nun klar den Wettbewerb mit geringen
Arbeitskosten auch über die Grenze, und damit bestimmte Mitgliedsstaaten. Diese Wir-
kung ist insbesondere in der Entsenderichtlinie angelegt, wenn man sie so versteht wie
der EuGH; diese Deutung ist aber nicht zwingend. Für das Verständnis der Urteile mag
daher relevant sein, dass nach den Erweiterungen zwölf neue Mitgliedstaaten vertreten
sind, auch im EuGH. Deren Mehrzahl wird erwarten, dass das Gemeinschaftsrecht ihre
Wettbewerbsposition zumindest bei Entsendungen stützt, wenn sie schon bereit sein
müssen Kapital, Waren und Manager einzulassen. Beobachter werden daher sagen, dass
die Urteile diesen Erwartungen entgegenkommen und auch dadurch Integration fördern. Der EuGH fördere den Binnenmarkt, auch auf Kosten traditioneller Rechte der Arbeit-
nehmer. Die Anerkennung eines Grundrechts, das konkret folgenlos bleibt, erscheint
dann als Mittel zur Erhöhung der Akzeptanz. Ein Primat des Kontexts vor der Rechtsdogmatik wirft die Frage nach der Rolle des
EuGH auf. Er wurde als Motor der Integration in Zeiten gerühmt, in denen politische
Integration und Rechtssetzung auf der Stelle traten. Von Zurückhaltung der Gemein-
schaftsgesetzgebung kann man aber seit Jahren nicht mehr sprechen. Es ist fraglich, ob
der EuGH noch immer bemüht sein soll, die Integration durch Urteile auch dann voran-
zutreiben, wenn das Vorantreiben nicht aus zwingender Ableitung folgt. Treibt er auch
dann voran, so läuft er Gefahr, von vielen nicht mehr als Garant des Rechts wahrge-
nommen zu werden, sondern als „mutiger“ Akteur der Politik. Als Gericht könnte er
Vorbehalte gegen die Europapolitik verringern, die viele Unionsbürger hegen. Als pri- 22 Verhältnis von Grundfreiheiten und Gemeinschaftsgrundrechten in der aktuellen EuGH Rechtsprechung
mär politischer Akteur stärkt er diese Bedenken. Er verringert dann jedenfalls bei einem
beträchtlichen Teil der Bevölkerung der EU Vertrauen anstatt Vertrauen aufzubauen. Verhältnis von Grundfreiheiten und Gemeinschaftsgrundrechten in der aktuellen EuGH Rechtsprechung mär politischer Akteur stärkt er diese Bedenken. Er verringert dann jedenfalls bei einem
beträchtlichen Teil der Bevölkerung der EU Vertrauen anstatt Vertrauen aufzubauen. mär politischer Akteur stärkt er diese Bedenken. ∗ Meiner Rechtsreferentin Frau Ioanna Dervisopoulos danke ich herzlich für die Unterstützung bei
der Erstellung des Manuskripts. IV. Stellungnahme und Ausblick Er verringert dann jedenfalls bei einem
beträchtlichen Teil der Bevölkerung der EU Vertrauen anstatt Vertrauen aufzubauen. 23 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb 783845219165, am 24.10.2024, 06:46:57
- https://www.nomos-elibrary.de/agb https://doi.org/10.5771/97838
Open Access –
- Die Auslegung der Entsenderichtlinie durch den EuGH
in den Entscheidungen Laval und Rüffert∗ Juliane Kokott 1 Siehe u.a. Urteile vom 3. Oktober 2000, Simap, C-303/98, Slg. 2000, I-7963; vom 9. September
2003, Jaeger, C-151/02, Slg. 2003, I-8389; vom 5. Oktober 2004, Pfeiffer u.a., C-397/01 bis
C-403/01, Slg. 2004, I-8835; und vom 7. September 2006, Kommission/Vereinigtes Königreich, Juliane Kokott Grundgedanke der Europäischen Integration ist es, den wirtschaftlichen Fortschritt
zu fördern, indem nationale Hürden für den Personen-, sowie Waren-, Dienstleistungs-
und Kapitalverkehr abgebaut werden und so ein Gemeinsamer Markt geschaffen wird. Von diesem Fortschritt profitieren im Ergebnis alle Bürgerinnen und Bürger in allen
Mitgliedstaaten. Das Funktionieren des Gemeinsamen Marktes bringt jedoch auch Spannungen mit
sich. Mit der Stärkung des innergemeinschaftlichen Wettbewerbs, der volkswirtschaft-
lich für die Union im Ganzen gewinnbringend ist, können für den Einzelnen – zumin-
dest vorübergehend – auch Nachteile verbunden sein. Der vergrößerte Wettbewerbs-
druck bringt für Arbeitnehmerinnen und Arbeitnehmer die Gefahr mit sich, geänderte
Arbeitsbedingungen akzeptieren zu müssen oder sogar ihre Stelle zu verlieren. Ihre Inte-
ressen gilt es daher in besonderem Maße zu schützen. Auch und gerade vor dem Hintergrund des derzeit noch bestehenden erheblichen
Wohlstandsgefälles in der Union stellt es eine bedeutsame Herausforderung für die
Fortentwicklung des Europarechts dar, einen gerechten Ausgleich zwischen der Ver-
wirklichung der Grundfreiheiten einerseits und dem Schutz und dem Ausbau sozialer
Standards andererseits herbeizuführen. Gemäß Artikel 2 des EU-Vertrages ist die För-
derung des wirtschaftlichen und des sozialen Fortschritts eines der grundlegenden Ziele
der Union. Ein wesentliches Anliegen des Lissabonner Vertrages ist es zudem, die sozi-
ale Dimension der Europäischen Union deutlicher zu betonen. I. Einleitung Die Bedeutung der sozialen Dimension im Recht der Europäischen Union und in der
Rechtsprechung des Europäischen Gerichtshofs ist ein aktuelles und wichtiges Thema. Gerade auch im Anschluss an einige Entscheidungen des EuGH wird darüber gegen-
wärtig vehement diskutiert. Dies überrascht nicht. Gilt es hier doch, die verschiedenen
miteinander im Wettstreit stehenden Interessen in einen gerechten Ausgleich zu brin-
gen. Der Einladung zum heutigen Symposium bin ich sehr gerne gefolgt – nicht nur
wegen der Bedeutung der aufgeworfenen Fragen, sondern auch weil ich meine, dass
sich die Vertreter der Europäischen Institutionen dem Dialog mit der Wissenschaft und
Öffentlichkeit stellen sollten. Mein Amt gebietet mir gleichwohl eine gewisse Zurückhaltung. Zwar war ich an den
Verfahren Rüffert und Laval, auf die ich näher eingehen werde, nicht als Generalanwäl-
tin beteiligt, doch künftig könnte ich möglicherweise mit ähnlichen Sachverhalten be-
fasst sein. Daher werde ich den Schwerpunkt meiner Ausführungen auf die Darstellung
der Grundlinien der Rechtsprechung des Europäischen Gerichtshofs legen und bei der
Kommentierung der Urteile Zurückhaltung wahren. 25 II. Schutz der Arbeitnehmerinnen und Arbeitnehmer in der Rechtsprechung
des Europäischen Gerichtshofs Bevor ich mich dem Gegenstand meines heutigen Vortrags zuwende, möchte ich zu-
nächst die vielfältigen Beiträge in Erinnerung rufen, die der Europäische Gerichtshof in
den vergangenen Jahrzehnten zum Schutz der Arbeitnehmerinnen und Arbeitnehmer
geleistet hat. Der Europäische Gerichtshof hat früh erkannt, dass es auf dem Weg zu einem Ge-
meinsamen Markt der Stärkung sozialer Rechte bedarf. Er hat mit der Betonung sozialer
Rechte als unverzichtbarem Bestandteil der Rechtsordnung der Europäischen Union ein
Gegengewicht zu den Kräften des Marktes gestärkt und damit die soziale Dimension der
Europäischen Union entscheidend mitgestaltet. Zu erwähnen ist insofern zunächst die umfassende Rechtsprechung zum Arbeitneh-
merschutz in den unterschiedlichsten Bereichen. Beispielhaft möchte ich die Entschei-
dungen zur Arbeitszeit1, zur Urlaubsgewährung2, zur befristeten Beschäftigung3 und 26 10.577
– Die Auslegung der Entsenderichtlinie durch den EuGH in den Entscheidungen Laval und Rüffert zur Teilzeitbeschäftigung4 nennen. Oder denken Sie an die Rechtsprechung des Ge-
richtshofs, mit der er die Gleichbehandlung von männlichen und weiblichen Arbeit-
nehmern durchgesetzt hat.5 Auch die Grundzüge der Behandlung entsandter Arbeitnehmerinnen und Arbeitneh-
mer wurden ursprünglich in der Rechtsprechung des Europäischen Gerichtshofs entwi-
ckelt. Danach verwehrt es das Gemeinschaftsrecht den Mitgliedsstaaten grundsätzlich
nicht, ihre Rechtsvorschriften oder die von den Sozialpartnern geschlossenen Tarifver-
träge hinsichtlich der Mindestlöhne auf jedermann zu erstrecken, der einer nichtselb-
ständigen Arbeit in ihrem Hoheitsgebiet nachgeht.6 Die Anwendung derartiger Vor-
schriften muss allerdings dem Schutz der Arbeitnehmer dienen und verhältnismäßig
sein.7 Diese Rechtsprechung hat Eingang gefunden in den 12. Erwägungsgrund der Ent-
senderichtlinie8, der ich mich nun zuwenden möchte. Bereits der langwierige Gesetzge-
bungsprozess dieser Richtlinie bestätigt nochmals, dass wir uns hier mit schwierigen
Fragen konfrontiert sehen: Die Verwirklichung der Dienstleistungsfreiheit9 will in Ein-
klang gebracht werden mit dem Schutz der Arbeitnehmer10 und einem fairen Wettbe-
werb11 und somit der Verhinderung von Sozialdumping. C-484/04, Slg. 2006, I-7471, sowie meine Schlussanträge vom 9. März 2006 in letzterer Rechtssa-
che. 2 Siehe u.a. Urteile vom 26. Juni 2001, BECTU, C-173/99, Slg. 2001, I-4881; vom 6. April 2006, Fe-
deratie Nederlandse Vakbeweging, C-124/05, Slg. 2006, I-3423, sowie meine Schlussanträge vom
12. Januar 2006 in letzterer Rechtssache. 3 Siehe u.a. Urteile vom 4. Juli 2006, Adeneler u.a., C-212/04, Slg. 2006, I-6057; und vom 15. April
2008, Impact, C-268/06, noch nicht in der amtlichen Sammlung veröffentlicht, sowie meine
Schlussanträge vom 27. Oktober 2005 bzw. vom 9. Januar 2008 in diesen Rechtssachen. 4 Siehe u.a. Urteile vom 13. Juli 1989, Rinner-Kühn, C-171/88, Slg. 1989, 2743; und vom 6. 2 Siehe u.a. Urteile vom 26. Juni 2001, BECTU, C-173/99, Slg. 2001, I-4881; vom 6. April 2006, Fe-
deratie Nederlandse Vakbeweging, C-124/05, Slg. 2006, I-3423, sowie meine Schlussanträge vom
12. Januar 2006 in letzterer Rechtssache. C-484/04, Slg. 2006, I-7471, sowie meine Schlussanträge vom 9. März 2006 in letzterer Rechtssa-
che. 12 Schlussanträge des Generalanwalts Bot vom 20. September 2006 in der Rechtssache Rüffert, C-
346/06, noch nicht in der amtlichen Sammlung veröffentlicht. 12 Schlussanträge des Generalanwalts Bot vom 20. September 2006 in der Rechtssache Rüffert, C-
346/06, noch nicht in der amtlichen Sammlung veröffentlicht.
13 Schlussanträge des Generalanwalts Mengozzi vom 23. Mai 2007 in der Rechtssache Laval un Part-
neri, C-341/05, Slg. 2007, I-11767, insbesondere Nrn. 147 und 151.
14 Schlussanträge des Generalanwalts Bot in der Rechtssache Rüffert (zitiert in Fn. 12, Nr. 70). g
14 Schlussanträge des Generalanwalts Bot in der Rechtssache Rüffert (zitiert in Fn. 12, Nr. II. Schutz der Arbeitnehmerinnen und Arbeitnehmer in der Rechtsprechung
des Europäischen Gerichtshofs Dezem-
ber 2007, Voß, C-300/06, Slg. 2007, I-10573. 5 Siehe u.a. Urteile vom 8. April 1976, Defrenne, 43/75, Slg. 1976, 455; und vom 21. Juni 2007,
Jonkman u.a., C-231/06 bis C-233/06, Slg. 2007, I-5149, sowie meine Schlussanträge vom 29. März
2007 in letzterer Rechtssache. 6 Vgl. u.a. Urteile vom 3. Februar 1982, Seco und Desquenne & Giral, 62/81 und 63/81, Slg. 1982,
223, Rdnr. 14; und vom 24. Januar 2002, Portugaia Construções, C-164/99, Slg. 2002, I-787, Rdnr. 21. 7 Vgl. in diesem Sinne Urteile vom 23. November 1999, Arblade u.a., C-369/96 und C-376/96, Slg. 1999, I-8453, Rdnr. 36, und vom 14. April 2005, Kommission/Deutschland, C-341/02, Slg. 2005,
I-2733, Rdnr. 24. 8 Richtlinie 96/71/EG des Europäischen Parlaments und des Rates vom 16. Dezember 1996 über die
Entsendung von Arbeitnehmern im Rahmen der Erbringung von Dienstleistungen, ABl. 1997, L 18,
S. 1, im Folgenden: Entsende-Richtlinie oder Richtlinie 96/71. 9 Siehe den ersten Erwägungsgrund der Richtlinie 96/71. 9 Siehe den ersten Erwägungsgrund der Richtlinie 96/71. 10 Siehe den fünften Erwägungsgrund der Richtlinie 96/71. 11 Siehe den fünften Erwägungsgrund der Richtlinie 96/71. 27 Juliane Kokott III. Auslegung der Entsenderichtlinie durch den Europäischen Gerichtshof g
p
,
346/06, noch nicht in der amtlichen Sammlung veröffentlicht.
13 Schlussanträge des Generalanwalts Mengozzi vom 23. Mai 2007 in der Rechtssache Laval un Part-
neri, C-341/05, Slg. 2007, I-11767, insbesondere Nrn. 147 und 151. 13 Schlussanträge des Generalanwalts Mengozzi vom 23. Mai 2007 in der Rechtssache Laval un Part-
neri, C-341/05, Slg. 2007, I-11767, insbesondere Nrn. 147 und 151. Schlussanträge des Generalanwalts Bot in der Rechtssache Rüffert (zitiert in Fn. 12, Nrn. 114 Schlussanträge des Generalanwalts Bot in der Rechtssache Rüffert (zitiert in Fn. 12, Nr. 94). 17 Falls die nationale Rechtsordnung kein System zur Allgemeinverbindlicherklärung kennt, können
die Mitgliedstaaten gem. Artikel 3 Absatz 8 der Richtlinie 96/71 folgende Mittel wählen: allgemein
wirksame Tarifverträge oder repräsentative Tarifverträge. III. Auslegung der Entsenderichtlinie durch den Europäischen Gerichtshof In der Rechtssache Rüffert hatte das vorlegende Gericht den Gerichtshof gefragt, ob
eine Tariftreueklausel wie sie das niedersächsische Vergabegesetz vorsah, eine nicht
gerechtfertigte Beschränkung der Dienstleistungsfreiheit darstellt. In der Rechtssache
Laval ging es abgesehen von der Streikproblematik um die Frage, ob das schwedische
System zur Festlegung der Löhne und weiterer Arbeitsbedingungen im Einklang mit
den Vorgaben der Entsenderichtlinie und mit der Dienstleistungsfreiheit steht. Gemäß Artikel 3 der Entsenderichtlinie sorgen die Mitgliedstaaten dafür, dass Unter-
nehmen den in ihr Hoheitsgebiet entsandten Arbeitnehmern bestimmte Arbeitsbedin-
gungen garantieren, die im Aufnahmemitgliedstaat durch Gesetz oder durch für allge-
meinverbindlich erklärte Tarifverträge festgelegt sind. Die Richtlinie zählt dann ver-
schiedene Arbeitsbedingungen auf, z.B. Höchstarbeitszeiten oder Mindestlöhne. Artikel
3 Absatz 7 der Entsenderichtlinie bestimmt, dass die Anwendung von für die Arbeit-
nehmer günstigeren Beschäftigungs- und Arbeitsbedingungen möglich ist. Wie ich eingangs bereits betont hatte, war ich als Generalanwältin nicht an den zu
besprechenden Fällen beteiligt. In der Rechtssache Rüffert hat mein Kollege General-
anwalt Bot die Schlussanträge vorgelegt. Generalanwalt Bot hatte dafür plädiert, die
Entsenderichtlinie so auszulegen, dass sie einem verstärkten nationalen Schutz der ent-
sandten Arbeitnehmer nicht entgegensteht.12 Vergleichbar argumentierte Generalanwalt
Mengozzi in der Rechtssache Laval.13 Auch ich habe eine große Sympathie für dieses
Verständnis der Entsenderichtlinie. Generalanwalt Bot versteht die Richtlinie als „harten Kern“ von Schutzbestimmun-
gen, die nur als Mindestgarantie zugunsten der entsandten Arbeitnehmer zu verstehen
sind. Die Richtlinie habe eine den Mitgliedsstaaten bis dahin zur Verfügung stehende
Regelungsmöglichkeit in eine Regelungsverpflichtung umgewandelt. Wie ich bereits
dargelegt habe, hatte der Gerichtshof ja schon vor Erlass der Entsende-Richtlinie festge-
stellt, dass die Mitgliedstaaten unter bestimmten Bedingungen ihre Rechtsvorschriften
auch auf entsandte Arbeitnehmer anwenden durften. Aus dieser Erlaubnis machte die
Entsende-Richtlinie nach Auffassung des Generalanwalts nun im Hinblick auf bestimm-
te Mindestbestimmungen zum Schutz der Arbeitnehmer eine Verpflichtung.14 Dass die
Mitgliedstaaten den entsandten Arbeitnehmern auch einen darüber hinaus gehenden
Schutz gewähren können, folgt nach Ansicht des Generalanwalts aus ihrem Artikel 3
Absatz 7. 28 10.577
– 15 Schlussanträge des Generalanwalts Bot in der Rechtssache Rüffert (zitiert in Fn. 12, Nr. 94).
16 Schlussanträge des Generalanwalts Bot in der Rechtssache Rüffert (zitiert in Fn. 12, Nrn. 114ff).
17 Falls die nationale Rechtsordnung kein System zur Allgemeinverbindlicherklärung kennt, können
die Mitgliedstaaten gem. Artikel 3 Absatz 8 der Richtlinie 96/71 folgende Mittel wählen: allgemein
wirksame Tarifverträge oder repräsentative Tarifverträge.
18 Urteil vom 18. Dezember 2007, Laval un Partneri, C-341/05, Slg. 2007, I-11767; und Urteil vom 3.
April 2008, Rüffert, C-346/06, noch nicht in der amtlichen Sammlung veröffentlicht, Rdnr. 33.
19 Urteil Laval un Partneri (zitiert in Fn. 18, Rdnr. 82) und Urteil vom 19. Juni 2008, Kommissi-
on/Luxemburg, C-319/06, noch nicht in der amtlichen Sammlung veröffentlicht, Rdnr. 27. 19 Urteil Laval un Partneri (zitiert in Fn. 18, Rdnr. 82) und Urteil vom 19. Juni 2008, Kommissi-
on/Luxemburg, C-319/06, noch nicht in der amtlichen Sammlung veröffentlicht, Rdnr. 27. g
p
g
18 Urteil vom 18. Dezember 2007, Laval un Partneri, C-341/05, Slg. 2007, I-11767; und Urteil vom 3.
April 2008, Rüffert, C-346/06, noch nicht in der amtlichen Sammlung veröffentlicht, Rdnr. 33. 16 Schlussanträge des Generalanwalts Bot in der Rechtssache Rüffert (zitiert in Fn. 12, Nrn. 114ff).
17 Falls die nationale Rechtsordnung kein System zur Allgemeinverbindlicherklärung kennt, können
die Mitgliedstaaten gem. Artikel 3 Absatz 8 der Richtlinie 96/71 folgende Mittel wählen: allgemein
wirksame Tarifverträge oder repräsentative Tarifverträge.
18 Urteil vom 18 Dezember 2007 Laval un Partneri C 341/05 Slg 2007 I 11767; und Urteil vom 3 Die Auslegung der Entsenderichtlinie durch den EuGH in den Entscheidungen Laval und Rüffert Die Auslegung der Entsenderichtlinie durch den EuGH in den Entscheidungen Laval und Rüffert Eine Tariftreueklausel stellt daher nach der Ansicht von Generalanwalt Bot einen zu-
lässigen verstärkten nationalen Schutz im Sinne von Artikel 3 Absatz 7 der Entsende-
Richtlinie dar.15 Eine gegenüber der Richtlinie verstärkte Schutzmaßnahme der Mit-
gliedstaaten muss allerdings auch mit den Grundfreiheiten vereinbar sein. Dass eine
solche Regelung eine Beschränkung des freien Dienstleistungsverkehrs ist, steht nach
Ansicht von Bot außer Zweifel. Er sieht diese Beschränkung jedoch aus Gründen des
Arbeitnehmerschutzes und zur Verhinderung von Sozialdumping als gerechtfertigt
an.16 Im Ergebnis hatte Generalanwalt Bot daher dem Gerichtshof vorgeschlagen, die
Entsende-Richtlinie und die Dienstleistungsfreiheit dahin auszulegen, dass sie eine Ta-
riftreueklausel erlauben, sofern diese das Verbot der Diskriminierung aus Gründen der
Staatsangehörigkeit sowie den Grundsatz der Transparenz beachtet. Der Gerichtshof ist den Schlussanträgen des Generalanwalts nicht gefolgt. Nach dem
Verständnis des Gerichtshofs sieht die Richtlinie ein begrenztes Regularium für den
Schutz entsandter Arbeitnehmer vor. Die Richtlinie zähle diejenigen Modalitäten auf,
mit denen der nationale Gesetzgeber seine Bestimmungen über Arbeitsbedingungen auf
entsandte Arbeitnehmer erstrecken kann. Nach diesem Verständnis kann ein Mindest-
lohn entweder gesetzlich festgelegt oder aber über den Weg der Allgemeinverbindlich-
keitserklärung eines Tarifvertrags bestimmt werden.17 Nach Auffassung des Gerichtshofs ermächtigt Artikel 3 Absatz 7 der Entsende-
Richtlinie die Mitgliedstaaten nicht, Arbeits- und Beschäftigungsbedingungen durchzu-
setzen, die nicht in Absatz 1 aufgelistet sind. Nur diese Auslegung der Richtlinie ver-
hindere, dass die Richtlinie leer laufe. Die Aufzählung von bestimmten Bereichen und
bestimmten Modalitäten, mit denen Arbeitsbedingungen auf entsandte Arbeitnehmer
erstreckt werden könnten, wäre bedeutungslos, wenn über den Absatz 7 auch günstigere
Arbeits- und Beschäftigungsbedingungen wie beispielsweise Tariflöhne, die über Min-
destlohnsätzen liegen, durch Tariftreueklauseln einseitig durchgesetzt werden könnten. Eine andere Auslegung liefe darauf hinaus, der Entsende-Richtlinie ihre praktische
Wirksamkeit zu nehmen.18 Ein über Artikel 3 Absatz 1 der Entsende-Richtlinie hinaus-
gehender Schutz scheint nach dem Verständnis des Gerichtshofs somit nur ausnahms-
weise nach Artikel 3 Absatz 10 zulässig zu sein, wenn es sich bei ihnen um Vorschrif-
ten im Bereich der öffentlichen Ordnung handelt.19 Die Entsende-Richtlinie berechtigt daher nach Ansicht des Gerichtshofs einen Mit-
gliedstaat nicht zum Erlass einer Tariftreueklausel. Im Anschluss hieran prüfte der Ge- 29 29 /10.5771/
– Juliane Kokott richtshof, ob diese Auslegung der Richtlinie durch deren Würdigung im Lichte der
Dienstleistungsfreiheit bestätigt werde.20 Der Gerichtshof prüft die Tariftreueklausel
noch einmal an der Grundfreiheit. Zwar bejaht er wie Generalanwalt Bot eine Be-
schränkung der Dienstleistungsfreiheit. 20 Urteil Rüffert (zitiert in Fn. 18, Rdnr. 36ff).
21 Urteil Rüffert (zitiert in Fn. 18, Rdnr. 40).
22 Urteil Kommission/Luxemburg (zitiert in Fn. 19, Rdnr. 26).
23 Urteil Kommission/Luxemburg (zitiert in Fn. 19, Rdnr. 47).
24 Urteil Kommission/Luxemburg (zitiert in Fn. 19, Rdnr. 54). Die Auslegung der Entsenderichtlinie durch den EuGH in den Entscheidungen Laval und Rüffert Anders als der Generalanwalt verneint der Ge-
richtshof aber die Rechtfertigung der Beschränkung durch zwingende Gründe des All-
gemeininteresses. Hierbei stützt er sich insbesondere darauf, dass die Tariftreueklausel
nur für öffentliche Auftraggeber gelte und nicht auch für private Auftraggeber.21 Diese
Argumentation ist sicherlich nicht zwingend. Die Ausführungen des Gerichtshofs in den Urteilen Laval und Rüffert deuteten be-
reits darauf hin, dass er die in der Entsende-Richtlinie enthaltenen Regelungsmöglich-
keiten als abschließend ansieht und darüber hinausgehende Maßnahmen grundsätzlich
für unzulässig erachtet. Für den Gerichtshof scheint mir somit die Richtlinie die ab-
schließend kodifizierte Abwägung zwischen Dienstleistungsfreiheit und Arbeitnehmer-
schutz darzustellen. In seinem Urteil Kommission/Luxemburg von letzter Woche scheint der Gerichtshof
seine Auffassung vom abschließenden Charakter der Entsende-Richtlinie zu verfesti-
gen.22 In dieser Rechtssache ging es unter anderem um eine luxemburgische Bestim-
mung, die die automatische Anpassung der Entlohnung an die Entwicklung der Lebens-
haltungskosten auch auf entsandte Arbeitnehmer erstreckte. Die Kommission beanstan-
dete nicht, dass die Mindestlöhne an die Lebenshaltungskosten angepasst werden. Dies
sei nach der Entsende-Richtlinie zulässig. Dass die Indexierung aber sämtliche Löhne
betreffe, also auch solche, die keine Mindestlöhne sind, verstoße gegen die Richtlinie. Dies hat der Gerichtshof bestätigt.23 Eine solche Maßnahme der Indexierung sei in der
Entsende-Richtlinie nicht vorgesehen, die abschließend die Aspekte nenne, hinsichtlich
derer die Mitgliedstaaten ihre nationalen Bestimmungen auf entsandte Arbeitnehmer
erstrecken können. Luxemburg habe auch nicht hinreichend nachgewiesen, dass es sich
bei dieser Bestimmung um eine Vorschrift im Bereich der öffentlichen Ordnung im
Sinne der Entsende-Richtlinie handele.24 In seinem Urteil prüft der Gerichthof die
Maßnahme nur mehr an der Richtlinie, anders als bei Rüffert nicht zusätzlich noch am
Maßstab der Grundfreiheit. Welcher Gestaltungsspielraum steht den Mitgliedstaaten nach dem Verständnis des
Gerichtshofs für den Schutz entsandter Arbeitnehmer folglich noch zur Verfügung? Unzweifelhaft zulässig sind nach der Rechtsprechung des Gerichtshofs Regelungen in
Bezug auf diejenigen Aspekte der Arbeitsbedingungen, die die Richtlinie explizit auf-
zählt. Bezüglich der Art und Weise wie diese Arbeitsbedingungen auf entsandte Arbeit-
nehmer erstreckt werden können, stehen den Mitgliedstaaten zwei Optionen zur Verfü- 30 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb Auslegung der Entsenderichtlinie durch den EuGH in den Entscheidungen Laval und Rüffert gung: die gesetzliche Festlegung oder ein für allgemeinverbindlich erklärter Tarifver-
trag.25 Für die entsendenden Unternehmen hat die Auslegung des Gerichtshofs sicher den
Vorteil einer größeren Rechtssicherheit und Vorhersehbarkeit der Löhne, die bei Dienst-
leistungen in einem anderen Mitgliedstaat zu entrichten sind. 25 Falls das nationale Rechtssystem keine Allgemeinverbindlicherklärung kennt, können die Mitglied-
staaten gem. Artikel 3 Absatz 8 der Richtlinie 96/71 folgende Mittel wählen: allgemein wirksame
Tarifverträge oder repräsentative Tarifverträge. Die Auslegung der Entsenderichtlinie durch den EuGH in den Entscheidungen Laval und Rüffert Gleichwohl besteht die
Gefahr einer Entwicklung der Löhne im Aufnahmemitgliedstaat in Richtung auf die
Mindestgewährleistungen. 26 Urteil vom 11. Dezember 2007, International Transport Workers’ Federation und Finnish Seamen’s
Union („Viking Line“), C-438/05, noch nicht in der amtlichen Sammlung veröffentlicht. 27 Urteil Laval un Partneri (zitiert in Fn. 18, Rdnr. 110). staaten gem. Artikel 3 Absatz 8 der Richtlinie 96/71 folgende Mittel wählen: allgemein wirksame
Tarifverträge oder repräsentative Tarifverträge.
26 Urteil vom 11. Dezember 2007, International Transport Workers’ Federation und Finnish Seamen’s
Union („Viking Line“), C-438/05, noch nicht in der amtlichen Sammlung veröffentlicht. 28 Wörtlich heißt es im Urteil Laval un Partneri (zitiert in Fn. 18) in Randnr. 103: „ … dass im Recht
auf Durchführung einer kollektiven Maßnahme, die den Schutz der Arbeitnehmer des Aufnahme-
mitgliedstaates gegen ein etwaiges Sozialdumping zum Ziel hat, ein zwingender Grund des Allge-
meininteresses“ liegen kann. Siehe hierzu bislang nur Urteil vom 19. Januar 2006, Kommissi-
on/Deutschland, C-244/04, Slg. 2006, I-885, Rdnr. 61. IV. Ausblick Die Urteile des Gerichtshofs Viking26, Laval und Rüffert werden gegenwärtig stark
kritisiert. Auch meine Vorredner haben heute verschiedene Bedenken formuliert. Be-
reits die Schlussanträge der Generalanwälte Bot und Mengozzi zeigen, dass auch inner-
halb des Gerichtshofs kein einhelliges Meinungsbild herrscht. Ich möchte in diesem Zusammenhang vor allem einen Gesichtspunkt betonen, der
mir von vielen kritischen Stimmen, die etwa durch das Urteil Laval das Ende des Streik-
rechts eingeläutet sehen, nicht hinreichend berücksichtigt wird. Man sollte sich bewusst
sein, dass der Gerichtshof in den betreffenden Urteilen nur über die jeweilige spezifi-
sche Vorlagefrage entschieden hat. Deren Ausgangspunkt sind jeweils ganz konkrete,
durch zahlreiche Besonderheiten gekennzeichnete Rechtsstreite. Eine genaue Lektüre
der Urteile zeigt, dass der Gerichtshof gerade diese Besonderheiten hervorhebt und an
ihnen sein Urteil festmacht. So betonte er im Urteil Laval den nationalen Kontext, in
den sich die kollektiven Maßnahmen einfügten. Für diesen spezifischen Kontext war
kennzeichnend, dass es keine Vorschriften gab, die hinreichend genau und zugänglich
waren, um einem Unternehmen zu ermöglichen, festzustellen, welche Verpflichtung es
hinsichtlich des Mindestlohns beachten muss.27 Der Gerichtshof hat keine allgemeine Aussage zum Verhältnis von Grundfreiheiten
und Grundrechten getroffen. Insbesondere hat er in den aktuellen Urteilen nicht ent-
schieden, dass das Streikrecht oder gar die Menschenwürde grundsätzlich hinter den
Grundfreiheiten zurückzustehen hat. Grundfreiheiten und Gemeinschaftsgrundrechte
stehen vielmehr auf einer Ebene und müssen miteinander in Einklang gebracht werden. Indem der Gerichtshof besonders die spezifischen Charakteristika der Fälle betont,
signalisiert er meines Erachtens unmissverständlich, anhand der Besonderheiten künfti-
ger Vorlageverfahren seine Rechtsprechung weiter ausdifferenzieren zu wollen. 25 Falls das nationale Rechtssystem keine Allgemeinverbindlicherklärung kennt, können die Mitglied-
staaten gem. Artikel 3 Absatz 8 der Richtlinie 96/71 folgende Mittel wählen: allgemein wirksame
Tarifverträge oder repräsentative Tarifverträge. 26 Urteil vom 11. Dezember 2007, International Transport Workers’ Federation und Finnish Seamen’s
Union („Viking Line“), C-438/05, noch nicht in der amtlichen Sammlung veröffentlicht. 31 Juliane Kokott Lassen Sie mich zum Abschluss betonen: Bei aller Kritik an den konkreten Urteilen
darf nicht übersehen werden, dass der Gerichtshof darin im Grundsatz aus sozialer Per-
spektive auch positiv zu bewertende Ausführungen macht: Er erkennt das Streikrecht
als Grundrecht an. Und bemerkenswerterweise greift er im Urteil Laval den Begriff des
Sozialdumpings auf und sieht im Streikrecht zum Schutz der Arbeitnehmer gegen Sozi-
aldumping ein zwingendes Allgemeininteresse.28 Ich komme zum Ende meiner Ausführungen. Meines Erachtens läuft insbesondere
die öffentliche Diskussion Gefahr, die Entscheidungen Rüffert und Laval überzubewer-
ten. Beide Entscheidungen sind kein Wendepunkt am Ende einer langen Kette von Ent-
scheidungen des Gerichtshofs, die sich im Spannungsfeld von Grundfreiheiten und so-
zialen Rechten bewegen. Wie sicherlich auch dieses Symposium zeigen wird, ist viel-
mehr festzuhalten, dass diese Entscheidungen Pendelausschläge der Rechtsprechung des
Europäischen Gerichtshofs darstellen, in dem Bemühen, in einer sich rasch entwickeln-
den Union die dargestellten schwierigen Abwägungsfragen auszudifferenzieren und von
Fall zu Fall weitere Aspekte zu berücksichtigen. Soviel ist sicher: Der Gerichtshof wird seine Rechtsprechung noch weiter entwickeln
und präzisieren. Auch der Diskurs in der Fachöffentlichkeit und Diskussionsveranstal-
tungen wie die heutige leisten insofern einen wichtigen Beitrag. Dass die heutige Ver-
anstaltung die Möglichkeit eröffnet, nicht lediglich die deutsche Perspektive, sondern
dank zahlreicher Teilnehmer aus anderen Mitgliedstaaten auch deren Sichtweise kennen
zu lernen, stellt eine wertvolle Chance dar. 32 /10.577
– Diskussionsbericht Diskussionsleitung: Christa Peutl Diskussionsleitung: Christa Peutl In der Diskussion wurde zunächst die Entscheidung Rüffert aufgegriffen und darauf
aufmerksam gemacht, dass vom EuGH und auch von Kokott zu Recht betont worden
sei, dass die Auslegung der Entsenderichtlinie als Maximalregelung nur damit erklärbar
sei, dass sie einen Eingriff in die Dienstleistungsfreiheit darstellt. Jedoch habe für die
betroffenen polnischen Arbeitnehmer damals noch gar keine Dienstleistungsfreiheit
bestanden, da Polen zu diesem Zeitpunkt noch nicht Mitglied der Europäischen Union
gewesen sei und die vollständige Freizügigkeit für polnische Arbeitnehmer erst im Jahre
2009 eingeführt werde. Insoweit wurde um Aufklärung gebeten. Kokott erklärte, dass
der EuGH im Rahmen eines Vorabentscheidungsverfahrens entschieden habe. Das vor-
legende OLG Celle habe den EuGH nach der Auslegung der Entsenderichtlinie gefragt
und plausibel dargelegt, dass seine Antwort für den nationalen Rechtsstreit entscheidend
sei. Für den EuGH seien keine Anhaltspunkte erkennbar gewesen, die der Zulässigkeit
entgegen gestanden hätten. Das Rüffert-Urteil gab außerdem zu der entscheidenden und zugleich schwierigen
Frage Anlass, wie sich der Mindestlohn definieren lässt. Kokott wies zunächst auf den
mit dem Urteil verfolgten Zweck hin, einen Ausgleich zwischen Dienstleistungsfreiheit
und Arbeitnehmerschutz zu erreichen. Im Rahmen dieses Ausgleichs sei zu berücksich-
tigen, dass die Dienstleistungsfreiheit nicht durch Mindestlöhne eingeschränkt werden
dürfe, die nur in bestimmten Regionen oder nur bei einzelnen privaten Arbeitgebern
gelten. Ein Kennzeichen für Mindestlöhne sei daher, dass sie durch Gesetz festgelegt
werden oder Allgemeingültigkeit besitzen. Im Rahmen weiterer Vorlagefragen könne
eine nähere Konkretisierung von Mindestlöhnen erfolgen. Rebhahn ergänzte, dass es
durchaus verschiedene Mindestlohnsätze geben könne, je nach der Art der Tätigkeit und
der Qualifikation der betroffenen Arbeitnehmer, jedoch ohne Berücksichtigung ihres
Dienstalters und ihrer Jahre der Betriebszugehörigkeit. Lyon-Caen war der Ansicht,
dass die von Kokott vorgeschlagene Abwägung die Problematik nicht vollständig erfas-
se, da Dienstleistungsfreiheit und Arbeitnehmerschutz keine einseitig protektionisti-
schen Regelungen, sondern selbst schon Kompromisse seien, die die Notwendigkeiten
eines funktionierenden Marktes berücksichtigten. Die Interessen der Unternehmen seien
bereits eingeflossen und würden bei der von Kokott geforderten Abwägung ein zusätzli-
ches Mal berücksichtigt werden, so dass einflussreiche Unternehmen gegenüber den
vom Markt geschwächten Arbeitnehmern noch mehr Macht erhielten. Die internationale Anerkennung des Grundrechts auf Kollektivvertragsfreiheit durch
den EuGH zog die Frage nach dem transnationalen Gehalt einer Kollektivvertragsfrei-
heit nach sich, zumal das Kollektivvertragssystem durch nationales Recht geregelt sei 33 33 Diskussionsbericht und Fragen des Arbeitskampfes in vielen nationalen Rechtsordnungen noch ungeklärt
seien. 1 EuGH vom 12.6.2003, Schmidberger, Rs. C-112/00, Slg. 2003, I-5659. Diskussionsleitung: Christa Peutl Kokott wies darauf hin, dass der EuGH das Streikrecht im Fall Laval relativ weit
ausgelegt habe, indem er sogar Boykotte in grenzüberschreitenden Konstellationen
durch das Streikrecht grundsätzlich gedeckt angesehen habe. Entscheidend sei letztlich
immer die Abwägung im konkreten Einzelfall. g
g
Von Zuhörerseite wurde angemerkt, dass eine solche Abwägung auffällig häufig zu-
gunsten der Grundfreiheiten und zulasten nationaler Grundrechte ausfalle. So hätte der
EuGH im Urteil Laval das Recht auf Durchführung einer kollektiven Maßnahme zwar
als nationales Grundrecht anerkannt, aber dieses hätte sich gegenüber der Dienstleis-
tungsfreiheit nicht durchsetzen können. Im EuGH-Urteil Schmidberger1 sei die Abwä-
gung lediglich zugunsten des nationalen Grundrechts auf Versammlungsfreiheit und
nicht zugunsten des freien Warenverkehrs ausgefallen, weil der deutsche Spediteur auf
einer anderen Autobahn hätte nach Italien fahren können. Diese Entwicklung könne zu
einer Legitimitätskrise der Europäischen Union, ja sogar zu einer fundamentalen Krise
Europas führen. Auch Rebhahn sah diese Problematik und sprach sich daher dafür aus, dass sich die
Gemeinschaft mit Eingriffen in nationale Regelungen zurückhalten sollte, solange noch
keine Gemeinschaftsgrundrechte mit einer eigenen Grundrechtsdogmatik existierten. Er
stellte klar, dass das von ihm in seinem Vortrag postulierte Gemeinschaftsgrundrecht
auf Tarifautonomie lediglich eine Perspektive darstelle, denn es dürfe nicht vergessen
werden, dass für noch kein Rechtsrahmen für transnationale Tarifverträge existiere. Lyon-Caen zeigte sich besorgt über das strukturelle Ungleichgewicht in der Gemein-
schaft: Während die unternehmerische Freizügigkeit und die individuelle Freizügigkeit
der Arbeitnehmer von Anfang an bestanden hätten, habe es bislang noch keine Freiheit
eines transnationalen kollektiven Handelns gegeben. Dem EuGH sei ein Ausgleich die-
ses Ungleichgewichts in den Fällen Laval und Viking nicht gelungen, obwohl es von
ihm erwartet worden sei. Was ist das für eine Europäische Union, in der die Unterneh-
men eine weite internationale Freizügigkeit genießen, die Rechte der Arbeitnehmer da-
gegen dahinter zurückbleiben? Aus Anlass eines aktuellen Verfahrens wurde die Verordnung (EU) Nr. 1370/2007
vom 23. Oktober 2007 angesprochen und nach ihrem Verhältnis zur Entsenderichtlinie
gefragt. Die Verordnung betrifft die Vergabe öffentlicher Verkehrsdienstleistungsauf-
träge auf Schiene und Straße und beinhaltet den Schutz spezieller Arbeitnehmerrechte. Rebhahn sah keine Parallele zum Fall Rüffert und nahm keinen Vorrang der Entsende-
richtlinie an, da die betreffende Verordnung eine gegenüber der Entsenderichtlinie spe-
ziellere und damit vorrangige Regelung darstelle. Dies gelte jedoch nur, wenn diese
Verordnung eine abschließende Regelung biete; ansonsten kämen die Grundfreiheiten
wieder zum Tragen. Es bliebe dann jedoch immer noch die nicht klar beantwortete Fra-
ge, inwieweit ein Sekundärrechtsakt am Primärrecht zu prüfen ist. Diskussionsleitung: Christa Peutl 34 10.5771
– Simone Gräfin von Hardenberg I. Hintergrund II. Zur Aufgabe des EuGH und den Besonderheiten des europäischen
Gemeinschaftsrechts III. Begegnung sozialer und wirtschaftlicher Ziele 1. Zielkonflikte 2. Verwirklichung sozialer Rechte 3. Diskriminierungsverbote IV. Zur Methode des EuGH 1. Die Rolle der Entscheidungsbegründung 2. Zwischen judicial activism und self-restraint 3. Orientierung am Gesamtinteresse und Erforderlichkeit des gesamt-
europäischen Diskurses Vielen Dank für die Einladung zu diesem sehr interessanten Thema. Bitte haben Sie
auch Verständnis, dass ich nicht direkt auf die hier genannten Urteile näher eingehen
werde. Diese werde ich vielmehr am Rande streifen, und in erster Linie etwas zum Ziel
des sozialen Europas aus der Perspektive der Europäischen Gerichtsbarkeit sagen. Diskussionsbericht Schließlich wurde über den praktischen Umgang mit der Entsenderichtlinie disku-
tiert. Es wurde davon ausgegangen, dass die Liste in Art. 3 Abs. 1 der Entsenderichtli-
nie nicht als abschließend zu betrachten ist. Der Fall Rüffert hätte dann allerdings zur
Konsequenz, dass jede Einzelregelung im Arbeitsrecht eines jedes Mitgliedstaates in die
Liste aufzunehmen wäre, damit nicht die Gefahr bestehe, dass über ein Fehlen in der
Liste ein Sozialdumping erfolgt. Selbst wenn jedoch eine entsprechende Regelung exis-
tiere, habe der Fall Rüffert auch gezeigt, dass nicht davon ausgegangen werden könne,
dass der EuGH diese dann tatsächlich in seine Abwägung einbezieht, da er für seine
Entscheidung ungünstige Regelungen gerne ausblende. Kokott gab zu bedenken, dass im Fall Rüffert ein Konflikt zwischen Entsenderichtli-
nie und Dienstleistungsfreiheit bestanden habe. Dieser sei auch in der Richtlinie selbst
zum Ausdruck gekommen, da diese laut Präambel eine Beseitigung der Hindernisse für
den freien Dienstleistungsverkehr zum Ziel habe. Während der EuGH die Richtlinie aus
der Perspektive der Dienstleistungsfreiheit ausgelegt habe, hätten die Generalanwälte
sie aus der Perspektive des Arbeitnehmerschutzes ausgelegt. Beides sei grundsätzlich
vertretbar, wobei der EuGH seine Entscheidung durchaus klarer hätte formulieren kön-
nen. Auch Rebhahn sprach sich für eine eingehendere juristische Begründung durch den
EuGH aus, da er dadurch mehr Vertrauen schaffen könne und seine Urteile dann eine
höhere Akzeptanz finden würden. Er wies außerdem darauf hin, dass es im Rat Stim-
men gegeben haben soll, die von einer abschließenden Regelung des Art. 3 Absatz 1 der
Entsenderichtlinie ausgegangen seien. Lyon-Caen war dagegen der Ansicht, dass man unter Berücksichtigung der Sozialge-
schichte nicht zur Annahme einer Maximalregelung gelangen könne. Gleichwohl könne
vom EuGH nicht verlangt werden, dass er die Sozialgeschichte sämtlicher Mitgliedstaa-
ten in seine Überlegungen einbeziehe. Lyon-Caen erwartete, dass in Zukunft dem sozia-
len Schutz im Verhältnis zu den Grundfreiheiten ein stärkeres Gewicht eingeräumt wer-
de, auch wenn dieser Schutz in der Europäischen Gemeinschaft eher mit Stillstand als
mit Fortschritt in Verbindung gebracht werde. Simone Gräfin von Hardenberg 35 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb 783845219165, am 24.10.2024, 06:46:57
- https://www.nomos-elibrary.de/agb https://doi.org/10.5771/97838
Open Access –
- Das Europäische Sozialrecht und die Rechtsprechung des EuGH Egils Levits I. Hintergrund
37
II. Zur Aufgabe des EuGH und den Besonderheiten des europäischen
Gemeinschaftsrechts
38
III. Begegnung sozialer und wirtschaftlicher Ziele
39
1. Zielkonflikte
39
2. Verwirklichung sozialer Rechte
39
3. Diskriminierungsverbote
40
IV. Zur Methode des EuGH
40
1. Die Rolle der Entscheidungsbegründung
40
2. Zwischen judicial activism und self-restraint
41
3. Orientierung am Gesamtinteresse und Erforderlichkeit des gesamt-
europäischen Diskurses
42 I. Hintergrund
II. Zur Aufgabe des EuGH und den Besonderheiten des europäi
Gemeinschaftsrechts
III. Begegnung sozialer und wirtschaftlicher Ziele
1. Zielkonflikte
2. Verwirklichung sozialer Rechte
3. Diskriminierungsverbote
IV. Zur Methode des EuGH
1. Die Rolle der Entscheidungsbegründung
2. Zwischen judicial activism und self-restraint
3. Orientierung am Gesamtinteresse und Erforderlichkeit des
europäischen Diskurses I. Hintergrund
37
II. Zur Aufgabe des EuGH und den Besonderheiten des europäischen
Gemeinschaftsrechts
38
III. Begegnung sozialer und wirtschaftlicher Ziele
39
1. Zielkonflikte
39
2. Verwirklichung sozialer Rechte
39
3. Diskriminierungsverbote
40
IV. Zur Methode des EuGH
40
1. Die Rolle der Entscheidungsbegründung
40
2. Zwischen judicial activism und self-restraint
41
3. Orientierung am Gesamtinteresse und Erforderlichkeit des gesamt-
europäischen Diskurses
42 Egils Levits Egils Levits chung des Binnenmarktes liegt. Das Ziel des sozialen Europas kommt insbesondere als
wichtiges Korrektiv zur Verwirklichung des Binnenmarktes zur Geltung, um soziale
Verwerfungen und andere unerwünschte Ergebnisse in Folge der Verwirklichung des
Binnenmarktes zu unterbinden. Im gegewärtigen Stadium der Integration gibt es aber
noch keine Europäische Sozialgemeinschaft, die in dieser Intensität mit der Wirt-
schaftsgemeinschaft vergleichbar wäre. Wir versuchen auf dem Wege der Rechtsprechung die soziale Dimension Europas
vorwärts zu bringen, aber in erster Linie ist dies eine Aufgabe der Politik. Aber auch der
Europäische Gerichtshof leistet seinen Beitrag dazu, insbesondere, wenn es im Rahmen
der primärrechtlich festgelegten Ziele – Verwirklichung des Binnenmarktes und sozia-
les Europa – zu Zielkonflikten kommt. Der Gerichtshof muss dabei einen gerechten
Ausgleich zwischen den beiden Zielen herstellen. I. Hintergrund Zunächst gilt es sich daran zu erinnern, dass die Europäische Gemeinschaft aus der
Europäischen Wirtschaftsgemeinschaft entstanden ist, was sich in unseren vertraglichen
Grundlagen bis heute widerspiegelt. Erst nach und nach hat man das Ziel des sozialen
Europas als ein wichtiges politisches Ziel erkannt und versucht dieses zu emanzipieren. Die Rechtsprechung ist an die bestehenden Rechtsnormen gebunden, aus denen sich
ergibt, dass die Verträge in erster Linie auf die Verwirklichung des Binnenmarktes zu-
geschnitten sind. Ferner gilt es festzuhalten, dass beide Ziele von den Verträgen nicht
als völlig gleichwertig angesehen werden, sondern der Schwerpunkt auf der Verwirkli- 37 37 1. Zielkonflikte Zunächst finden wir als umstrittenste und meist diskutierte Konstellation einen Kon-
flikt zwischen den beiden Zielen vor. Dieser Konflikt kann sich dergestalt manifestie-
ren, dass die Verwirklichung des sozialen Europas eine Beschränkung des Binnenmark-
tes zur Folge hätte oder umgekehrt. Das heißt, dass durch die Verwirklichung des Bin-
nenmarktes das soziale Europa nicht zur Geltung kommen würde. Dies ist die schwie-
rigste oder jedenfalls am häufigsten diskutierte, konfliktbehaftete Begegnung der beiden
Zielvorstellungen. Bei genauer Betrachtung gibt es innerhalb dieser Konstellation viele verschiedene
Interessen, die zum Ausgleich gebracht werden müssen. Ein Beispiel dafür geben die
hier bereits angesprochenen Entsendefälle. In jenen Fällen können die Interessenlagen
zweier Mitgliedstaaten, aber auch die Interessen der Arbeitgeber und Arbeitnehmer im
Entsende- und Aufnahmeland konträr sein. Wiederum können in beiden betroffenen
Ländern die Interessen von Arbeitgebern und Arbeitnehmern miteinander kollidieren. Auf der anderen Seite kann die Interessenlage der Arbeitgeber im Aufnahmeland durch-
aus ähnlich oder gar deckungsgleich mit dem Interesse der Arbeitnehmer aus dem Ent-
sendeland sein. Auf diese Art und Weise ergeben sich durchaus interessante, aber
gleichzeitig sehr komplizierte Interessenlagen, die in Einklang gebracht werden müssen. Bei diesem Ausgleich muss der Europäische Gerichtshof als supranationale Instanz
immer das gesamte Europa im Blick haben. So gilt es zum Beispiel die Interessen von
zwei Arbeitnehmern, der eine aus dem Entsendeland, der andere aus dem Aufnahme-
land, zum Ausgleich zu bringen Wie meine Vorrednerin und Kollegin Frau Kokott erklärt hat, gibt es dafür das juris-
tische Instrument der Abwägung. Genauso wie jedes andere Gericht wägen wir eben-
falls ab. Das Ergebnis einer solchen Abwägung kann natürlich kritisiert werden, wobei
die Kritik üblicherweise von der Seite kommt, zu deren Ungunsten der individuelle
Abwägungsprozess ausgefallen ist. III. Begegnung sozialer und wirtschaftlicher Ziele Bei Betrachtung der Ziele Verwirklichung des Binnenmarktes und soziales Europa
fällt auf, dass diese Ziele in drei verschiedenen Konstellationen aufeinander treffen. II. Zur Aufgabe des EuGH und den Besonderheiten des europäischen Ge-
meinschaftsrechts Die Aufgabe des Europäischen Gerichtshofs ist die Wahrung des Rechts der Europä-
ischen Gemeinschaft. Dieses Recht zeichnet sich im Vergleich zum nationalen Recht
durch zwei Eigenschaften aus. Einerseits gibt es auf einem sehr hohen Abstraktionsniveau formulierte Rechtsnor-
men, die das Resultat politischer Uneinigkeiten darstellen und daher nicht näher kon-
kretisiert sind. So zeichnen sich zum Beispiel die zwei Ziele – Verwirklichung des Bin-
nenmarktes und soziales Europa – durch eine derartige Abstraktheit und Unbestimmt-
heit aus, dass der Gerichtshof oft gezwungen ist, rechtspolitische Entscheidungen oder
Wertentscheidungen zu treffen. Schließlich kann sich der Europäische Gerichtshof an
dieser Stelle keine Rechtsverweigerung leisten, sondern muss, bei gegebener Zustän-
digkeit, einen ihm vorgelegten Fall auch entscheiden. Auf der anderen Seite versucht insbesondere das Sekundärrecht, wenn auch auf einer
Ebene mit geringerer Bedeutung, sehr präzise und sehr detailliert zu sein. Daher bringen
wir mitunter sehr kleinteilige Entscheidungen zu Sachverhalten von eher beschränkter
Bedeutung hervor. Wir haben es also einerseits mit großen Entscheidungen zu tun, die eine Rechtsaus-
legung und –fortbildung auf einem hohen Abstraktionsniveau leisten, und andererseits
mit sehr präzisen Entscheidungen, die auf konkreterem Niveau Auslegungsschwierig-
keiten klären. 38 Das Europäische Sozialrecht und die Rechtsprechung des EuGH Egils Levits Egils Levits heit verwirklicht werden kann und muss. Als Beispiel lässt sich hier das Urteil im Fall
Watts anführen, worin entschieden worden ist, dass sich Patienten, die in einem Mit-
gliedstaat zur medizinischen Behandlung berechtigt sind, zur Inanspruchnahme dieser
medizinischen Behandlung in einen anderen Mitgliedstaat begeben dürfen und dann
Ausgleich der Kosten von der entsprechenden Einrichtung ihres Heimatstaates verlan-
gen können.1 Diese Konstellation birgt, im Gegensatz zum zuvor Erläuterten, keinen
Konflikt der beiden Ziele in sich. Die Förderung des einen Ziels verwirklicht zugleich
auch das andere. Dies ist ebenfalls eine Situation, in der der Gerichtshof die Stärkung
des sozialen Aspektes in Europa vorantreibt. 3. Diskriminierungsverbote Drittens gibt es Fälle, in denen das soziale Europa durch die Verwirklichung eines
im Primärrecht oder Sekundärrecht verankerten Diskriminierungsverbotes zur Geltung
kommt. So verhält es sich etwa bei der Entscheidung in der Rechtssache Mangold, die
im übrigen gerade wegen der Stärkung des sozialen Aspekts einer gewissen Kritik aus-
gesetzt war.2 Hier kam der soziale Aspekt in der Verwirklichung des Diskriminierungs-
verbotes wegen Alters zum Vorschein. 1 EuGH v. 16.05.2006, Watts, Rs. C-372/04, Slg. 2006, I-4325.
2 EuGH v. 22.11.2005, Mangold, Rs. C–144/04, Slg. 2005, I-9981. 2 EuGH v. 22.11.2005, Mangold, Rs. C–144/04, Slg. 2005, I-9981. 1 EuGH v. 16.05.2006, Watts, Rs. C-372/04, Slg. 2006, I-4325. 2. Verwirklichung sozialer Rechte Interessanterweise gibt es auch Konstellationen, in denen der Aspekt des sozialen
Europas gerade durch die Beseitigung der Beschränkung einer Europäischen Grundfrei- 39 1. Die Rolle der Entscheidungsbegründung Im Folgenden möchte ich noch auf die Methoden eingehen, die der Europäische Ge-
richtshof anwendet, um in den zuvor beschriebenen Konstellationen den sozialen As-
pekt Europas zu stärken. An erster Stelle stehen dabei die allgemeinen Rechtsgrundsätze, die vom Gerichtshof
zu den Grundrechten und insbesondere den sozialen Rechten entwickelt worden sind. Wir ziehen jedoch auch die Rechtsvergleichung der verschiedenen mitgliedstaatlichen
Rechtsordnungen heran, was zugegebenermaßen wenig transparent erscheinen mag. Ein
Vorredner sagte, dass die Begründung der Urteile des Europäischen Gerichtshofs oft zu
wünschen übrig lässt, dass oftmals nicht alle die Entscheidung tragenden Erwägungen
dargestellt werden. Das ist auch aus meiner Sicht bedauerlich und rührt nach meiner
Einschätzung wohl auch daher, dass sich der Europäische Gerichtshof von Beginn an
der französischen Tradition verpflichtet sah, in der die Begründung seit jeher sehr kurz 40 https://doi.org/10
Open Access – 10.577
– Das Europäische Sozialrecht und die Rechtsprechung des EuGH und sehr oberflächlich gehalten war. Daran hat sich aber nach meiner Anschauung
schon seit geraumer Zeit viel geändert, so dass die Urteile jetzt deutlich tiefgehender
begründet sind im Vergleich zu früheren Entscheidungen. Weiterhin ist auf die beson-
dere Rolle der Schlussanträge der Generalanwälte für das Verständnis der Urteile hin-
zuweisen. Dennoch besteht in der Urteilsbegründungspraxis des EuGH aus meiner per-
sönlichen Sicht nach wie vor ein gewisser Unterschied zur deutschen Tradition. Dies
führt dazu, dass viele Erwägungen und Abwägungen, die der Gerichtshof tatsächlich
anstellt, sich nicht in der Urteilsbegründung wiederfinden lassen, so dass es am Ende
aussehen könnte, als habe sich der Gerichtshof überhaupt nicht damit befasst. Das kann
zu Missverständnissen führen. Dennoch ist aus meiner Sicht eine durchaus positive
Entwicklung hin zu sehr eingehenden Urteilsbegründungen zu verzeichnen, in denen
die Überlegungen des Gerichtshofs – auch die verworfenen – ausführlich dargestellt
werden. Gesondert möchte ich in diesem Zusammenhang noch auf die rechtsvergleichende
Analyse hinweisen, die oftmals einen wichtigen Bestandteil in den das jeweilige Urteil
tragenden Überlegungen darstellt. Lediglich in der früheren Rechtsprechung taucht die-
se rechtsvergleichende Analyse auch tatsächlich im Urteil auf. Gegenwärtig wird oft-
mals nur das Ergebnis einer derartigen ausführlichen Analyse, die intern angefertigt
wurde, lapidar in zwei Sätzen im Urteil festgestellt. 3 EuGH v. 18.12.2007, Laval, Rs. C-341/05, Slg. 2007, I-11767. 2. Zwischen judicial activism und self-restraint Was die Rechtsfortentwicklung des europäischen Gemeinschaftsrechts angeht, sollte
ich noch erwähnen, dass der Europäische Gerichtshof nur notdürftig in den Verträgen
wurzelnde Mängel durch Anwendung gewisser rechtlicher Instrumente, Hilfskonstruk-
tionen und der allgemeinen Rechtsgrundsätze beheben bzw. abmildern kann. Denn stets
muss er den Vorwurf des judicial activism fürchten, wenn er sich zu sehr in die eigentli-
che Sphäre des Gemeinschaftsgesetzgebers einmischt oder einzumischen scheint. Der
EuGH ist daher sehr vorsichtig in seinen Formulierungen. Auf der Skala zwischen judi-
cial activism und self-restraint befindet sich der EuGH meiner Meinung nach näher am
self-restraint. Das ist jedoch meine subjektive Einschätzung und mir ist sehr wohl be-
wusst, dass man das von außerhalb auch anders beurteilen kann. Wenn wir beispiels-
weise eine notwendige Abwägung vornehmen, ist es klar, dass man eine solche Abwä-
gung kritisieren kann und auch das aus ihr folgende Ergebnis durchaus nicht kritiklos
hingenommen werden muss. Jedoch halte ich es doch für fragwürdig, dass man in dem
Abwägungsergebnis oft einen Fall von judicial activism sieht, nur weil man nach seiner
Einschätzung die Abwägung für unrichtig hält. Der tiefere Grund für dieses Problem liegt freilich darin, dass der Gemeinschaftsge-
setzgeber viele Entscheidungen, auch auf dem Gebiet der sozialen Gestaltung Europas, 41 Egils Levits wegen Uneinigkeit nicht selbst trifft, was seine Aufgabe wäre, sondern diese Arbeit auf
die Rechtsprechung abwälzt. 3. Orientierung am Gesamtinteresse und Erforderlichkeit des gesamteuropäischen
Diskurses 3. Orientierung am Gesamtinteresse und Erforderlichkeit des gesamteuropäischen
Diskurses Jene Arbeit, derer sich der Gemeinschaftsgesetzgeber in der dargestellten Weise ent-
ledigt, ist für den Gerichtshof wiederum schwierig. Man darf hier nicht aus den Augen
verlieren, dass es nicht nur in den Mitgliedstaaten unterschiedliche Meinungen, sondern
dass es in Europa auch unterschiedliche nationale Diskurse gibt. Wir zum Beispiel neh-
men hier in Berlin an einem nationalen Diskurs teil, in dem die Urteile des Gerichtshofs
auch aus einem nationalen Blickwinkel heraus beurteilt werden. Es gibt aber 27 solcher
Diskurse und die Schwerpunkte können durchaus unterschiedlich sein. So ist zum Bei-
spiel der Meinungsstand im Diskurs zu Laval3 in Polen und in Lettland, aber auch in
Großbritannien ganz anders als hier in Deutschland. Mein Wunsch wäre es, einen stär-
ker integrierten, öffentlichen wie juristischen gesamteuropäischen Diskurs zu führen,
der bereits in der Lage ist, die unterschiedlichen Interessenlagen der Mitgliedstaaten zu
berücksichtigen. Dies wäre für den Gerichtshof von herausragender Bedeutung. Ein
solcher Diskurs existiert zwar bereits, ist jedoch von relativ geringer Intensität. Das ist
eine Frage der stärkeren Integration der Öffentlichkeit in Europa, die bisher nur lang-
sam vorangekommen ist. In der Praxis stellt sich das oft so dar, dass der Gerichtshof versucht so wenig wie
möglich weitgehende Grundsatzentscheidungen zu treffen. Es wird wirklich nur das
entschieden, was in dem konkreten Fall unbedingt nötig ist und der Rest bleibt den Mit-
gliedstaaten und den anderen Gemeinschaftsorganen überlassen. Das führt zu Weilen zu einer erschwerten Lesbarkeit des Urteilstenors, aber wir ver-
suchen zum Beispiel in den Vorabentscheidungsverfahren stets soviel als möglich den
nationalen Gerichten zu überlassen, da gerade die nationalen Gerichte die primären
Gemeinschaftsgerichte sind. Soweit wir jedoch nicht auf die nationale Ebene zurück-
verweisen können, sind wir gezwungen doch zu entscheiden, zum Beispiel um einen
Ausgleich zwischen verschiedenen Zielrichtungen einer Gemeinschaftsnorm zu errei-
chen. Dabei sind wir als Gericht natürlich an die Verträge gebunden und wir versuchen
mit Augenmaß einen möglichst gerechten Ausgleich zu finden. Eine Aufgabe, die
manchmal besser und manchmal vielleicht schlechter gelingt. Wir wollen uns jedoch keinesfalls einer in der Öffentlichkeit stattfindenden Kritik
verschließen und haben auch durchaus Anlass das eine oder andere selbstkritisch zu
betrachten. Nicht nur, dass wir, wie jedes andere Gericht auch, mit Kritik leben können 42 Das Europäische Sozialrecht und die Rechtsprechung des EuGH müssen, wir sind im Gegenteil auch dankbar für eine sachgerechte Kritik. Denn sie er-
möglicht es das Problembewusstsein zu schärfen. 3. Orientierung am Gesamtinteresse und Erforderlichkeit des gesamteuropäischen
Diskurses Ich möchte jedoch an dieser Stelle nochmals betonen, dass der Gerichtshof immer
das gesamte Europa im Blick hat und haben muss und daher auf einen Ausgleich auf
gesamteuropäischer Ebene hinwirken muss. Diese Tatsache gilt es, meiner persönlichen
Überzeugung nach, bei nationalen Diskursen und Streitständen stets zu berücksichtigen. Denn der Ausgleich muss ein Ausgleich sein, der dann in allen 27 Mitgliedstaaten als
mehr oder weniger gerecht empfunden werden kann. Fokussiert man eine Diskussion
jedoch lediglich auf eine nationale Ebene, gelangt man im Rahmen des Findungsprozes-
ses für einen gerechten Ausgleich häufig zu einem anderen Ergebnis, das, vom europäi-
schen Standpunkt aus gesehen, verzerrt wäre. Da wir als Europäischer Gerichtshof immer Gesamteuropa im Blick haben, wäre es
für uns daher auch sehr nützlich, ein gesamteuropäisches Feedback zu bekommen. Es wird schließlich häufig angeführt, dass Europa an einem Mangel an politischer
Öffentlichkeit leidet. Das ist in der Tat ein wichtiges politisches und legitimatorisches
Problem für die Europäische Union. Aber es wäre meiner Meinung nach auch wichtig
und ein Schritt in die richtige Richtung, wenn es uns gelänge, wenigstens die juristische
Öffentlichkeit auf europäischer Ebene zu stärken und einen entsprechenden integrierten
juristischen Diskurs zu schaffen, was für die europäische Juristenwelt und für unseren
Gerichtshof ein großer Fortschritt wäre. 43 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb 783845219165, am 24.10.2024, 06:46:57
- https://www.nomos-elibrary.de/agb https://doi.org/10.5771/97838
Open Access –
- 1 Auf Grund der mir zur Verfügung stehenden Zeit sind lediglich die Ausführungen zur Zweiten The-
se - und diese auch nur in einer verkürzten Form - vorgetragen worden. Das soziale Europa aus der Perspektive der
deutschen Gerichtsbarkeit Ulrich Koch rste These
46
weite These
47
1. Vorbemerkung
48
2. Fehlende Verankerung in den gemeinsamen Verfassungstraditionen der
Mitgliedstaaten
48
3. Mögliche Berücksichtigung des nationalen Rechts bei der
gemeinschaftsrechtlichen Ausgestaltung des Streikrechts
49
4. Widersprüche in der Ausgestaltung des Streikrechts durch den europäischen
Gerichtshof gegenüber dem nationalem Recht
50
5. Die Abwägung zwischen Grundfreiheiten und Grundrechten
50
6. Die Prüfung unter dem Gesichtspunkt der Verhältnismäßigkeit
51
7. Die Konkurrenzsituation zwischen Gemeinschaftsrecht und nationalem Recht
52
ritte These
53
1. Die Entscheidung Werhof
54
2. Der Super-GAU – der Konflikt zwischen Gemeinschafts- und nationalem
Verfassungsrecht
54 Ulrich Koch besser noch - zu einem Miteinander von Gemeinschaftsrecht und nationalem Recht -
werden sowohl der Europäische Gerichtshof wie auch die deutschen Arbeitsgerichte
immer wieder vor neue Herausforderungen gestellt. Über drei aus meiner Sicht aktuelle
Bauvorhaben im Rahmen der Großbaustelle „Gemeinschaftsrecht und Arbeitsgerichts-
barkeit“ möchte ich gerne sprechen. Es geht zunächst um die Frage der Anwendbarkeit der allgemeinen Grundsätze des
Gemeinschaftsrechts im nationalen Recht. Anschließend werde ich im Bereich des kol-
lektiven Rechts auf mögliche Friktionen zwischen den beiden Rechtsquellen eingehen
und mit Ausführungen zu zwei aktuellen Konfliktfällen zwischen dem Gemeinschafts-
recht und dem nationalen Recht schließen. Den Schwerpunkt der Betrachtungen werden
dabei die Entscheidungen des Gerichtshofs in den Rechtssachen Mangold, Viking und
Werhof bilden. Die Urteile in den Verfahren Rüffert und teilweise auch Laval mögen
zwar für die Politik einen Handlungsbedarf begründen, sie haben aber – anders als die
erstgenannten Entscheidungen – für die Gerichtsbarkeit keine herausragende Wirkung. Erste These 1. Vorbemerkung g
2. Fehlende Verankerung in den gemeinsamen Verfassungstraditionen der
Mitgliedstaaten
48 g
3. Mögliche Berücksichtigung des nationalen Rechts bei der
gemeinschaftsrechtlichen Ausgestaltung des Streikrechts g
g
g
4. Widersprüche in der Ausgestaltung des Streikrechts durch den europäischen
Gerichtshof gegenüber dem nationalem Recht 6. Die Prüfung unter dem Gesichtspunkt der V 1. Die Entscheidung Werhof
d 2. Der Super-GAU – der Konflikt zwischen Gemeinschafts- und nationalem
Verfassungsrecht Mit meinen Ausführungen werde ich nicht in das teilweise in der Politik und bei den
Verbänden zu vernehmende Klagelied darüber einstimmen, dass der Europäische Ge-
richtshof einem Sozialdumping Vorschub leistet. Eine solche Bewertung der Rechtspre-
chung des Gerichtshofs, noch dazu im Rahmen dieser Veranstaltung, steht mir nicht zu. Ich werde mich im Folgenden auf drei Themen mit einem Bezug zu meiner richterli-
chen Tätigkeit beschränken, die ich auch wegen der mir zur Verfügung stehenden Zeit
nur kurz anreißen kann, von denen ich aber hoffe, dass sie interessant genug sind, von
Ihnen in der Diskussion aufgegriffen zu werden.1 Der Anwendungsvorrang des Gemeinschaftsrechts eröffnet der deutschen Arbeitsge-
richtsbarkeit die Möglichkeit, das nationale Recht und seine vermeintlich nicht mehr
zeitgemäßen Regelungen unbeachtet zu lassen. Kommt es aber zu einem Nebeneinander 45 /10.5771/97
– Ulrich Koch Ulrich Koch Erste These Erste These Es fehlen präzise Kriterien, nach denen ein nationales Gericht beurteilen kann, ob
ein allgemeiner Grundsatz des Gemeinschaftsrechts auf den von ihm zu beurteilenden
Sachverhalt anzuwenden ist. Ausgangspunkt für diese These ist der Sachverhalt, der der Entscheidung des Ge-
richtshofs in der Rechtssache Mangold zugrunde lag. Zwei offenbar gut miteinander
bekannte Rechtsanwälte hatten einen befristeten Arbeitsvertrag geschlossen und seine
Rechtfertigung ausschließlich auf § 14 Abs. 3 Satz 4 TzBfG in der bis zum 30. April
2007 geltenden Fassung gestützt, und damit auf eine Vorschrift, die sie beide für un-
wirksam hielten. Das Arbeitsgericht München hatte zumindest Zweifel an der Verein-
barkeit von § 14 Abs. 3 TzBfG mit dem Gemeinschaftsrecht und legte diese Frage dem
Gerichtshof im Rahmen eines Vorabentscheidungsverfahrens vor. Der Gerichtshof hat auf den Sachverhalt, in der es ausschließlich um die Rechtferti-
gung einer sachgrundlosen Befristung zwischen zwei Privatpersonen ging, den primär-
rechtlichen Grundsatz der Gleichbehandlung in Beschäftigung und Beruf herangezogen,
der Diskriminierungen in Hinblick auf das Alter entgegensteht. Der Gerichtshof hat sich
bei seinen Aussagen zum Anwendungsbereich des Gemeinschaftsrechts auf die Feststel-
lung beschränkt, dass die in § 14 Abs. 3 Satz 4 TzBfG aF enthaltene Regelung in den
Geltungsbereich des Gemeinschaftsrechts fällt, weil der nationale Gesetzgeber mit dem
TzBfG die Vorgaben der Richtlinie 1999/70 umsetzen wollte. Der Gerichtshof hat dazu
weiter ausgeführt, dass damit alle nationalen Maßnahmen erfasst seien, die die Errei-
chung des mit der (Befristungs-)Richtlinie verfolgten Zieles gewährleisten sollen, ein- 46 Das soziale Europa aus der Perspektive der deutschen Gerichtsbarkeit schließlich derjenigen, mit denen nach der eigentlichen Umsetzung die bereits erlasse-
nen nationalen Rechtsvorschriften ergänzt oder geändert werden. istungsrichtlinie dient aber nur dazu, Die Befristungsrichtlinie dient aber nur dazu, rch die Anwendung des Grundsatzes der Nichtdiskriminierung die Qualität be − durch die Anwendung des Grundsatzes der Nichtdiskriminierung die Qualität be-
fristeter Arbeitsverhältnisse zu verbessern; − durch die Anwendung des Grundsatzes der Nichtdiskriminierung die Qualität be-
fristeter Arbeitsverhältnisse zu verbessern; − einen Rahmen zu schaffen, der den Missbrauch durch aufeinander folgende be-
fristete Arbeitsverträge oder -verhältnisse verhindert. − einen Rahmen zu schaffen, der den Missbrauch durch aufeinander folgende be-
fristete Arbeitsverträge oder -verhältnisse verhindert. Keines der beiden Ziele steht mit dem in der Entscheidung Mangold behandelten
Sachverhalt in Beziehung. Bei dem zur Prüfung gestellten befristeten Arbeitsvertrag
handelte es sich um den erstmaligen Abschluss eines befristeten Arbeitsvertrags, so dass
ein Missbrauch durch aufeinander folgende befristete Arbeitsverträge ausgeschlossen
war. 2 Zweifel an der Reichweite des Grundsatzes der Gleichbehandlung in Beschäftigung und Beruf ha-
ben den Dritten Senat des BAG zu einem Vorabentscheidungsersuchen veranlasst, BAG vom 27.
Juni 2006, 3 AZR 352/05 (A), BAGE 118, 340 = AP BetrAVG § 1b Nr. 6 = EzA Richtlinie 2000/78
EG-Vertrag 1999 Nr. 2, Rn. 29. 3 EuGH vom 11. Dezember 2007, C-438/05 (Viking), EzA GG Art. 9 Arbeitskampf Nr. 141 = NZA
2008, 124, Rn. 44. Erste These Auch eine Diskriminierung von Herrn Mangold gegenüber einem unbefristet be-
schäftigten Arbeitnehmer ist nach dem mitgeteilten Sachverhalt nicht geltend gemacht
worden. Die einzige Verbindung, die ich sehe, ist der Abschluss eines befristeten Ar-
beitsvertrags, dessen Rechtfertigung sich nach den Bestimmungen des TzBfG bestimm-
te.2 Wie hätte ein nationales Gericht bis zum Ablauf der Umsetzungsfrist der Gleichbe-
handlungsrichtlinie entscheiden müssen, wenn die Bundesrepublik innerhalb der Um-
setzungsfrist der Gleichbehandlungsrichtlinie § 1 des Kündigungsschutzgesetzes um
einen Absatz ergänzt hätte, nach dem für die Zeit von drei Jahren unbefristet eingestellte
Arbeitnehmer, die das 52. Lebensjahr vollendet haben, erst nach Ablauf von 24 Mona-
ten Kündigungsschutz erwerben? Wäre eine solche Regelung auch in den Anwendungs-
bereich des Gemeinschaftsrechts gefallen? Zweite These 4 Rebhahn ZESAR 2008, 109, 114.
5 An dieser Stelle sei ausdrücklich auf die im Rahmen des Symposiums gehaltenen Ausführungen
von Frau Kokott (S. 23 ff.) und Herrn Rebhahn (S. 17 ff.) verwiesen.
6 EuGH vom Dezember 2007, Viking, C-438/05, EzA GG Art. 9 Arbeitskampf Nr. 141 = NZA 2008,
124, Rn. 44.
7 EuGH 14. Mai 1974, Nold, Rs. 4/73, EuGHE 1974, 491. 1. Vorbemerkung Die Anerkennung eines Grundrechts auf kollektive Maßnahmen einschließlich des
Streikrechts ist meines Erachtens zu begrüßen. Dies gilt aber nur eingeschränkt für seine
dogmatische Herleitung und die inhaltliche Ausgestaltung des Grundrechts. Allerdings
können die genanten Entscheidungen wegen der Besonderheiten in den zugrunde lie-
genden Sachverhalten wohl nur ein erster Fingerzeig auf die zukünftige gemeinschafts-
rechtliche Ausgestaltung des Arbeitskampfrechts sein. In beiden Entscheidungen ging
es nicht um einem gewerkschaftlich getragenen Streik von Arbeitnehmern gegen ihren
eigenen Arbeitgeber zur Verbesserung ihrer Arbeitsbedingungen. Bei den Kollektiv-
maßnahmen handelte es sich ausschließlich oder zumindest in der Hauptsache um Boy-
kottmaßnahmen, die sich gegen grenzüberschreitende Marktaktivitäten eines Dritten
richteten.4 und die nachfolgende Entscheidung Laval für die deutschen Arbeitsgerichte enthält und
ob dieses „Rüstzeug“ für eine Streitentscheidung ausreichend bemessen ist. und die nachfolgende Entscheidung Laval für die deutschen Arbeitsgerichte enthält und
ob dieses „Rüstzeug“ für eine Streitentscheidung ausreichend bemessen ist. Ulrich Koch und die nachfolgende Entscheidung Laval für die deutschen Arbeitsgerichte enthält und
ob dieses „Rüstzeug“ für eine Streitentscheidung ausreichend bemessen ist. Zweite These Der Einfluss des nationalen Rechts auf Arbeitskämpfe mit Gemeinschaftsbezug ist für
die deutschen Arbeitsgerichte durch die Entscheidung des Europäischen Gerichtshofs
nur teilweise geklärt. Der Europäische Gerichtshof hat in der Entscheidung in der Rechtssache Viking das
Recht auf Durchführung einer kollektiven Maßnahme einschließlich des Streikrechts als
Grundrecht anerkannt, welches fester Bestandteil der allgemeinen Grundsätze des Ge-
meinschaftsrechts sei.3 Ich werde nachfolgend darauf eingehen, welche Vorgaben diese 2 Zweifel an der Reichweite des Grundsatzes der Gleichbehandlung in Beschäftigung und Beruf ha-
ben den Dritten Senat des BAG zu einem Vorabentscheidungsersuchen veranlasst, BAG vom 27. Juni 2006, 3 AZR 352/05 (A), BAGE 118, 340 = AP BetrAVG § 1b Nr. 6 = EzA Richtlinie 2000/78
EG-Vertrag 1999 Nr. 2, Rn. 29. 47 /10.5771/
– Ulrich Koch Ulrich Koch 8 Der dabei gebotene Rekurs auf die Ausgestaltung des Arbeitskampfrechts in den Mitgliedstaaten
hätte möglicherweise auch keinen gemeinschaftsweit anerkannten Mindestanstandart ergeben, auf
dessen Grundlage ein Gemeinschaftsgrundrecht hätte hergeleitet werden können. 9 Dazu ausführlich Rebhahn ZESAR 2008, 109, 110 ff.; die Herleitung und die inhaltliche Ausgestal-
tung eines so bedeutenden Gemeinschaftsgrundrechts, dass sich nicht mehr – und insbesondere
nicht in der vom Gerichtshof gegebenen Form – an gemeinsamen Verfassungstraditionen der Mit-
gliedstaaten und den von ihnen abgeschlossenen völkerrechtlichen Verträgen orientiert, wird – wie
schon die Herleitung des Grundsatzes der Gleichbehandlung in Beschäftigung und Beruf – die Fra-
ge nach einer entsprechenden Kompetenz des Gerichtshofs aufwerfen. 2. Fehlende Verankerung in den gemeinsamen Verfassungstraditionen der Mitglied-
staaten5 Trotz des Vorbehalts in Art. 137 Abs. 5 des EG-Vertrags, der von der Regelungs-
kompetenz der Gemeinschaft ua. das Koalitionsrecht, das Streikrecht und das Aussper-
rungsrecht ausnimmt, hat der Europäische Gerichtshof das Recht auf Durchführung
einer kollektiven Maßnahme einschließlich des Streikrechts als Grundrecht anerkannt,
welches fester Bestandteil der allgemeinen Grundsätze des Gemeinschaftsrechts sei.6 Der Gerichtshof hat in der Vergangenheit ungeschriebene Gemeinschaftsgrundrechte
aus den gemeinsamen Verfassungstraditionen der Mitgliedstaaten und den völkerrecht-
lichen Verträgen insbesondere der EMRK abgeleitet. Dies beruht letztlich auf Art. 6
Abs. 2 des Vertrags über die Europäische Union, mit dem die vorangegangene Recht-
sprechung des Gerichtshofs zur Herleitung von Gemeinschaftsgrundrechten übernom-
men worden ist.7 Von diesen Vorgaben scheint sich der Gerichtshof in seinen Urteilen
in den Rechtssachen Viking und Laval zumindest teilweise gelöst zu haben. Er hat das
Grundrecht nicht auf die gemeinsamen Verfassungstraditionen der Mitgliedstaaten ge- 48 Das soziale Europa aus der Perspektive der deutschen Gerichtsbarkeit stützt,8 sondern auf vier internationale Abkommen, von denen eines – die Charta der
Grundrechte der Europäischen Union – noch nicht rechtsverbindlich ist. Auch in den
weiteren vom Gerichtshof angeführten Abkommen haben nicht alle Mitgliedstaaten das
Streikrecht für sich als verbindlich anerkannt.9 3. Mögliche Berücksichtigung des nationalen Rechts bei der gemeinschaftsrechtli-
chen Ausgestaltung des Streikrechts 3. Mögliche Berücksichtigung des nationalen Rechts bei der gemeinschaftsrechtli-
chen Ausgestaltung des Streikrechts Weil der Gerichtshof das Streikrecht ua. aus Art. 28 der Grundrechtscharta herleitet,
der neben dem Gemeinschaftsrecht auf die einzelstaatlichen Rechtsvorschriften und
Gepflogenheiten Bezug nimmt, geht er in den Entscheidungen Viking und Laval auf
den Inhalt des finnischen10 bzw. schwedischen11 Rechts ein und stellt fest, dass sich
das Gemeinschaftsrecht bei gemeinschaftsbezogenen Arbeitskämpfen wegen des je-
weils im nationalen Recht vorhandenen Vorbehalts durchsetzt. Dass man einen solchen
Vorbehalt auch für das deutsche Recht anerkennen muss, kann vom Ergebnis her
schwerlich in Frage gestellt werden.12 Allerdings ist der Gerichtshof – stärker als bisher
erkennbar – gehalten, bei der Ausgestaltung des Grundrechts auf kollektive Maßnahmen
dem Arbeitskampfrecht des jeweiligen Mitgliedstaats einen größeren Einfluss einzu-
räumen. Dies folgt aus dem in Art. 137 Abs. 5 EG ausdrücklichen enthaltenen Vorbe-
halt zu Gunsten der Mitgliedstaaten.13 8 Der dabei gebotene Rekurs auf die Ausgestaltung des Arbeitskampfrechts in den Mitgliedstaaten
hätte möglicherweise auch keinen gemeinschaftsweit anerkannten Mindestanstandart ergeben, auf
dessen Grundlage ein Gemeinschaftsgrundrecht hätte hergeleitet werden können. 9 Dazu ausführlich Rebhahn ZESAR 2008, 109, 110 ff.; die Herleitung und die inhaltliche Ausgestal-
tung eines so bedeutenden Gemeinschaftsgrundrechts, dass sich nicht mehr – und insbesondere
nicht in der vom Gerichtshof gegebenen Form – an gemeinsamen Verfassungstraditionen der Mit-
gliedstaaten und den von ihnen abgeschlossenen völkerrechtlichen Verträgen orientiert, wird – wie
schon die Herleitung des Grundsatzes der Gleichbehandlung in Beschäftigung und Beruf – die Fra-
ge nach einer entsprechenden Kompetenz des Gerichtshofs aufwerfen. 10 EuGH vom 11. Dezember 2007, Viking, C-438/05, EzA GG Art. 9 Arbeitskampf Nr. 141 = NZA
2008, 124, Rn. 44: „Außerdem kann das Streikrecht, wie aus Randnr. 5 des vorliegenden Urteils
hervorgeht, nach finnischem Recht u. a. dann nicht ausgeübt werden, wenn der Streik gegen die gu-
ten Sitten, das innerstaatliche Recht oder das Gemeinschaftsrecht verstoßen würde.“ 10 EuGH vom 11. Dezember 2007, Viking, C-438/05, EzA GG Art. 9 Arbeitskampf Nr. 141 = NZA
2008, 124, Rn. 44: „Außerdem kann das Streikrecht, wie aus Randnr. 5 des vorliegenden Urteils
hervorgeht, nach finnischem Recht u. a. dann nicht ausgeübt werden, wenn der Streik gegen die gu-
ten Sitten, das innerstaatliche Recht oder das Gemeinschaftsrecht verstoßen würde.“ 11 EuGH vom 18. Dezember 2007, Laval, C-341/05, EzA GG Art. 9 Arbeitskampf Nr. 142 = NZA
2008, 159, Rn. 10 EuGH vom 11. Dezember 2007, Viking, C-438/05, EzA GG Art. 9 Arbeitskampf Nr. 141 = NZA
2008, 124, Rn. 44: „Außerdem kann das Streikrecht, wie aus Randnr. 5 des vorliegenden Urteils
hervorgeht, nach finnischem Recht u. a. dann nicht ausgeübt werden, wenn der Streik gegen die gu-
ten Sitten, das innerstaatliche Recht oder das Gemeinschaftsrecht verstoßen würde.“ 4. Widersprüche in der Ausgestaltung des Streikrechts durch den europäischen Ge-
richtshof gegenüber dem nationalem Recht Nach der Entscheidung des Gerichtshofs in der Rechtssache Viking ist die durch das
Grundrecht auf kollektive Maßnahmen geschützte gemeinschaftsrechtliche Position
teilweise weiter gehend als nach nationalem Recht. Der Gerichtshof hat weder die
Übernahme der Arbeitskampfmaßnahmen durch eine Gewerkschaft gefordert noch her-
vorgehoben, dass die kollektiven Maßnahmen auf ein tariflich regelbares Ziel gerichtet
sein müssen. Daher müssten – anders als nach nationalem Recht – auch ein wilder, d.h. gewerkschaftlich nicht getragener Streik und andere kollektive Maßnahmen zur Verbes-
serung der Arbeitsbedingungen wie zB. die Ausübung eines kollektiven Zurückbehal-
tungsrechts oder ein kollektiver Widerspruch gegen einen Betriebsübergang von dem
Gemeinschaftsgrundrecht geschützt sein. Gleiches könnte für einen reinen Sympathie-
oder einen politischen Streik gelten. Insoweit ist die Anerkennung des Streikrechts für die Verbände durchaus ambiva-
lent: die Ausgestaltung des Arbeitskampfrechts durch Art. 9 Abs. 3 GG schützt den Ar-
beitgeber vor unzulässigen Kampfformen und -mitteln der Gewerkschaft. Er müsste
aber einen auf ein tariflich nicht regelbares Ziel gerichteten Arbeitskampf hinnehmen,
wenn der Arbeitskampf wegen der gewerkschaftlichen Forderung in den Anwendungs-
bereich des Gemeinschaftsrechts fällt und die geltend gemachte Forderung den Anfor-
derungen des gemeinschaftsrechtlichen Verhältnismäßigkeitsprinzips genügt. Zu diesen Friktionen gegenüber dem nationalen Recht kommt eine weitere Proble-
matik hinzu, die der dogmatischen Herleitung des Streikrechts geschuldet ist. Art. 28
der Grundrechtscharta gewährt das Recht auf Kollektivmaßnahmen ua. nach den einzel-
staatlichen Rechtsvorschriften und Gepflogenheiten. Wird daher das Streikrecht bei
gemeinschaftsrechtsrelevanten Arbeitskämpfen einer doppelten Schranke unterworfen? Müssen die kämpfenden Verbände bei der Wahrnehmung des „Grundrechts“ danach
sowohl die gemeinschaftsrechtlichen Vorgaben als auch die des nationalen Rechts be-
achten? 3. Mögliche Berücksichtigung des nationalen Rechts bei der gemeinschaftsrechtli-
chen Ausgestaltung des Streikrechts 92: „Es trifft zwar zu, wie die schwedische Regierung in Erinnerung ruft, dass das
Recht auf Durchführung kollektiver Maßnahmen in Schweden ebenso wie in anderen Mitgliedstaa-
ten den Schutz der Verfassung genießt, doch ändert dies nichts daran, dass, wie sich aus Randnr. 10
des vorliegenden Urteils ergibt, dieses Recht, das in diesem Mitgliedstaat die Blockade von Baustel-
len umfasst, nach der schwedischen Verfassung ausgeübt werden kann, wenn gesetzliche oder ver-
tragliche Bestimmungen dem nicht entgegenstehen.“ 11 EuGH vom 18. Dezember 2007, Laval, C-341/05, EzA GG Art. 9 Arbeitskampf Nr. 142 = NZA
2008, 159, Rn. 92: „Es trifft zwar zu, wie die schwedische Regierung in Erinnerung ruft, dass das
Recht auf Durchführung kollektiver Maßnahmen in Schweden ebenso wie in anderen Mitgliedstaa-
ten den Schutz der Verfassung genießt, doch ändert dies nichts daran, dass, wie sich aus Randnr. 10
des vorliegenden Urteils ergibt, dieses Recht, das in diesem Mitgliedstaat die Blockade von Baustel-
len umfasst, nach der schwedischen Verfassung ausgeübt werden kann, wenn gesetzliche oder ver-
tragliche Bestimmungen dem nicht entgegenstehen.“ g
g
g g
12 Dafür auch Zwanziger, DB 2008, 294, 295, der Art. 23 Abs. 1 GG als ausreichende Ermächtigungs-
norm ansieht. 13 Im Rahmen meines Vortrags habe ich hierzu die Frage nach der dogmatischen Verankerung des
Vorbehalts und seiner Existenz im nationalen Recht aufgeworfen. 13 Im Rahmen meines Vortrags habe ich hierzu die Frage nach der dogmatischen Verankerung des
Vorbehalts und seiner Existenz im nationalen Recht aufgeworfen. 49 49 Ulrich Koch 4. Widersprüche in der Ausgestaltung des Streikrechts durch den europäischen Ge-
richtshof gegenüber dem nationalem Recht 4. Widersprüche in der Ausgestaltung des Streikrechts durch den europäischen Ge-
richtshof gegenüber dem nationalem Recht 16 Blanke, Die Entscheidungen des EuGH in den Fällen Viking, Laval und Rueffert – Domestizierung
des Streikrechts und europaweite Nivellierung der industriellen Beziehungen, in: Oldenburger Stu-
dien zur Europäisierung und zur transnationalen Regulierung, S. 12, abzurufen unter
http://www.uni‐ oldenburg.de/cetro/31251.htm; Kocher ArbuR 2008, 13, 14 f.; Zwanziger, DB
2008, 294, 295 f. 5. Die Abwägung zwischen Grundfreiheiten und Grundrechten Zu den zu Recht als problematisch angesehenen Teilen der Entscheidung Viking ge-
hört das Verhältnis der Grundfreiheiten zu den Gemeinschaftsgrundrechten. Dabei
wählt der Gerichtshof bei seiner Prüfung einen gegenüber seiner Entscheidung in der
Rechtssache Schmidberger veränderten Ansatz. Dort hatte er noch ausgeführt, dass die
Anwendung der dort betroffenen Grundrechte der Meinungs- und Versammlungsfrei-
heit mit den Erfordernissen hinsichtlich der durch den Vertrag geschützten Rechte in 50 Das soziale Europa aus der Perspektive der deutschen Gerichtsbarkeit Einklang gebracht werden und dem Verhältnismäßigkeitsgrundsatz entsprechen muss.14
Dies konnte als Anzeichen für die Gleichrangigkeit der widerstreitenden Interessen an-
gesehen werden. Ähnliche Formulierungen verwendet er zwar auch in der Rechtssache
Viking,15 eine Einschränkung der Grundfreiheiten im Lichte der Grundrechte und eine
Abwägung zwischen „wirtschaftlichen Freiheiten“ und „sozialen Grundrechten“ im
Sinne einer praktischen Konkordanz unterbleibt jedoch.16 Ein solcher Ansatz zu Guns-
ten der Grundfreiheiten wirkt sich jedoch notwendigerweise auf die inhaltliche Prüfung
des Eingriffs und ihr Ergebnis aus. Ist die Tarifforderungen erhebende Arbeitnehmersei-
te bei einem Arbeitskampf in Zukunft nach Europarecht stets gezwungen, ihre Forde-
rungen und Kampfmittel – ggf. vor den Arbeitsgerichten – zu rechtfertigen? 14 EuGH vom 12. Juni 2003, Schmidberger, C-112/00, EuGHE I 2003, 5659, Rn. 74. 15 EuGH vom 11. Dezember 2007, Viking, C-438/05, EzA GG Art. 9 Arbeitskampf Nr. 141 = NZA
2008, 124, Rn. 79. 17 EuGH vom 18. Dezember 2007, Laval, C-341/05, EzA GG Art. 9 Arbeitskampf Nr. 142 = NZA
2008, 159, Rn. 104 f.; 11. Dezember 2007, Viking, C-438/05, EzA GG Art. 9 Arbeitskampf Nr. 141
= NZA 2008, 124, Rn. 78 f. 14 EuGH vom 12. Juni 2003, Schmidberger, C-112/00, EuGHE I 2003, 5659, Rn. 74.
15 EuGH vom 11. Dezember 2007, Viking, C-438/05, EzA GG Art. 9 Arbeitskampf Nr. 141 = NZA 18 EuGH 11. Dezember 2007, Viking, C-438/05, EzA GG Art. 9 Arbeitskampf Nr. 141 = NZA 2008,
124, Rn. 85. 20 Dazu Rebhahn, ZESAR 2008, 109, 116 f.
21 EuGH vom 11. Dezember 2007, Viking, C-438/05, EzA GG Art. 9 Arbeitskampf Nr. 141 = NZA
2008, 124, Rn. 72.
22 EuGH 18. Dezember 2007, Laval, C-341/05, EzA GG Art. 9 Arbeitskampf Nr. 142 = NZA 2008,
159, Rn. 99.
23 Ebenso Rebhahn, ZESAR 2008, 109, 115; anders wohl Temming, ZESAR 2008, 231, 241. Ulrich Koch schränkung ist für Tarifauseinandersetzungen um bessere Arbeitsbedingungen oder den
Erhalt von gefährdeten Arbeitsplätzen nicht von Bedeutung. schränkung ist für Tarifauseinandersetzungen um bessere Arbeitsbedingungen oder den
Erhalt von gefährdeten Arbeitsplätzen nicht von Bedeutung. 6. Die Prüfung unter dem Gesichtspunkt der Verhältnismäßigkeit Steht fest, dass der Arbeitskampf in eine oder vielleicht sogar mehrere Grundfreihei-
ten eingreift, ist nach der Rechtsprechung des Gerichtshofs über die „Rechtfertigung“
dieses Eingriffs nach Verhältnismäßigkeitsgesichtspunkten zu entscheiden. Die Rechte
aus den Grundfreiheiten müssen gegen die mit der Sozialpolitik verfolgten Ziele abge-
wogen werden.17 Im Rahmen dieser Abwägung wird vom Gerichtshof der Inhalt des
Gemeinschaftsgrundrechts nicht mehr erwähnt. Diese Beurteilung obliegt dem nationa-
len Gericht, das „allein für die Beurteilung des Sachverhalts und die Auslegung der na-
tionalen Rechtsvorschriften zuständig ist, zu bestimmen, ob und inwieweit die genannte
kollektive Maßnahme diesen Anforderungen entspricht“.18 Für die Prüfung der Verhältnismäßigkeit macht der Gerichtshof dem nationalen Ge-
richt in der Entscheidung Viking nur wenige Vorgaben, lediglich dass es an einem legi-
timen Ziel für die Arbeitskampfmaßnahmen fehlt, wenn „die fraglichen Arbeitsplätze
oder Arbeitsbedingungen nicht gefährdet oder ernstlich bedroht waren“.19 Diese Ein- 17 EuGH vom 18. Dezember 2007, Laval, C-341/05, EzA GG Art. 9 Arbeitskampf Nr. 142 = NZA
2008, 159, Rn. 104 f.; 11. Dezember 2007, Viking, C-438/05, EzA GG Art. 9 Arbeitskampf Nr. 141
= NZA 2008, 124, Rn. 78 f. 19 EuGH 11. Dezember 2007, Viking, C-438/05, EzA GG Art. 9 Arbeitskampf Nr. 141 = NZA 2008,
124, Rn. 81. 19 EuGH 11. Dezember 2007, Viking, C-438/05, EzA GG Art. 9 Arbeitskampf Nr. 141 = NZA 2008,
124, Rn. 81. 51 /
/
– Ulrich Koch 7. Die Konkurrenzsituation zwischen Gemeinschaftsrecht und nationalem Recht Nach den Entscheidungen Viking und Laval wird zukünftig zwischen Arbeitskämp-
fen zu differenzieren sein, in denen die Grundfreiheiten der betroffenen Unternehmen
beeinträchtigt werden und die deshalb in den Anwendungsbereich des Gemeinschafts-
rechts fallen und solchen ohne Gemeinschaftsbezug, die sich wie bisher nach nationa-
lem Recht beurteilen. Maßgeblich ist danach, ob auf Seiten der betroffenen Unterneh-
men durch die erhobenen Forderungen in die Grundfreiheiten des EG-Vertrags einge-
griffen werden soll. Als solche kommen nach der Entscheidung des Gerichtshofs insbesondere die
Dienstleistungsfreiheit (Art. 49 EG), die Niederlassungsfreiheit (Art. 43 EG) und wohl
auch die Warenverkehrsfreiheit des Art. 28 EG in Betracht.20 Die Niederlassungsfrei-
heit (Art. 43 EG) wird nach der Rechtsprechung des Gerichtshofs beeinträchtigt, wenn
es sich um eine kollektive Maßnahme handelt, die im Fall ihrer Durchsetzung geeignet
wäre, es für das betroffene Unternehmen weniger attraktiv und sogar zwecklos zu ma-
chen, von seiner Niederlassungsfreiheit Gebrauch zu machen.21 Die Dienstleistungs-
freiheit des Art. 49 EG ist betroffen, wenn die geforderten Arbeitsbedingungen für die
entsandten Arbeitnehmer über die in dem anderen Mitgliedstaat für die dort beschäftig-
ten Arbeitnehmer geltenden Mindestarbeitsbedingungen hinausgehen.22 Zumindest die vom Gerichtshof verwandte Formulierung des Eingriffs in die Nieder-
lassungsfreiheit erlaubt keine trennscharfe Abgrenzung von Arbeitskämpfen, die sich
nach Gemeinschaftsrecht bzw. nach nationalem Recht beurteilen. So dürfte die Nieder-
lassungsfreiheit noch nicht bei einer gewerkschaftlichen Forderung auf Abschluss eines
Tarifsozialplans betroffen sein, der die Verlagerung eines Unternehmens in einen ande-
ren Mitgliedstaat „verteuert“.23 Eindeutig ist das aber nicht. Ebenso bleibt fraglich, ob
eine gegenüber einer ausländischen Gesellschaft mit Sitz in einem anderen Mitgliedstaat
oder ihrer deutschen Tochter erhobene Tarifforderung zu einem Gemeinschaftsbezug
führt, weil der mit einem Tarifvertragsabschluss verbundene Anstieg der Personalkosten
die Attraktivität des hiesigen Standorts schmälert. Ist für ein nationales Arbeitsgericht zweifelhaft, ob ein Arbeitskampf, dessen Recht-
mäßigkeit es zu beurteilen hat, in den Anwendungsbereich des Gemeinschaftsrechts 52 0.5
– Das soziale Europa aus der Perspektive der deutschen Gerichtsbarkeit fällt, ist es im Rahmen des Art. 243 EG berechtigt bzw. verpflichtet, über die Reichwei-
te der Grundfreiheiten den Gerichtshof im Rahmen eines Vorabentscheidungsverfahrens
anzurufen. Dieses könnte zB. die Frage betreffen, ob ein Streik bei Eisenbahngesell-
schaften die Warenverkehrsfreiheit beeinträchtigt oder auch ein politischer Streik von
dem Grundrecht auf kollektive Maßnahmen geschützt wird. 24 Auch dieser Teil konnte aus Zeitgründen nicht mündlich vorgetragen werden. 7. Die Konkurrenzsituation zwischen Gemeinschaftsrecht und nationalem Recht g
Korrigiert der Gerichtshof seine umstrittene Dogmatik zum Verhältnis von Grund-
freiheiten und „Grundrechten“ nicht, so bleibt für die Berücksichtigung der bisherigen
Grundsätze des nationalen Arbeitskampfrechts nur die Verhältnismäßigkeitsprüfung im
Rahmen der Rechtfertigung des Eingriffs in die Grundfreiheiten. An dieser Stelle ist –
spätestens – der Vorbehalt des Art. 137 Abs. 5 EG von Bedeutung. Dieser ermöglicht
den nationalen Gerichten eine gemeinschaftsrechtsgeleitete Prüfung unter Heranziehung
der in der Vergangenheit von Bundesverfassungsgericht und Bundesarbeitsgericht ent-
wickelten Grundsätze. Die gerichtliche Kontrolle des Umfangs der erhobenen Tariffor-
derungen oder die Wahl der zu ihrer Durchsetzung eingesetzten Mittel würde danach –
von Extremfällen abgesehen – auch zukünftig unterbleiben. 25 EuGH vom 9. März 2006, Werhof, C-449/04, EuGHE I 2006, 2397 = AP Richtlinie 77/187/EWG
Nr. 2 = EzA BGB 2002 § 613a Nr. 44, Rn. 34.
26 Bauer/Arnold, NJW 2006, 6, 8; wohl auch Nicolai,DB 2005, 2641, 2642.
27 BAG 26. April 2006, 7 AZR 500/04, BAGE 118, 76 = AP TzBfG § 14 Nr. 23 = EzA TzBfG § 14
Nr. 28, Rn. 17.
28 Az. 2 BvR 2661/06. Dritte These Dritte These Der Europäische Gerichtshof sollte in Entscheidungen, die das Recht der Mitglied-
staaten erstmals dem Anwendungsbereich des Gemeinschaftsrechts unterwerfen, einen
nachvollziehbaren Begründungsansatz wählen.24 Der Europäische Gerichtshof sollte in Entscheidungen, die das Recht der Mitglied-
staaten erstmals dem Anwendungsbereich des Gemeinschaftsrechts unterwerfen, einen
nachvollziehbaren Begründungsansatz wählen.24 Die teilweise auf die Mitteilung des Ergebnisses und eine aus sich heraus nur schwer
nachvollziehbare Begründung beschränkte Darstellung der Entscheidungen des Ge-
richtshofs stellen die nationalen Gerichte vor Probleme. Eine etwas ausführlicher be-
gründete Entscheidung des Gerichtshofs würde vielfach ihre Akzeptanz erhöhen und
erneute Vorabentscheidungsverfahren vermeiden, die für alle an einem solchen Verfah-
ren beteiligten Personen und Stellen einen beträchtlichen Aufwand bedeuten. Dies gilt
insbesondere, wenn der Gerichtshof den Anwendungsbereich des Gemeinschaftsrechts
ausweitet oder neue und ausfüllungsbedürftige Rechtsgrundsätze aufstellt. Ansonsten
läuft die betreffende Entscheidung des Gerichtshofs Gefahr, von den Gerichten der Mit-
gliedstaaten nicht oder nicht zutreffend gewürdigt zu werden. Dazu zwei aktuelle Bei-
spiele: 24 Auch dieser Teil konnte aus Zeitgründen nicht mündlich vorgetragen werden. 53 1. Die Entscheidung Werhof Das Vorabentscheidungsersuchen des Landesarbeitsgerichts Düsseldorfs betraf –
kurz zusammengefasst – die Auslegung einer vertraglichen Bezugnahmeklausel auf
einen Tarifvertrag im Zusammenhang mit einem Betriebsübergang. In diesem Zusam-
menhang führte der Gerichtshof sinngemäß und ohne weitere Begründung aus, dass
eine Auslegung der vertraglichen Verweisklausel als „dynamisch“ für den Betriebser-
werber zu einer Beeinträchtigung seines Grundrechts auf negative Koalitionsfreiheit
führen könnte.25 Die Vorstellung von einem Verstoß gegen die negative Koalitionsfrei-
heit des Arbeitgebers durch Bindung eben dieses Arbeitgebers an den von ihm vorfor-
mulierten Arbeitsvertrag erscheint – nicht nur mir – nicht mehr nachvollziehbar. Mögli-
cherweise hat der Gerichtshof bei seiner Entscheidungsfindung nicht hinreichend be-
dacht, dass es im Streitfall nicht um eine besondere Form von Tarifbindung, sondern
schlicht um Vertragsfreiheit geht. Dass diese Verpflichtung aus dem Arbeitsvertrag
dann auch den Betriebserwerber trifft, ist Folge des Art. 3 Abs. 1 der Betriebsüber-
gangsrichtlinie. So stehen weitere Nachfragen der deutschen Gerichte zu erwarten. 2. Der Super-GAU – der Konflikt zwischen Gemeinschafts- und nationalem
Verfassungsrecht Ulrich Koch Ulrich Koch 1. Die Entscheidung Werhof 1. Die Entscheidung Werhof 1 EuGH vom 27.3.1990, Rush Portuguesa/Office national d’immigration, Rs. C-113/89, Slg. 1990, I-
1417. 2. Der Super-GAU – der Konflikt zwischen Gemeinschafts- und nationalem
Verfassungsrecht Die gleichermaßen in der Begründung knapp abgefasste Entscheidung des Gerichts-
hofs in der Rechtssache Mangold ist auf breite Kritik gestoßen, die dem Gerichtshof ua. eine Überschreitung seiner Kompetenzen vorwarf und sich für die Unverbindlichkeit
des Unanwendbarkeitsausspruchs aussprach.26 Der Siebte Senat des Bundesarbeitsge-
richts ist dem nicht gefolgt. Er hat versucht, die Entscheidungsgründe seinerseits auszu-
legen und hat angenommen, dass der Gerichtshof die ihm durch die deutschen Zustim-
mungsgesetze zu den Europäischen Verträgen übertragenen Kompetenzen nicht über-
schritten hat.27 Vor der Entscheidung des Bundesarbeitsgerichts ist diskutiert worden,
ob der erkennende Senat den Europäischen Gerichthof erneut anzurufen sollte, um ihn
Gelegenheit zur Ergänzung seines primärrechtlichen Prüfungsansatzes zu geben. Bei
einer etwas breiteren und nachvollziehbareren Darstellung des primärrechtlichen Prü-
fungsansatzes wäre diese Diskussion sicherlich nicht oder nicht in dieser Schärfe ge-
führt worden. Gegen diese Entscheidung des Bundesarbeitsgerichts hat die unterlegene
Arbeitgeberin Verfassungsbeschwerde beim Bundesverfassungsgericht eingelegt.28 Ich 54 10.5771
– Das soziale Europa aus der Perspektive der deutschen Gerichtsbarkeit bin mir bis heute nicht im Klaren darüber, welchen Inhalt ein erneutes Vorabentschei-
dungsverfahren hätte haben können. Über die Frage, ob der Europäische Gerichtshof
eine von den deutschen Zustimmungsgesetzen zu den Europäischen Verträgen nicht
mehr gedeckte Rechtsfortbildung vorgenommen hat, kann nach meinem Dafürhalten –
wenn überhaupt – nur das Bundesverfassungsgericht verbindlich entscheiden. Art. 100
des Grundgesetzes lässt aber nach seinem Wortlaut eine so begründete Richtervorlage
an das Bundesverfassungsgericht nicht zu. Dieser Zustand ist aus meiner Sicht unbe-
friedigend. Möglicherweise hat das Bundesverfassungsgericht bei seiner Entscheidung über die
gegen die Entscheidung des Siebten Senats eingelegte Verfassungsbeschwerde Veran-
lassung, für die Konfliktfälle bei der Abgrenzung von Gemeinschaftsrecht und nationa-
lem Recht klare Vorgaben aufzustellen. Dann allerdings wäre der fragwürdige befristete
Arbeitsvertrag zwischen den Rechtsanwälten Werner Mangold und Rüdiger Helm nicht
nur durch die denkwürdige Entscheidung des Europäischen Gerichtshofs, sondern auch
noch durch eine Entscheidung des Bundesverfassungsgerichts, durch die das Verhältnis
zwischen Gemeinschaftsrecht und nationalem Recht austariert würde, ein weiteres Mal
geadelt. 55 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
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Open Access –
- Diskussionsbericht Diskussionsleitung: Ulrich Becker Diskussionsbericht Wahrung solcher nationaler Besonderheiten müsse ein offener Diskurs geführt werden,
der die wirtschaftliche und sozialpolitische Situation in den Mitgliedstaaten einbeziehe. Wahrung solcher nationaler Besonderheiten müsse ein offener Diskurs geführt werden,
der die wirtschaftliche und sozialpolitische Situation in den Mitgliedstaaten einbeziehe. p
g
Wenn das Gleichgewicht zwischen Wirtschaftsverfassung auf Europaebene und So-
zialverfassung auf nationaler Ebene gestört sei, komme es zu einer erneuten Diskussion
über das Demokratiedefizit Europas. Dieses Gleichgewicht sei Teil der demokratischen
Verfassung, denn die nationale Zuordnung des Sozialen sei Teil der politischen Kon-
struktion Europas insgesamt. Levits sprach die Problematik des Demokratiedefizits an,
konnte im Rahmen der gegebenen Zeit jedoch nicht auf dieses große Thema näher ein-
gehen. Er gab zu bedenken, dass der Vertrag in bestimmten Bereichen zwar durchaus
ein Gleichgewicht zwischen Binnenmarkt und nationaler Sozialordnung vorgesehen
habe, dabei seien die Grundfreiheiten auf die Verwirklichung des Binnenmarktes ausge-
richtet. Die sozialen Aspekte dienten dabei als Korrektive, wobei sie im Einzelfall auch
überwiegen könnten. Es handle sich um einen Zielkonflikt, der zum Ausgleich gebracht
werden müsse. Dem EuGH wurde in der weiteren Diskussion vorgeworfen, in den Fällen Viking
und Laval – im Gegensatz zur Entscheidung Schmidberger – „judicial activism“ betrie-
ben zu haben. Im Urteil Viking nehme der EuGH eine Verhältnismäßigkeitsprüfung
vor, die theoretisch dazu führen könne, dass nicht einmal ein politischer Streik zulässig
wäre. Darüberhinaus gebe es für den EuGH nur die Grundrechtsausübung soweit sie
dem Arbeitnehmerschutz diene. Diese Rechtsprechung stelle „judicial activism“ dar und
stehe im massiven Gegensatz zum Schmidberger-Urteil, in dem immer wieder von ei-
nem weiten Ermessen der Mitgliedstaaten gesprochen worden sei. Ein zweites Beispiel für „judicial activism“ sei die Entsenderichtlinie, die eigentlich
dem Arbeitnehmerschutz dienen solle. Der Richtlinie werde vom EuGH jedoch nur
dann praktische Wirksamkeit zugesprochen, wenn und soweit sie den freien Dienstleis-
tungsverkehr gewähre. Dieser sei zwar bei der Konzeption der Richtlinie zu beachten
gewesen, jedoch sei seine Verwirklichung nicht das erklärte Ziel der Richtlinie. Die
Entsenderichtlinie sei daher in ihr absolutes Gegenteil verkehrt worden. Levits verstand unter „judical activism“ das Erschließen neuer Kompetenzen oder
das Aufstellen neuer Grundsätze, z. B. die Einführung von neuen Rechtsgrundsätzen –
oder auch Grundrechten – auf dem Wege allgemeiner Rechtsprinzipien. Dies sei auch
manchmal erforderlich, wenn die Politik versage. So habe der EuGH Grundrechte über
allgemeine Rechtsgrundsätze einführen müssen. Er sah in der EuGH-Rechtsprechung
jedoch weder bei der Abwägung zwischen zwei konkurrierenden Zielen noch bei der
Verhältnismäßigkeitsprüfung Anlass, von „judical activism“ zu sprechen. 2 EuGH vom 16.6.2008, Kommission/Luxemburg, Rs. C-319/06, noch nicht in der amtlichen Samm-
lung veröffentlicht. Diskussionsleitung: Ulrich Becker Die erste Frage richtete sich an Levits und bezog sich darauf, ob die in den einzelnen
Mitgliedstaaten geführten Diskussionen über die ergangenen Urteile des EuGH bei den
EuGH-Richtern Beachtung fänden. Levits betonte, dass der EuGH allen 27 Mitglied-
staaten die gleiche Beachtung schenke. Politische Diskussionen, die nur in einzelnen
Mitgliedstaaten geführt werden, spielten für die Richter bei ihrer Entscheidung keine
gewichtige Rolle, da die EuGH-Richter streng nach juristischer Methode entscheiden
würden und dabei der europäischen Gesamtperspektive verpflichtet seien. Sie seien kei-
ne Vertreter ihrer Staaten. Jedoch würden die verschiedenen fachlichen Diskurse, die
durch die unterschiedliche Interessenlage der einzelnen Mitgliedsstaaten oder auch der
betroffenen Sozialpartner bedingt seien, den EuGH-Richtern wertvolle Anregungen
geben. In der Diskussion wurde Levits‘ These aufgegriffen, dass die Europäische Gemein-
schaft zunächst eine Wirtschaftsgemeinschaft gewesen sei und sich das Ziel eines sozia-
len Europas erst nach und nach entwickelt habe. Dem wurde entgegengehalten, dass
soziale Fragestellungen von Anfang an Beachtung gefunden hätten, nur eben im Rah-
men der nationalen Sozialpolitik. Auch der sehr differenzierte Zuständigkeitskatalog im
heutigen Artikel 137 EG-V zum kollektiven Arbeitsrecht solle nicht bedeuten, dass kol-
lektives Arbeitsrecht für die Gemeinschaft unwichtig sei, sondern dass dieses in erster
Linie weiterhin durch die Mitgliedstaaten gewährleistet werden solle. Die Mitgliedstaa-
ten hätten sich zu dieser Konzeption bekannt. Der EuGH müsse daher diese Aufteilung
achten und den Mitgliedstaaten ausreichend Raum für ihre nationale Sozialpolitik las-
sen. Dies gelte umso mehr, als der EuGH als Erster in der Entscheidung Rush Portugue-
sa1 im Fall der Entsendung ausländischer Arbeitnehmer die Bedeutung nationaler Rege-
lungen hervorgehoben und der Entsenderichtlinie damit den Weg bereitet habe. Daher
sei es nicht akzeptabel und mit der Tradition des EuGH auch nicht vereinbar, wenn Art. 3 Absatz 7 der Entsenderichtlinie, der eine weitergehende nationale Sozialpolitik er-
mögliche, nicht ausreichend Beachtung geschenkt werde. Auch die Diskussion um ei-
nen Mindestlohn zeige, dass nationale Besonderheiten berücksichtigt werden müssten. Während das Tarifentgelt in manchen Staaten gleichzeitig das Höchstentgelt sei, gebe es
in Deutschland zusätzlich zum Tarifentgelt übertarifliche Zulagen, so dass in Deutsch-
land ein Tariftreuegesetz ein Gesetz nur über den tariflichen Mindestlohn sei. Unter 57 /10.5771/97
– Diskussionsbericht In die Diskussion wurde das einige Tage zuvor ergangene Luxemburg-Urteil2 einge-
bracht. Die nationale Kompetenz zur Definition öffentlicher Ordnung sei darin ausge-
sprochen eng ausgelegt worden, da diese nur auf den Bereich Polizei und Sicherheit
bezogen worden sei. 58 10.577
– Simone Gräfin von Hardenberg Diskussionsbericht Nach Auffassung von Levits relativiert sich diese Feststellung aber dadurch, dass das
Urteil weniger auf einer inhaltlichen als vielmehr auf einer prozessualen Entscheidung
beruhe, da in diesem Verfahren keine Tatsachen angeführt worden seien, die eine Beur-
teilung der Erforderlichkeit und der Verhältnismäßigkeit der erlassenen Maßnahme er-
möglichten. Das Urteil mache deutlich, dass eine Einschränkung der Grundfreiheiten
nur dann unter Berufung auf die öffentliche Ordnung gerechtfertigt sein könne, wenn
diese mit Tatsachen hinreichend begründet worden sei. Die alleinige Berufung auf die
öffentliche Ordnung könne nicht per se eine Grundfreiheit beseitigen. Simone Gräfin von Hardenberg Simone Gräfin von Hardenberg 59 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
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Open Access –
- Scope of Action at the European Level Brian Bercusson † I. Executive Summary
62
1. Strategies in the short-term
63
2. Strategies in the medium-term
63
3. Strategies for the long-term
64
II. Introduction: The price of not remembering history is to repeat it
64
III. The ECJ invents a new EU law on collective action
66
1. Uncertain criteria for the legality of collective action
66
2. Horizontal direct effect against trade unions
67
3. Unequal treatment of workers on grounds of nationality
68
4. Violation of international labour law standards
68
5. Violation of the principle of subsidiarity
69
6. A charter for “social dumping”
70
7. In sum…
70
IV. Scope of action at European level after the Lisbon Treaty
73
V. Strategies in the short-term
73
1. Legal form
73
2. A Protocol
74
3. Enhanced cooperation
74
4. The “Schengen” model
74
5. A non-binding Social Declaration
74
6. An interpretative instrument (inverse “opt-out”)
75
7. Content
75
8. An “anti-social dumping principle” against abuse of the exercise of economic
freedoms
76
9. A non-regression clause
77
10. A more favourable treatment clause
77
11. An interpretative framework for economic freedoms
78
a) A rebuttable presumption in favour of collective action
78
b) Interpreting “proportionality” in light of the acquis communautaire social
79
c) Human rights are not mere derogations from economic freedoms; rather
the reverse
79
12. The Agency Workers Directive: Equal treatment of agency workers includes
cross-border workers
79
VI. Strategies in the medium-term
80
1. “Update” the “explanations” to the EU Charter
80
2. A standard social safeguard clause in directives
81
61
https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb 3. Monitoring transposition of the Services Directive 3. Monitoring transposition of the Services Directive
i l i 4. Social impact assessments 5. Operationalising the Lisbon Treaty’s horizontal social clause 6. Revise the Posting Directive VII. Strategies for the long-term VII. Strategies for the long-term g
g
1. Revise Treaty provisions on economic freedoms 1. Revise Treaty provisions on economic freedoms 1. Revise Treaty provisions on 2. Revise the Treaty to prevent employers using economic freedoms against
trade unions taking collective action (horizontal direct effect) 4. Reform the European Court of Justice
85
a) Structure of the ECJ
85
b) Competences of the ECJ
85
c) Procedures of the ECJ
86
d) Composition of the ECJ
86 4. Reform the European Court of Justice a) Structure of the ECJ )
b) Competences of the ECJ c) Procedures of the ECJ d) Composition of the ECJ )
p
5. Reinforce international labour law in the Treaties a) The European Social Charter )
p
b) ILO Conventions c) Interpreting “proportionality” in light of international labour law VIII. Conclusion 1. National labour law 2. The balance of power in the EU economy 3. The law on workers’ collective action 4. Transnational collective action and EU law I. Executive Summary I. Executive Summary y
1. Strategies in the short-term 2. Strategies in the medium-term 3. Strategies for the long-term II. Introduction: The price of not remembering history is to repeat it II. Introduction: The price of not remembering history is to repeat it III. The ECJ invents a new EU law on collective action III. The ECJ invents a new EU law on collective action 1. Uncertain criteria for the legality of collective action 2. Horizontal direct effect against trade unions g
3. Unequal treatment of workers on grounds of nationality 4. Violation of international labour law standards 5. Violation of the principle of subs 6. A charter for “social dumping” 7. In sum… IV. Scope of action at European level after the Lisbon Treaty V. Strategies in the short-term 1. Legal form g
2. A Protocol
h
d 3. Enhanced cooperation 4. The “Schengen” model 5. A non-binding Social Declaration 6. An interpretative instrument (inverse “opt-out”) 8. An “anti-social dumping principle” against abuse of the exercise of economic
freedoms
7 9. A non-regression clause 10. A more favourable treatment clause 11. An interpretative framework for economic freedoms
78
a) A rebuttable presumption in favour of collective action
78
b) Interpreting “proportionality” in light of the acquis communautaire social
79
c) Human rights are not mere derogations from economic freedoms; rather
the reverse
79 12. The Agency Workers Directive: Equal treatment of agency workers includes
cross-border workers
79 VI. Strategies in the medium term
80
1. “Update” the “explanations” to the EU Charter
80
2 A
d d
i l
f
d l
i di
i
81 1. “Update” the “explanations” to the EU Charter 2. A standard social safeguard clause in directives 61 Brian Bercusson 3. Monitoring transposition of the Services Directive
81
4. Social impact assessments
81
5. Operationalising the Lisbon Treaty’s horizontal social clause
82
6. Revise the Posting Directive
82
VII. Strategies for the long-term
83
1. Revise Treaty provisions on economic freedoms
83
2. Revise the Treaty to prevent employers using economic freedoms against
trade unions taking collective action (horizontal direct effect)
84
3. Revise the subsidiarity principle to exclude EC competence to regulate
collective action
85
4. Reform the European Court of Justice
85
a) Structure of the ECJ
85
b) Competences of the ECJ
85
c) Procedures of the ECJ
86
d) Composition of the ECJ
86
5. I. Executive Summary Reinforce international labour law in the Treaties
87
a) The European Social Charter
87
b) ILO Conventions
87
c) Interpreting “proportionality” in light of international labour law
87
VIII. Conclusion
88
1. National labour law
88
2. The balance of power in the EU economy
88
3. The law on workers’ collective action
89
4. Transnational collective action and EU law
89 1. Strategies in the short-term 1. Strategies in the short-term In light of this new law, not foreseen by the Lisbon Treaty, Member States may now
undertake a commitment to a stronger social dimension. Suggestions are made as to the
legal form and the substantive content of such a commitment. As to legal form: Some Member States could adopt a Protocol on a stronger social
dimension as a condition to final ratification of the Lisbon Treaty. All Member States
could agree to this Protocol binding only those Member States who accept the Protocol. If at least 14 Member States agreed, in the course of ratifying the Lisbon Treaty, that
they wanted stronger social provisions, the mechanism of enhanced cooperation (Arti-
cle 43 TEU) allows this, leaving it open to other Member States to join later. A “Social Schengen” agreement between an initially small number of Member
States, which more may gradually join, could be adopted in the course of ratification of
the Lisbon Treaty, eventually to be incorporated into the legal framework of the EU. A commonly agreed “social declaration”, though not legally binding, could have a
future impact on the political agenda of the EU institutions. An interpretative instrument could be attached to the ratification in the form of an
“opt-out” opposite to that of the UK and Poland: prohibiting any court from holding that
national laws or practices regulating collective bargaining and collective action are in-
consistent with the economic freedoms in the Treaties. As to the content: An “anti-social dumping principle”: exercise of economic free-
doms without a guarantee of jobs and working conditions is an abuse which justifies
collective action. The social policy objective of “improved living and working conditions” (Article 136
EC) allows for a general principle of “non-regression” in EC law and extends to the
freedom of Member States to require more favourable treatment of workers. An interpretative framework for economic freedom to include the freedom of action
of collective labour (trade unions) would include a rebuttable presumption in favour of
collective action, and a criterion of “proportionality” based on the acquis communau-
taire social protecting workers. The exercise of economic freedoms derogating from
fundamental rights is only permitted if justified as “proportionate”. The proposed Agency Workers Directive should guarantee equal treatment of cross-
border agency workers. I. Executive Summary The paper begins with an analysis of the decisions of the European Court of Justice
in Viking, Laval and Rüffert. The decisions are criticised as inventing an EU law on
workers’ collective action. This new law establishes uncertain criteria for the legality of
collective action, introduces horizontal direct effect against trade unions, allows for un-
equal treatment of workers on grounds of nationality, violates international labour law
standards, infringes the principle of subsidiarity and is tantamount to a charter for “so-
cial dumping”. This new EU law is already being invoked by employers at national
level to block otherwise lawful collective action. It poses a threat to the stability of na-
tional industrial relations systems and to European integration. The paper then explores the scope for action at European level to address this new
EU law created by the ECJ which was not envisaged when the Lisbon Treaty was pro-
posed. It suggests short, medium and long-term strategies in the context of the Irish re-
jection of the Lisbon Treaty. 62 Scope of Action at the European Level Scope of Action at the European Level Brian Bercusson Brian Bercusson A standard social safeguard clause for directives could include protection of funda-
mental rights and collective agreements, and a clearer non-regression principle allowing
more favourable provisions. Transposition of the Services Directive may allow for challenges to the ECJ’s inter-
pretation of Article 49 in Laval by requiring service providers to respect fundamental
rights to collective action, collective bargaining and collective agreements. Commission
proposals should include a “social impact assessment”. The Lisbon Treaty’s horizontal social clause, (new Article 5a EC) should be elabo-
rated to make it effectively operational, given more precise meaning and adapted to spe-
cific Commission proposals. For example, to revise the Posting Directive to reverse the
ECJ’s interpretation in Laval. 3. Strategies for the long-term The Treaty provisions on economic freedoms need to be re-drafted: to reduce their
negative impact on fundamental rights of collective action and to protect workers; to
prevent employers using economic freedoms against trade unions taking collective ac-
tion (no horizontal direct effect), and the principle of subsidiarity should be reworked to
reflect explicitly the protection of collective action. Reform of the European Court of Justice includes establishment of a specialist tribu-
nal: the chambre social, excluding competence to override fundamental rights (to collec-
tive action) protected in Member States, authorising the social partners to intervene in
cases before the ECJ, and reconsidering the composition of the Court. Reinforcing international labour law in the Treaties would mean ratification by the
EU of the European Social Charter, explicit recognition of specific ILO Conventions as
interpreted by the Freedom of Association Committee, and interpreting “proportional-
ity” in light of international labour law. 2. Strategies in the medium-term The “explanations” to the EU Charter should be “updated” to reflect a stronger so-
cial dimension. 63 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb Scope of Action at the European Level The decisions of the European Court of Justice (ECJ) in Viking,1 Laval2 and Rüf-
fert3 share a common premise: collective action which restricts the economic freedom
of employers may violate EU law (on free movement, on posted workers and on public
procurement). Although recognising the right to collective action as a fundamental
right, the reasoning of the ECJ reflects doctrines long superseded in national legal dis-
course. Nineteenth century doctrinal ghosts of the dominance of market freedoms, long
since revised to reflect the social model of industrial relations in twentieth century
European welfare states, have returned to haunt EU labour law of the twenty-first cen-
tury. The decisions reveal a conflict between the ECJ’s doctrine on collective action and
the express policy choices of the EU legislature. In contrast to the decision in Viking,
the EU legislature in Council Regulation (EC) No. 2679/98 of 7 December 1998 de-
clared that free movement of goods was not to affect the right to strike.4 In the Services
Directive, the EU legislature explicitly provided that fundamental rights to collective
action, collective bargaining and collective agreements were not to be subject to EU law
on free movement of services.5 In contrast to the decision in Laval, the EU legislature
adopted the Posting Directive 96/71/EC in which Recital 22 states: “…this Directive is
without prejudice to the law of the Member States concerning collective action to defend
the interests of trades and professions”.6 In contrast to the ECJ decision in Rüffert, the
EC directives on public procurement, revised in 2004, reflected the policy allowing 4 The “Monti” Regulation. Council Regulation (EC) No. 2679/98 of 7 December 1998 on the functi-
oning of the internal market in relation to the free movement of goods among the Member States. OJ L337/8 of 12.12.98; Article 2: “This Regulation may not be interpreted as affecting in any way
the exercise of fundamental rights as recognised in Member States, including the right or freedom to
strike. These rights may also include the right or freedom to take other actions covered by the speci-
fic industrial relations systems in Member States”. 5 Directive 2006/123/EC of the European Parliament and of the Council of 12 December 2006 on
services in the internal market, OJ L376/26 of 27.12.2006. Article 1(7) provides: “This Directive
does not affect the exercise of fundamental rights as recognised in the Member States and by Com-
munity law. 1 Case C-438/05, International Transport Workers’ Federation, Finnish Seamen’s Union v Viking Li-
ne ABP, OÜ Viking Line Eesti, decided 11 December 2007. II. Introduction: The price of not remembering history is to repeat it A crucial element in maintaining a balance of economic power within EU Member
States is the legal right of workers and their organisations to take collective action. A
specific legal problem arises where national laws on collective action encounter EU law
on free movement of goods, services, capital or workers. 64 2 Case C-341/05, Laval un Partneri Ltd v Svenska Byggnadsarbetareförbundet, Svenska Byggnadsar-
betareförbundet, avd. 1, Svenska Elektrikerförbundet, decided 18 December 2007. 3 Case C-346/06, Rechtsanwalt Dr. Dirk Rüffert v. Land Niedersachsen, decided 3 April 20 7 Paras. 58-60. The Preamble of Directive 2004/18/EC confirms that: “Contract performance condi-
tions are compatible with this Directive provided that they are not directly or indirectly discrimina-
tory and are indicated in the contract notice or in the contract documents (Recital 33). The Advocate
General concluded that there was little doubt that a restriction on the freedom to provide service ex-
ists (para. 102). But the public authority was not violating Article 49 because it sought to ensure the
protection of posted workers (para. 118). This was an appropriate means of preventing social dump-
ing as it ensures that local workers and posted workers on the same site will be treated equally (para.
119). Scope of Action at the European Level Nor does it affect the right to negotiate, conclude and enforce collective agreements and
to take industrial action in accordance with national law and practices which respect Community
law”. Recital 15 provides: “This Directive respects the exercise of fundamental rights applicable in
the Member States and as recognised in the Charter of fundamental Rights of the European Union
and the accompanying explanations, reconciling them with the fundamental freedoms laid down in
Articles 43 and 49 of the Treaty. Those fundamental rights include the right to take industrial action
in accordance with national law and practices which respect Community law”. 6 Directive 96/71/EC concerning the posting of workers in the framework of the provision of services. OJ 1996, L18/1. 6 Directive 96/71/EC concerning the posting of workers in the framework of the provision of services. OJ 1996, L18/1. 65 Brian Bercusson public authorities to make compliance with collective agreements a contract perform-
ance condition, duly acknowledged by Advocate General Bot in his Opinion.7 Unless the ECJ’s doctrines are rejected and the decisions reversed, the EU will pay
the price of years of painful struggle to lay these doctrinal ghosts to rest. The price of
not remembering history is to repeat it. Scope of Action at the European Level In contrast, the same ECJ in Laval looks to the Posting Directive for the criterion of
lawful collective action. Collective action is lawful only to secure standards stipulated
in the Posting Directive: legislation and collective agreements declared universally ap-
plicable in the construction sector. These are maximum standards. Transnational collec-
tive action to impose higher standards is unlawful as violating the Treaty’s economic
freedoms. Member States may extend this to include other collective agreements, other
terms of employment and other sectors. But not negotiation at the workplace, which
prevents transnational undertakings from ascertaining labour standards with certainty. A superficially common criterion of “proportionality” masks real differences. Advocate General Mengozzi in Laval explicitly invokes “proportionality”, but this is
not to be applied to primary collective action in support of pay claims, even if “exces-
sive”, though such action may be unlawful if linked to other terms of collective agree-
ment. Similarly for solidarity action. This is contradicted by the ECJ in Laval, for whom the criterion of “proportionality”
is implicit: collective action is lawful only up to the maximum standards allowed by the
Posting Directive. But not if standards are uncertain, as in negotiation at the workplace. For Advocate General Maduro in Viking, the criterion of “proportionality” is, at best,
implicit: primary action is lawful if before relocation; solidarity action is lawful if vol-
untary. These concepts disappear in the ECJ’s judgment in Viking, which evokes the crite-
rion of “proportionality” explicitly. Primary action is lawful if suitable to meet a serious
threat, is not more than necessary and less restrictive means available are exhausted. Solidarity action is not proportionate, though it may protect workers. In sum, there are two broadly opposing principles in the judgments of Laval and Vi-
king, positing two different legal outcomes for the legality of collective action. In Viking, legality depends on whether the action satisfies a general anti-social dump-
ing principle (but subject to “proportionality”). In Laval, legality is subject to the maximum standards principle of the Posting Direc-
tive (which may be extended, but subject to “certainty”). So much for predictability… The outcome is deeply unsatisfactory. 1. Uncertain criteria for the legality of collective action The European Court has spectacularly failed to establish clear rules of European la-
bour law governing collective action. The Opinions of each of the Advocates General
and each of the judgments of the ECJ in Viking and Laval prescribe very different prin-
ciples for determining the legality of collective action. This does not produce certainty,
nor inspire confidence in a judicial role in the regulation of collective action. The
widely different propositions may be summarised very succinctly as follows. For Advocate General Mengozzi in Laval, the lawfulness of primary collective ac-
tion is subject to the criterion of “proportionality”, which requires a real advantage
which contributes significantly to workers’ protection. The lawfulness of solidarity ac-
tion depends on the lawfulness of primary action. In complete contrast, for Advocate General Maduro in Viking, to be lawful, primary
collective action must be taken before relocation. Solidarity action depends on whether
it is voluntary. The ECJ in Viking disregards Maduro’s Opinion and, like Mengozzi, invokes the cri-
terion of “proportionality”. This requires a serious threat to jobs and conditions (not
present where there is a legally binding guarantee of statutory provisions and collective
agreements). But also, collective action must be suitable to achieve the objective, not go
beyond what is necessary and any other less restrictive means have been exhausted. Solidarity action cannot be objectively justified, though it is to be balanced with work-
ers’ protection. 66 /10.5771
– 3. Unequal treatment of workers on grounds of nationality The fundamental principle of equal treatment regardless of nationality means that
workers are entitled to the same terms and conditions regardless of nationality. The de-
cision in Viking proposes restrictive criteria for lawful collective action against service
providers aimed at ensuring the application to their employees in the host country of
collective agreements equally applicable to workers in the host country. Laval and Rüf-
fert go even further in restricting the kinds of collective agreements which may be ap-
plied only to agreements prescribing legally binding national minimum standards. The
decisions of the ECJ challenge the principle of equal treatment of workers by distin-
guishing migrant workers from those employed temporarily by service providers. 2. Horizontal direct effect against trade unions Trade unions are singled out by the Court as subject to complaints that their collec-
tive action violates the economic freedoms of others. The formulation is extremely
wide: the economic freedoms may be invoked by “any individual who has an interest in
compliance with the obligations laid down and… applies in particular to all agreements 67 Brian Bercusson intended to regulate paid labour collectively”.8 Moreover, the Court focuses such com-
plaints on trade unions. intended to regulate paid labour collectively”.8 Moreover, the Court focuses such com-
plaints on trade unions. intended to regulate paid labour collectively”.8 Moreover, the Court focuses such com-
plaints on trade unions. intended to regulate paid labour collectively”.8 Moreover, the Court focuses such com-
plaints on trade unions. Doctrinal objections to horizontal direct effect include: Doctrinal objections to horizontal direct effect include:
− collective agreements are not restrictions on freedom to provide services (as de-
clared explicitly in the Services Directive); − collective agreements are not restrictions on freedom to provide services (as de-
clared explicitly in the Services Directive); − collective action is not a regulatory measure; − trade unions are not regulatory bodies as “emanations of the State”; − horizontal direct effect violates freedom of association; and − horizontal direct effect harmonises EC law on collective action. Practical objections to horizontal direct effect include: − a potential flood of complaints against collective agreements, and − there are no practical criteria which distinguish agreements having regulatory ef-
fect. − there are no practical criteria which distinguish agreements having regulatory ef-
fect. 8 Viking, para. 58. 9 The criterion of “proportionality” is indeterminate. For example, according to the ECJ in Viking, it
disaggregates into a number of indicators: (i) there must be a “serious threat” to jobs and working
conditions; (ii) there is no legally binding undertaking by the employer to maintain existing collec-
tive agreements; (iii) the collective action must be suitable to achieve its ends; (iv) no other means
less restrictive of economic freedoms is available; and (v) all alternative methods of achieving the
union’s objective have been exhausted. 4. Violation of international labour law standards It is questionable whether the international labour law obligations of Member States
are compatible with the constraints imposed by the ECJ on the right to collective action. These obligations are incumbent on them not only as signatories to a number of interna-
tional law instruments (inter alia, ILO Conventions 87 and 98, the European Social
Charter, Article 6(4)), but as a general principle of Community law. 68 – Scope of Action at the European Level Scope of Action at the European Level Scope of Action at the European Level Scope of Action at the European Level 10 Providing an overarching historical perspective, the ETUC stated in its letter attached to the ITF
written submission in Viking: (paras. 4 and 6): “The precise contours of the rules governing collec-
tive action in each Member State are the outcome of different national historical experience… In the
Member States of the EU the rules governing collective industrial action reflect an established equi-
librium in the balance of forces between the social partners. It would produce a shock of incalcu-
lable magnitude if this equilibrium, carefully constructed over time in different Member States, we-
re to be destabilised by an intervention reflecting Viking’s interpretation of Community law”. 11 The ECJ condemns the ITF’s Flags-of-Convenience (FOC) policy which: (para. 88) “results in shi-
powners being prevented from registering their vessels in a State other than that of which the bene-
ficial owners of those vessels are nationals, the restrictions on freedom of establishment resulting
from such action cannot be objectively justified”. This is either a misunderstanding or a misrepre-
sentation of the FOC policy which was previously clearly stated to concern negotiating rights and
not registration (para. 8). This is offset by positive statement that immediately follows: “the objec-
tive of that policy is also to protect and improve seafarers’ terms and conditions of employment”. 5. Violation of the principle of subsidiarity The ECJ’s decisions deal a potentially mortal blow to the national industrial relations
systems of the EU Member States. The decisions restrict the right to collective action,
restrict national collective bargaining systems and restrict labour standards to the mini-
mum. Taken together, the decisions in Laval and Rüffert define a uniform model of
industrial relations mandatory for all Member States. The ECJ’s model of national in-
dustrial relations has the following characteristics as regards collective action, collective
bargaining and collective agreements: − national constitutional protection of the fundamental right of workers to take col-
lective action is subordinate to economic freedoms of employers protected by the
EC Treaty; the legality of collective action is subject to the criterion of “propor-
i
li
9 − national constitutional protection of the fundamental right of workers to take col-
lective action is subordinate to economic freedoms of employers protected by the
EC Treaty; the legality of collective action is subject to the criterion of “propor-
tionality”;9 p y
p
y
EC Treaty; the legality of collective action is subject to the criterion of “propor-
tionality”;9 − only legally binding collective agreements fixing national minimum standards
can be enforced against employers (from other Member States and, probably, to
avoid discrimination, also against domestic employers); − flexible collective bargaining arrangements at local level are not enforceable
against employers from other Member States as setting standards so uncertain as
to constitute obstacles to free movement. Few national systems currently conform to this model. Nobody can have expected
the EU, let alone the ECJ, to arrogate to itself the competence to impose a uniform
model of industrial relations radically altering systems developed over centuries in ac-
cordance with very different national histories, cultures and social partnership institu-
tions.10 10 Providing an overarching historical perspective, the ETUC stated in its letter attached to the ITF
written submission in Viking: (paras. 4 and 6): “The precise contours of the rules governing collec-
tive action in each Member State are the outcome of different national historical experience… In the
Member States of the EU the rules governing collective industrial action reflect an established equi-
librium in the balance of forces between the social partners. It would produce a shock of incalcu-
lable magnitude if this equilibrium, carefully constructed over time in different Member States, we-
re to be destabilised by an intervention reflecting Viking’s interpretation of Community law”. 6. A charter for “social dumping” 6. A charter for “social dumping” 6. A charter for “social dumping” Beyond this devastation wrought on national industrial relations systems, the ECJ in
Viking also condemns transnational solidarity action which interferes with free move-
ment of employers, despite acknowledging its objective to protect workers.11 It thereby
established a legal framework for a balance of power which decidedly favours transna-
tional employers against the protection of workers. The proposed re-configuration of the legal framework of Member States’ industrial
relations systems by restricting collective action and collective agreements to limit la-
bour standards to the minimum amount to a charter for “social dumping”. 12 As suggested above, it is questionable whether the international labour law obligations of Member
States are compatible with the constraints imposed by the ECJ on the right to collective action. 5. Violation of the principle of subsidiarity 69 69 Brian Bercusson 7. In sum… The ECJ’s doctrine on the EU law on collective action: The ECJ’s doctrine on the EU law on collective action: − subordinates collective action to the economic freedoms of employers, − requires collective action to be justified by public policy, − requires collective action to be justified by public policy, − in accordance with a criterion of “proportionality” which requires, among other
conditions, − in accordance with a criterion of “proportionality” which requires, among other
conditions, − a “serious threat” to jobs and working conditions, − suitability to achieve its ends, − suitability to achieve its ends, − lack of other less restrictive means of action, − exhaustion of all alternative methods, and − is enforceable against trade unions through the doctrine of horizontal direct ef-
fect. − is enforceable against trade unions through the doctrine of horizontal direct ef-
fect. The Court’s doctrine adopts the premise that fundamental social rights are to be in-
terpreted narrowly where they restrict economic freedoms guaranteed by the Treaty. This doctrinal approach is fatal to the purpose and function of collective action of work-
ers, which is precisely to restrict the economic freedom of employers.12 The question is not whether the fundamental right to take collective action can be
justified where it restricts employers’ economic freedom. Rather, the Treaty’s provi- 12 As suggested above, it is questionable whether the international labour law obligations of Member
States are compatible with the constraints imposed by the ECJ on the right to collective action. 12 As suggested above, it is questionable whether the international labour law obligations of Member
States are compatible with the constraints imposed by the ECJ on the right to collective action. 70 Scope of Action at the European Level sions on economic freedoms should be interpreted to ensure respect for fundamental
rights of workers, protected as a general principle of Community law. The decisions threaten to overturn national industrial systems in Member States. The
immediate victims are Finland, Sweden and Germany. But the same fate awaits others. The future of national industrial relations systems in the EU depends on whether these
decisions and their consequences are reversed. Aggressive employers have already sought to exploit the new EU legal doctrine to
stop collective action protected by national laws. 7. In sum… Early in 2008, the uncertain applica-
tion of the criteria laid down by the ECJ to assess whether collective action which re-
stricts economic freedom is lawful enabled British Airways to exploit this uncertainty to
threaten legal action against Balpa, the pilots’ trade union, which had voted by a large
majority to take collective action.13 13 The Financial Times (12 March 2008, page 2) included the following report (Kevin Done), "BA
uses EU law to prevent strike by pilots": "British Airways is looking to use competition law and a
threat to seek 'unlimited damages' against the UK pilots' union in order to stop them going on strike. An overwhelming majority of BA's 3,200 pilots voted last month in favour of taking strike action
over BA's plans to set up OpenSkies, a new airline subsidiary, with a pilot workforce separate to its
mainline operations, which fly to and from Heathrow and Gatwick airports. Conciliation talks aimed
at resolving the bitter dispute over future pilot staffing at BA subsidiary airlines in Europe collapsed
on Friday. Balpa, the pilots' union, had planned to issue dates for its first strikes, which would
ground the airline, but was forced to postpone the move when BA warned the union it had 'a valid
legal claim' against it, if it 'took the disproportionate step of calling a strike'. The union said yester-
day that BA was claiming its pilots could not legally pursue their concerns over job security because
of European legislation. Balpa said BA was claiming it had a fundamental right under article 43 of
the EC Treaty to establish operations in another European Union member state. The right included
both establishing new airline services in other EU states, as well as acquiring existing opera-
tions from other airlines. BA was claiming that Balpa was seeking to limit that right by insisting that
there should be a single pilot workforce with a shared seniority list to determine pilot employment. BA has already begun to recruit pilots for OpenSkies. Jim McAuslan, Balpa general-secretary, said
BA 'should be at the negotiating table' and not using European legislation, designed to ensure free
competition between companies, in order to restrict the freedoms of trades unions in industrial dis-
putes. 7. In sum… He said that Balpa was seeking a High Court hearing to clarify whether the European legisla-
tion could be used in an industrial dispute and whether the union could rely on the strike ballot to
avoid a claim for unlimited damages. The union has won court backing for the strike vote to remain
valid beyond the normal 28-day limit until applicability of European law is clarified. It rules out tak-
ing any strike action during the Easter holiday period. BA said yesterday it was 'pleased' Balpa had
recognised it had 'a strong legal case'. It said that 'any strike action would be unlawful', and the un-
ion had therefore decided not to issue strike dates. OpenSkies is BA's most ambitious attempt to
take advantage of the US/European Union 'open skies' treaty liberalising transatlantic aviation". On
15 March 2008, more than 1,000 British Airways pilots marched on BA's Heathrow headquarters
over plans to use non-BA pilots in a new subsidiary after pilots had voted for industrial action, but
BA applied for a legal injunction to prevent it going ahead. A spokesman said given that roughly
2,000 of BA's 3,000 pilots are usually either on duty or resting before or after flights, the turnout
was very high. "Every pilot who could be there was there, which was quite remarkable," the
spokesman said. 71 71 Brian Bercusson Brian Bercusson The potential cost of defending against such legal threats forced Balpa to withdraw
from collective action.14 The lesson is clear. The uncertain application of the criteria
laid down by the ECJ to assess whether collective action which restricts economic free-
dom is lawful enables employers (here British Airways) to exploit this uncertainty to
threaten and thereby possibly obtain an interim injunction.15 The cost and delay en-
tailed in defending against such legal threats effectively deters collective action. In prac- 14 The union’s legal action was heard by the High Court in London over four days, from 19-22 May
2008. The outcome was immediately reported in the on-line edition of the Financial Times of 22
May 2008: “British Airways’ new transatlantic airline has been cleared for take-off after the car-
rier’s pilots on Thursday unexpectedly abandoned a High Court review of their right to strike. BA’s
launch of OpenSkies – an upmarket carrier that will fly between Europe and the US – had been
marred by the threat of a crippling work stoppage at its mainline operations during the peak summer
travel season. Members of the British Air Line Pilots Association overwhelmingly voted to strike
over the initiative in February, claiming that it could be used as a ‘Trojan horse’ to usher less fa-
vourable working conditions across the airline. The union’s decision to withdraw its challenge over
the legality of industrial action means that OpenSkies remains on track to operate its first flight from
Paris to New York on June 19. Balpa has agreed not to reballot its members to authorise a strike. The volte face came on the fourth day of a judicial review hearing over whether a walkout by pilots
were to violate Article 43 of the Treaty of Rome, which gives any European Union-based company
the right to set up a business in other EU states. John Hendy QC, for Balpa, on Thursday told the
High Court the union simply could not afford to pursue the case through an exhaustive series of ap-
peals. “It is very clear that win, lose or draw this case, there will be further litigation by way of ap-
peals and further appeals, the House of Lords and perhaps the European Court of Justice as well,
which will have the effect of increasing exposure to costs of whichever party ultimately loses”, he
said. Brian Bercusson “It would plainly be madness to embark on industrial action with the risk that, in the end, it
would be declared unlawful”. The first tickets for OpenSkies flights between Paris Orly airport and
New York’s JFK went on sale on Thursday. BA said it ‘welcomed’ Balpa’s decision to withdraw
the proceedings, reaffirming its stance that the new carrier posed no threat to the jobs, pay or work-
ing conditions of its mainline pilots. Balpa said it now planned to embark on an EU-wide campaign
to clarify the purpose of Article 43, to ensure it could not be used to undermine workers’ right to
strike. The High Court tussle was being closely watched by unions throughout Europe, who feared
that a ruling against Balpa would give multinationals another weapon to block industrial action. “We shall be pressing for a review of this law which has prevented British trade union members
from protecting their careers”, said Jim McAuslan, Balpa general secretary. BA set up the
OpenSkies subsidiary to take advantage of the liberalisation of transatlantic aviation agreed last year
by the US and the EU. The carrier’s Paris-New York flights this June will be the first occasion when
BA has operated long-haul services that do not begin or end in the UK”. 15 15 The option of Balpa seeking to refer the legal issues to the ECJ was not pursued. Questions could
include, e.g.: (i) Does compliance with domestic law on collective industrial action constitute a cri-
terion for national courts assessing whether the fundamental right to take collective action restricts
the economic freedom of employers in accordance with EC law? (ii) What further criteria does EC
law provide to enable judges to determine whether the fundamental right to take collective action
restricts the economic freedom of employers in accordance with EC law? (iii) What, if any, are the
circumstances in which domestic courts may issue injunctions restraining the exercise of the funda-
mental right to take collective action which allegedly restricts the economic freedom of employers
in accordance with EC law? (iv) Is it consistent with EC law for a domestic court to provide an in-
terim remedy on the basis of the “proportionality” of collective action in changing circumstances
and future and unforeseeable events? 15 The option of Balpa seeking to refer the legal issues to the ECJ was not pursued. 16 The following draws on some of the ideas prepared for the ETUC Congress in Seville in May 2007
by a group of European labour law professors, the Research Group on Transnational Trade Union
Rights of the ETUI-REHS. B. Bercusson (ed) Manifesto for a Social Constitution: 8 options for the
European Union, European Trade Union Institute (ETUI-REHS), Brussels, 2007, (133 pp.) also
available in French and German. IV. Scope of action at European level after the Lisbon Treaty This new EU law on collective action was not envisaged when the Lisbon Treaty was
proposed. In the aftermath of the rejection of the Lisbon Treaty in the Irish referendum
of 12 June 2008, the main objective of this paper is to explore the scope for action at
European level, looking to short, medium and long-term strategies. Brian Bercusson Questions could
include, e.g.: (i) Does compliance with domestic law on collective industrial action constitute a cri-
terion for national courts assessing whether the fundamental right to take collective action restricts
the economic freedom of employers in accordance with EC law? (ii) What further criteria does EC
law provide to enable judges to determine whether the fundamental right to take collective action
restricts the economic freedom of employers in accordance with EC law? (iii) What, if any, are the
circumstances in which domestic courts may issue injunctions restraining the exercise of the funda-
mental right to take collective action which allegedly restricts the economic freedom of employers
in accordance with EC law? (iv) Is it consistent with EC law for a domestic court to provide an in-
terim remedy on the basis of the “proportionality” of collective action in changing circumstances
and future and unforeseeable events? 72 Scope of Action at the European Level tice, the exercise of the fundamental right to strike, ostensibly protected as a general
principle of EU law, is denied. V. Strategies in the short-term Apart from giving legally binding effect to the EU Charter of Fundamental Rights,
the Lisbon Treaty did not contribute greatly to the social dimension of the EU. And, of
course, the new EU law on collective action was not foreseen. Some Member States
would have preferred a stronger social dimension. It is possible to reverse some of the
negative consequences of the ECJ’s decisions and to enable those Member States to
undertake a commitment to a stronger social dimension.16 Two issues arise: − what would be the legal form of such a commitment; − what would be the substantive content of such a commitment? 17 See B. Bercusson, “Option 2: A ‘Social Protocol’ to the Constitutional Treaty”, ibid., pp.30-31 and
63-73.
18 See Antoine Jacobs, “Option 3: Enhanced cooperation”, ibid., pp.32-33 and 75-63.
19 See Isabelle Schömann, “Option 4: The Schengen Model: ‘Variable Geometry’”, ibid., pp.33-34 and
85-94.
20 See Yota Kravaritou, “Option 6: A Non-Binding Social Declaration’”, ibid., pp.36-37and 105-110. 1. Legal form A number of alternatives may be envisaged. A number of alternatives may be envisaged. 73 /10.5771/
– Brian Bercusson 4. The “Schengen” model19 A stronger social dimension could follow the road of the Schengen model. The
Schengen Agreement harmonising border controls was made initially by only 5 Member
States in 1985. More Member States gradually joined, so that it now includes 25 Mem-
ber States (except Ireland and the UK, but including also Iceland, Norway and Switzer-
land). In 1997, a Protocol attached to the Treaty of Amsterdam incorporated these ad-
vances into the legal framework of the EU. A “Social Schengen Agreement” could be
adopted alongside the Lisbon Treaty. 2. A Protocol17 A number of Member States could adopt a Protocol (attached to the Lisbon Treaty)
on a stronger social dimension. These Member States would undertake to persuade all
Member States to agree to attach this Protocol to the Treaties, though it would only bind
those Member States which adopted it. 3. Enhanced cooperation18 Article 43 TEU allows for enhanced cooperation by a majority of the Member States. If at least 14 Member States agreed that, alongside the Lisbon Treaty, they wished to
adopt provisions for a stronger social dimension, the mechanism of enhanced coopera-
tion could allow this, leaving it open to other Member States to join later. 4. The “Schengen” model19 4. The “Schengen” model19 Scope of Action at the European Level Scope of Action at the European Level 22 Cf. Council Regulation (EC) No. 2679/98 of 7 December 1998 on the functioning of the internal
market in relation to the free movement of goods among the Member States. OJ L337/8 of 12.12.98;
Article 2, quoted above. 21 See B. Bercusson, “Option 7: An Interpretative Instrument”, ibid., pp.37-38 and 111-123. 6. An interpretative instrument (inverse “opt-out”) 21 The UK and Poland attached an “opt-out” to the Lisbon Treaty excluding application
of the EU Charter to their laws. An inverse “social opt-out” could be modelled on Arti-
cle 1 of this opt-out: “The Treaties do not extend the ability of the Court of Justice or
any court or tribunal to find that the laws or practices of the Member States regulating
collective bargaining and collective action are inconsistent with the economic freedoms
that they affirm”.22 Member States desiring a stronger social dimension could attach
this formula to their ratification of the Lisbon Treaty as an interpretative guide. 7. Content23 24 Replacement Article 2(3) TEU. “The Union shall establish an internal market. It shall work for the
sustainable development of Europe based on balanced economic growth and price stability, a highly
competitive social market economy, aiming at full employment and social progress, and a high level
of protection and improvement of the quality of the environment. It shall promote scientific and
technological advance. It shall combat social exclusion and discrimination, and shall promote social
justice and protection, equality between women and men, solidarity between generations and pro-
tection of the rights of the child”. (italics added) Formerly in the Constitutional Treaty, Part I, Artic-
le I-3(3). 27 Sophie Robin-Olivier, “Son raisonnement est, dans les grandes lignes, empreint du plus grand clas-
sicisme”. “Liberté de l’action syndicale vs liberté d’établissement”, Revue de Droit du Travail, Jan-
vier 2008, p. 8. 23 What follows are only a few illustrative suggestions among many possibilities. 5. A non-binding Social Declaration20 Member States desiring a stronger social dimension could attach to their ratification
of the Lisbon Treaty a commonly agreed “social declaration”. Though not legally bind-
ing, this could have a future impact on the political agenda of the EU institutions, in-
cluding the Commission’s action programme and the Court’s interpretation of the EU
Charter’s social provisions. 74 10.577
– Scope of Action at the European Level 7. Content23 The Lisbon Treaty proposes to replace the present Article 2 of the Treaty on Euro-
pean Union with a new Article 2, including references to “social progress” and “social
justice”.24 The new Article 2 affirms that “The Union shall establish an internal mar-
ket”.25 But the internal market is only a means towards achieving a “social market
economy, aiming at full employment and social progress” and the Union “shall pro-
mote social justice and protection”.26 It may be necessary to redress the imbalance in Community law, as presently inter-
preted by the ECJ (and also the present Commission), by challenging the primacy given
to the internal market over all other Community policies. y p
In Laval and Viking, the ECJ regarded collective action primarily in terms of its im-
pact on the economic freedoms of the common market.27 What was shocking, if not 22 Cf. Council Regulation (EC) No. 2679/98 of 7 December 1998 on the functioning of the internal
market in relation to the free movement of goods among the Member States. OJ L337/8 of 12.12.98;
Article 2, quoted above. 24 Replacement Article 2(3) TEU. “The Union shall establish an internal market. It shall work for the
sustainable development of Europe based on balanced economic growth and price stability, a highly
competitive social market economy, aiming at full employment and social progress, and a high level
of protection and improvement of the quality of the environment. It shall promote scientific and
technological advance. It shall combat social exclusion and discrimination, and shall promote social
justice and protection, equality between women and men, solidarity between generations and pro-
tection of the rights of the child”. (italics added) Formerly in the Constitutional Treaty, Part I, Artic-
le I-3(3). 25 Ibid. 27 Sophie Robin-Olivier, “Son raisonnement est, dans les grandes lignes, empreint du plus grand clas-
sicisme”. “Liberté de l’action syndicale vs liberté d’établissement”, Revue de Droit du Travail, Jan-
vier 2008, p. 8. 27 Sophie Robin-Olivier, “Son raisonnement est, dans les grandes lignes, empreint du plus grand clas-
sicisme”. “Liberté de l’action syndicale vs liberté d’établissement”, Revue de Droit du Travail, Jan-
vier 2008, p. 8. 75 Brian Bercusson surprising, is how little weight was given by the ECJ to other, arguably equal if not
more prominent, EU policies. 28 For example, economic freedoms may be invoked against the many directives requiring information
and consultation of workers and their representatives. Such requirements restrict the economic free-
dom of management to make speedy decisions in re-structuring undertakings. An item in the Finan-
cial Times of 28 February 2008 (page 24) reported: "A new power struggle has broken out between
Gaz de France and Suez, this time between the two companies' European works councils, which are
crucial to breaking the union impasse that has repeatedly delayed the creation of the E 75bn
($113bn) energy giant. GdF's European unions are demanding that they represent the enlarged
group when the merger is complete before they will give the non-binding opinion that is required
under French law before the companies' board can approve the deal. Suez is resisting the demand
and both sides are seeking agreement ahead of the next works council meeting on March 11. Failure
to find a compromise could throw the merger timetable off track, further delaying a deal that has al-
ready been two years in the making. GdF's French unions also have yet to give their opinion and
have been using efficient delaying tactics against the merger. Jean-Francois Cirelli, GdF chief ex-
ecutive, said he remained 'reasonably confident' that the deadline of June 30 could still be met, and
that the European unions would give their opinion at the March meeting, Discussions with unions
had entered a more positive phase, he said". 7. Content23 These include fundamental rights (the EU Charter’s free-
dom of association, collective bargaining and collective action), improvement of work-
ing conditions (Article 136 EC), the public policy of protection of workers against un-
fair competition (social dumping), and so on. Each of these was mentioned in the judg-
ments in Laval and Viking, but ultimately subordinated to the economic freedoms of
employers in the common market. This is a threat to the entire acquis communautaire social. If economic freedoms
override collective action, they could be invoked to circumscribe the rest of the acquis
communautaire social.28 The following are only a few proposals which could be re-
flected in one of the legal forms suggested above to accompany ratification of the Lis-
bon Treaty. 10. A more favourable treatment clause The ECJ adopted a bizarre interpretation in Laval of Article 3(7) of the Posting Di-
rective to restrict the freedom of Member States to require more favourable treatment of
workers. This was in order to support its view of the Directive as a stipulating a “maxi-
mum standard”.30 This could be addressed by amending the Posting Directive. But this 29 The proposed revision of the Working Time Directive exemplifies the need for such a principle. Amended proposal for a Directive of the European Parliament and of the Council amending Direc-
tive 2003/88/EC concerning certain aspects of the organisation of working time – Political agree-
ment on a common position. SOC 357, CODEC 758, Brussels, 11 June 2008. 0 Article 3(7) of the Posting Directive 96/71/EC appeared to support this principle: “Paragraphs 1 to 6
shall not prevent the application of terms and conditions of employment which are more favourable
to workers”. The ECJ interpreted this provision as follows (paras. 79-80; italics added): “It is true
that Article 3(7) of Directive 96/71 provides that ‘paragraphs 1 to 6 are not to prevent the applicati-
on of terms and conditions of employment which are more favourable to workers’. In addition, ac-
cording to recital 17, the mandatory rules for minimum protection in force in the host country must
not prevent the application of such terms and conditions. Nevertheless, Article 3(7) of Directive
96/71 cannot be interpreted as allowing the host Member State to make the provision of services in
its territory conditional on the observance of terms and conditions of employment which go beyond
the mandatory rules for minimum protection. As regards the matters referred to in Article 3(1), first
paragraph, (a) to (g), Directive 96/71 expressly lays down the degree of protection for workers of
undertakings established in other Member States who are posted to the territory of the host Member
State which the latter State is entitled to require those undertakings to observe. Moreover, such an
interpretation would amount to depriving the directive of its effectiveness”. Reiterated in Rüffert,
paras. 32-33. Contrast the Opinion of Advocate General Bot in Rüffert: (paras. 9. A non-regression clause The references in the social policy objectives listed in Article 136 EC to “improved
living and working conditions” and “harmonisation while the improvement is being
maintained” argue for a general principle of “non-regression” in Community social pol-
icy. This could be made more explicit and elaborate as a principle of Community law.29 29 The proposed revision of the Working Time Directive exemplifies the need for such a principle.
Amended proposal for a Directive of the European Parliament and of the Council amending Direc-
tive 2003/88/EC concerning certain aspects of the organisation of working time – Political agree-
ment on a common position. SOC 357, CODEC 758, Brussels, 11 June 2008. Scope of Action at the European Level Scope of Action at the European Level 9. A non-regression clause 8. An “anti-social dumping principle” against abuse of the exercise of economic
freedoms Laval and Viking both contain expressions of concern by the Advocates General and
the ECJ about “social dumping”. The unrestrained exercise of economic freedom of
movement may threaten existing jobs and working conditions. This may be character-
ised as an “abuse” of the exercise of economic freedoms. A condition of the exercise of
economic freedom is that there is a guarantee of existing jobs and working conditions. Failure to provide such a guarantee justifies collective action against such an abuse. 28 For example, economic freedoms may be invoked against the many directives requiring information
and consultation of workers and their representatives. Such requirements restrict the economic free-
dom of management to make speedy decisions in re-structuring undertakings. An item in the Finan-
cial Times of 28 February 2008 (page 24) reported: "A new power struggle has broken out between
Gaz de France and Suez, this time between the two companies' European works councils, which are
crucial to breaking the union impasse that has repeatedly delayed the creation of the E 75bn
($113bn) energy giant. GdF's European unions are demanding that they represent the enlarged
group when the merger is complete before they will give the non-binding opinion that is required
under French law before the companies' board can approve the deal. Suez is resisting the demand
and both sides are seeking agreement ahead of the next works council meeting on March 11. Failure
to find a compromise could throw the merger timetable off track, further delaying a deal that has al-
ready been two years in the making. GdF's French unions also have yet to give their opinion and
have been using efficient delaying tactics against the merger. Jean-Francois Cirelli, GdF chief ex-
ecutive, said he remained 'reasonably confident' that the deadline of June 30 could still be met, and
that the European unions would give their opinion at the March meeting, Discussions with unions
had entered a more positive phase, he said". 76 Scope of Action at the European Level Scope of Action at the European Level 11. An interpretative framework for economic freedoms The market economy is not limited to the economic freedoms of enterprises only. Market freedom includes the freedom of action of collective actors both of capital (en-
terprises) and labour (trade unions).31 The economic freedom of movement of one side
should not be invoked to restrain the economic freedom of action of the other side, as
the ECJ has done in Viking and Laval.32 This should be remedied by amendments pro-
viding a more even-handed interpretative framework for economic freedoms. For ex-
ample: 10. A more favourable treatment clause 81-83, italics added)
“In addition, I would remind the Court that the 17th recital in the preamble to that Directive provides
that ‘the mandatory rules for minimum protection in the host country must not prevent the applicati-
on of terms and conditions of employment which are more favourable to the workers’. The first
subparagraph of Article 3(7) of the directive translates this intention of the Community legislature
by stating that ‘[p]aragraphs 1 to 6 shall not prevent the application of terms and conditions of
employment which are more favourable to workers“. In my view, there are two aspects to this last
provision. First, it means that the mandatory nature of the protective rules in force in the Member
State where the services are performed may be eclipsed by application of the rules in force in the
State in which the provider is established if those rules provide for terms and conditions of
employment that are more favourable to the posted workers. Secondly, and it is this aspect which is
relevant in the present case, Article 3(7) of Directive 96/71 also, in my view, permits the Member
State where the services are performed to improve, for the matters referred to in Article 3(1) of the 77 77 Brian Bercusson principle is found in many other legislative provisions and the ECJ’s interpretation may
be dangerous more generally. An attempt could be made to clarify and extend the prin-
ciple. principle is found in many other legislative provisions and the ECJ’s interpretation may
be dangerous more generally. An attempt could be made to clarify and extend the prin-
ciple. a) A rebuttable presumption in favour of collective action A rebuttable presumption that collective action is presumed to be in response to a se-
rious threat to jobs and working conditions. This may be rebutted when challenged by
the employer.33 directive, the level of social protection which it wishes to guarantee to workers employed in its terri-
tory and which it can therefore apply to workers posted there. Hence in principle, this provision
authorises the implementation of enhanced national protection”. See also paragraphs 90-98 of the
Opinion. 31 As stated by Miguel Poiares Maduro: “…the system requires a set of social rights that can be said to
guarantee participation and representation in market decisions and, by internalizing costs which tend
to be ignored in those decisions, increase efficiency... rights of participation and representation such
as the freedom of association, the right to collective bargaining, and the right to collective action
should be considered as instrumental to a fully functioning integrated market which can increase ef-
ficiency and wealth maximization”. “Striking the Elusive Balance Between Economic Freedom and
Social Rights in the EU”, in P. Alston (ed), The EU and Human Rights (Oxford University Press,
1999), pp. 449-472, at 470. 31 As stated by Miguel Poiares Maduro: “…the system requires a set of social rights that can be said to
guarantee participation and representation in market decisions and, by internalizing costs which tend
to be ignored in those decisions, increase efficiency... rights of participation and representation such
as the freedom of association, the right to collective bargaining, and the right to collective action
should be considered as instrumental to a fully functioning integrated market which can increase ef-
ficiency and wealth maximization”. “Striking the Elusive Balance Between Economic Freedom and
Social Rights in the EU”, in P. Alston (ed), The EU and Human Rights (Oxford University Press,
1999), pp. 449-472, at 470. 32 In a parallel case of alleged conflict between the Treaty’s provisions on competition and collective
agreements, the ECJ had refused to outlaw collective agreements in order to preserve competition. Albany International BV v. Stichting Bedrijfspensioenfonds Textielindustrie, Case C-67/96; with Joined
cases C-115/97, C-116/97 and C-117/97; [1999] ECR I-5751. Unfortunately, the ECJ rejected this ap-
proach in Laval and Viking, distinguishing the Treaty’s competition provisions from free movement
provisions. 33 As stated in Viking, the presumption may be rebutted if the employer gives a legally binding under-
taking providing guarantees in the form of a collective agreement. 31 As stated by Miguel Poiares Maduro: “…the system requires a set of social rights that can be said to
guarantee participation and representation in market decisions and, by internalizing costs which tend
to be ignored in those decisions, increase efficiency... rights of participation and representation such
as the freedom of association, the right to collective bargaining, and the right to collective action
should be considered as instrumental to a fully functioning integrated market which can increase ef-
ficiency and wealth maximization”. “Striking the Elusive Balance Between Economic Freedom and
Social Rights in the EU”, in P. Alston (ed), The EU and Human Rights (Oxford University Press,
1999), pp. 449-472, at 470. directive, the level of social protection which it wishes to guarantee to workers employed in its terri-
tory and which it can therefore apply to workers posted there. Hence in principle, this provision
authorises the implementation of enhanced national protection”. See also paragraphs 90-98 of the
Opinion. 34 In doctrinal terms, this is a specifically EU criterion based on the acquis communautaire social re-
flected in Articles 27 and 28 of the EU Charter: protection by EU law of the transnational economic
freedom of employers is balanced with protection of transnational collective action by workers who
should be properly informed and consulted before decisions affecting them are made. The common
element is the prevention of “social dumping”. 35 This approach, also applied by the ECJ to freedom of assembly and association in Schmidberger
(Case C-112/00, Eugen Schmidberger, Internationale Transporte und Planzuge v. Republic of Aus-
tria, [2003] ECR I-5659, 12 June 2003) and to human dignity in Omega (Case C-36/02, Omega
Spielhallen- und Automatenaufstellungs-GmbH v. Oberburgermeisterin der Bundesstadt Bonn,
[2004] ECR I-9609, 14 October 2004) is offensive to fundamental human rights. 36 This is the approach proposed by Advocate General Stix-Hackl in her Opinion in Omega. b) Interpreting “proportionality” in light of the acquis communautaire social The lawfulness of collective action is conditional on “proportionality” (Viking) and is
explicitly linked to the Posting Directive (Laval). Why the Posting Directive? Collective
action is proportionate where the employer fails to comply with obligations under the
general acquis communautaire social protecting the rights of workers as an objective of
general interest recognised by the Union.34 a) A rebuttable presumption in favour of collective action In the absence of such a collecti-
ve agreement, judges should not intervene to restrain collective action in the course of collective
bargaining. As stated by Advocate General Mengozzi in his Opinion in Laval. 33 As stated in Viking, the presumption may be rebutted if the employer gives a legally binding under-
taking providing guarantees in the form of a collective agreement. In the absence of such a collecti-
ve agreement, judges should not intervene to restrain collective action in the course of collective
bargaining. As stated by Advocate General Mengozzi in his Opinion in Laval. 78 Scope of Action at the European Level Brian Bercusson Brian Bercusson the user employer.37 This could apply to temporary agencies who post workers to other
Member States.38 The principle of equal treatment could be applied to posted workers
generally. For example, where posted by employers to work alongside comparable
workers employed by other employers, or in sectors regulated by collective agreements
covering comparable workers. the user employer.37 This could apply to temporary agencies who post workers to other
Member States.38 The principle of equal treatment could be applied to posted workers
generally. For example, where posted by employers to work alongside comparable
workers employed by other employers, or in sectors regulated by collective agreements
covering comparable workers. VI. Strategies in the medium-term39 The viability of strategies in the medium-term depends on the political context; not
least, the elections to the European Parliament and of a new Commission in 2009. The following proposals aiming for a stronger social dimension may involve new or
revised directives, but may have wider institutional implications. 37 Commission of the European Communities, Proposal for a Directive of the European Parliament
and the Council on working conditions for temporary workers, COM(2002) 149 final, Brussels, 20
March 2002; Amended Proposal, COM(2002) 701 final, Brussels, 28 November 2002. See now the
Amended proposal for a Directive of the European Parliament and the Council on working condi-
tions for temporary workers - Political agreement on a common position, Brussels, 11 June 2008.
SOC 358, CODEC 761. 38 For a discussion of transnational temporary agency work (excluded from the Services Directive) and
the need for a directive, see B. Bercusson and N. Bruun, “Free movement of services, transnational
temporary agency work and the acquis communautaire”, in K. Ahlberg, B. Bercusson, N. Bruun, H.
Kountouros, C. Vigneau, L. Zappalà, Transnational Labour Regulation: A Case Study of Tempo-
rary Agency Work, Peter Lang, Brussels, 2008. c) Human rights are not mere derogations from economic freedoms; rather the re-
verse The ECJ regards the fundamental right to collective action as a derogation from the
Treaty’s guarantee of economic freedoms. As such, collective action must be justi-
fied.35 The ECJ also declares the fundamental right to collective action to be protected
as a general principle of Community law. The Lisbon Treaty provides that the EU Char-
ter is to have the same legal status as the Treaties and proposes that the EU ratifies the
European Convention on Human Rights. The ECJ’s approach should be reversed: eco-
nomic freedoms may exceptionally derogate from fundamental rights guaranteed by
Community law, but this has to be justified, and any derogation from fundamental rights
is only permitted if “proportionate” (necessary and no alternative available).36 12. The Agency Workers Directive: Equal treatment of agency workers includes
cross-border workers The decisions in Laval and Viking threaten the principle of equal treatment of cross-
border workers. The Commission’s proposal on Temporary Agency Workers included a
requirement that agency workers be treated equally to comparable workers employed by 79 /10.5771/9
– Brian Bercusson 2. A standard social safeguard clause in directives Directives already contain standard clauses; for example, regarding obligations of
Member States as regards effective enforcement. A standard social safeguard clause
could be formulated for inclusion in directives.41 This could include the following pro-
visions: − explicitly protecting national standards (e.g. as in the “Monti” Regulation − explicitly protecting national standards (e.g. as in the “Monti” Regulation); − substantive provisions protecting fundamental rights and collective agreements
per se (as in the Services Directive); − a non-regression principle, following Article 136 EC, made more explicit and
elaborate; or − allowing national provisions more favourable to workers;42 − specifying obligations on Member States to provide information to employers on
labour standards.43 3. Monitoring transposition of the Services Directive Laval, by asserting the power of Article 49 EC, implicitly challenges the attempt in
the Services Directive to protect fundamental rights to collective action and collective
bargaining and collective agreements from liberalisation of services.44 Member States
transposing the Services Directive may offer an opportunity to promote legislation chal-
lenging the ECJ’s interpretation of Article 49. This may allow for litigation enabling the
ECJ to reconsider its decision in Laval. 1. “Update” the “explanations” to the EU Charter These “explanations” were “updated” by the Convention on the Future of Europe and
again by the Member States at the summit of June 2004. In particular, the explanations
accompanying Article 28 on collective action were “updated” at the initiative of the UK
government in an attempt to reduce its scope and impact. These “explanations” could be
revisited, not least since the UK has opted out of the Charter. They could be revised to
reflect a more satisfactory equilibrium between the fundamental right “to take collective
action” and economic freedoms.40 38 For a discussion of transnational temporary agency work (excluded from the Services Directive) and
the need for a directive, see B. Bercusson and N. Bruun, “Free movement of services, transnational
temporary agency work and the acquis communautaire”, in K. Ahlberg, B. Bercusson, N. Bruun, H. Kountouros, C. Vigneau, L. Zappalà, Transnational Labour Regulation: A Case Study of Tempo-
rary Agency Work, Peter Lang, Brussels, 2008. 39 Of course, some of the proposed short-term strategies become medium-term if they do not succeed
in the short-term, and so on… 39 Of course, some of the proposed short-term strategies become medium-term if they do not succeed
in the short-term, and so on… 40 Revision of the “explanations” to Article 28’s “right to negotiate and conclude collective agree-
ments at the appropriate levels” might also assist the social dialogue, not least at EU level. There is
no need to limit this to Article 28. The ECJ’s reasoning in Schmidberger might attract support from
human rights lobbies for a revision of the “explanations” to Article 12: Freedom of assembly and of 40 Revision of the “explanations” to Article 28’s “right to negotiate and conclude collective agree-
ments at the appropriate levels” might also assist the social dialogue, not least at EU level. There is
no need to limit this to Article 28. The ECJ’s reasoning in Schmidberger might attract support from
human rights lobbies for a revision of the “explanations” to Article 12: Freedom of assembly and of 80 Scope of Action at the European Level association. Similarly, Article 27: Workers’ right to information and consultation within the under-
taking could also be revisited. g
41 The proposed Lisbon Treaty includes a horizontal social clause, Article 5A. y
42 Overriding the ECJ’s bizarre interpretation of Article 3(7) of the Posting Directive. 44 E.g. in Articles 1(7)), 4(7) 16(3), Recital 15…. 43 A requirement in the public procurement directives, said to promote the transparency of the single
market. 44 E.g. in Articles 1(7)), 4(7) 16(3), Recital 15…. 46 This could introduce criteria of decentralisation and flexibility to provide for articulation between
national/sectoral and local/enterprise/workplace agreements. This could accommodate the flexible
qualities of the Nordic systems advocated by the Commission. 45 Indeed, it might be a subject for social dialogue. This extends the Interinstitutional Agreement on
Better Lawmaking of December 2003 between the Commission, Council and Parliament to include
the social partners, recognising their privileged position. The “opinion” foreseen in Article 138(3)
EC could include a social impact assessment Brian Bercusson Brian Bercusson systems and, in particular, collective bargaining and collective agreements. The formu-
lation of this assessment could engage the social partners.45 systems and, in particular, collective bargaining and collective agreements. The formu-
lation of this assessment could engage the social partners.45 5. Operationalising the Lisbon Treaty’s horizontal social clause The Lisbon Treaty’s horizontal social clause, Article 5A, provides: “In defining and implementing its policies and activities, the Union shall take into
account requirements linked to the promotion of a high level of employment, the guar-
antee of adequate social protection, the fight against social exclusion, and a high level
of education, training and protection of human health”. This could be elaborated so as to make it more effectively operational. Its terms
could acquire more precise meaning and adapted to specific Commission proposals. Examples follow. 47 Not least in anticipation of the pending decision in Case C-319/06, Commission v. Luxembourg,
Opinion of Advocate-General Trstenjak, 13 September 2007. 4. Social impact assessments Commission proposals should include a “social impact assessment”. The content
should assess the proposal not only in terms of specific social benchmarks, such as
minimum standards required, but in terms of its potential impact on industrial relations association. Similarly, Article 27: Workers’ right to information and consultation within the under-
taking could also be revisited. 81 81 6. Revise the Posting Directive − making the equal treatment principle applicable to migrant workers applicable to
posted workers; if necessary, by declaring that posted workers are to be treated
equally to domestic workers after working for a specified (short) period in the
host Member State. By legitimizing collective agreements specifically applicable to transnational move-
ment of labour, this would reinforce the principle of non-discrimination/equal treatment
of workers, collective bargaining systems which are decentralised but articulated, legis-
lative mechanisms available to enforce these standards and the right to collective action
to support these standards. VII. Strategies for the long-term The ECJ in Viking and Laval relied on the EC Treaty’s provisions guaranteeing eco-
nomic freedoms. These were characterised as equivalent, and indeed potentially supe-
rior to fundamental rights.48 Action at European level is required for a stronger social
dimension in the long-term by revising the Treaties. The attack could be on the substance of the ECJ’s approach to EC law (market pri-
macy), or on the exercise by the EU of its competences (subsidiarity), and hence the
scope of the ECJ’s jurisdiction; or indeed both.49 Three specific proposals may be sug-
gested. 6. Revise the Posting Directive The ECJ’s interpretation of the Posting Directive in Laval is unorthodox, if not
wrong as contradicting the aim of the EU legislator. The EU legislator could amend the − extending the scope of collective agreements establishing standards binding for-
eign enterprises which post workers, and making mandatory what are currently
only options under Article 3(8) of the Directive (amending the definitions of ap-
plicable collective agreements determining conditions applicable to foreign ser-
vice providers);46 − make clear that both legislative sanctions and collective action are available to
enforce these standards; − make clear that both legislative sanctions and collective action are available to
enforce these standards; − expanding the scope of “public policy” provisions which may be extended to ap-
ply to foreign services providers under Article 3(10) of the Directive;47 − making clear the power of Member States to require more favourable conditions,
reversing the ECJ’s bizarre interpretation of Article 3(7) of the Directive; 45 Indeed, it might be a subject for social dialogue. This extends the Interinstitutional Agreement on
Better Lawmaking of December 2003 between the Commission, Council and Parliament to include
the social partners, recognising their privileged position. The “opinion” foreseen in Article 138(3)
EC could include a social impact assessment 46 This could introduce criteria of decentralisation and flexibility to provide for articulation between
national/sectoral and local/enterprise/workplace agreements. This could accommodate the flexible
qualities of the Nordic systems advocated by the Commission. 46 This could introduce criteria of decentralisation and flexibility to provide for articulation between
national/sectoral and local/enterprise/workplace agreements. This could accommodate the flexible
qualities of the Nordic systems advocated by the Commission. 47 Not least in anticipation of the pending decision in Case C-319/06, Commission v. Luxembourg,
Opinion of Advocate-General Trstenjak, 13 September 2007. 47 Not least in anticipation of the pending decision in Case C-319/06, Commission v. Luxembourg,
Opinion of Advocate-General Trstenjak, 13 September 2007. 82 10.577
– Scope of Action at the European Level − making the equal treatment principle applicable to migrant workers applicable to
posted workers; if necessary, by declaring that posted workers are to be treated
equally to domestic workers after working for a specified (short) period in the
host Member State. 48 Unfortunately, at present, the formal supremacy of EU law is often translated into, and reinforces,
the substantive primacy of promoting economic freedoms in the common market. 49 Or a combination, focussing on the limitation to transnational matters? The ECJ may determine
rules on transnational collective action, but not on matters purely internal to Member States not af-
fected by EC law. What is the consequence if there are radically different rules on transnational and
national collective action, and between the national rules in different Member States? Do such dif-
ferences inherently impede free movement, deterring employers, and hence violate the Treaty? Such
differences might impede free movement of workers, as some might avoid moving to Member
States where their rights to take collective action are more restricted. What are the criteria of
“transnationality”? Is national action caught only if there is actual obstruction of a transnational
market relationship, or is it sufficient if it merely (potentially) impedes market access? 1. Revise Treaty provisions on economic freedoms The task is to re-draft the provisions on economic freedoms in such a way as to re-
duce or eliminate their negative impact on fundamental rights; specifically, on collective
action, and so as to protect workers. For example, the primacy of economic freedoms could be attacked by reformulating
the provisions to include the statement that economic freedoms may not be interpreted 48 Unfortunately, at present, the formal supremacy of EU law is often translated into, and reinforces,
the substantive primacy of promoting economic freedoms in the common market. 49 Or a combination, focussing on the limitation to transnational matters? The ECJ may determine
rules on transnational collective action, but not on matters purely internal to Member States not af-
fected by EC law. What is the consequence if there are radically different rules on transnational and
national collective action, and between the national rules in different Member States? Do such dif-
ferences inherently impede free movement, deterring employers, and hence violate the Treaty? Such
differences might impede free movement of workers, as some might avoid moving to Member
States where their rights to take collective action are more restricted. What are the criteria of
“transnationality”? Is national action caught only if there is actual obstruction of a transnational
market relationship, or is it sufficient if it merely (potentially) impedes market access? 83 83 Brian Bercusson as affecting in any way the exercise of fundamental rights based on the “common con-
stitutional traditions” of the Member States; specifically, as regards protection of the
right to collective action.50 This also removes the primary obligation of Member States
(and national law) to comply with the EU internal market objective.51 The fundamental economic freedoms of employers are usually exercised through the
collective organisational form of corporate capital. The Treaty’s economic freedom of
workers applies only to individual workers. Another approach would be to draft a fifth
fundamental economic freedom of collective organisations of workers.52 This would
promote an interpretation of the Treaty recognising that collective action by workers is
consistent with the effective functioning of the internal market. Workers and trade un-
ions, as market participants, may take collective action to ensure their voice is heard and
their interests are taken into account, a feature essential to the effective functioning of
the internal market. Collective bargaining, collective agreements and collective action
are essential to the effective and equitable functioning of the labour market. 50 As in the “Monti” Regulation, Council Regulation (EC) No. 2679/98 of 7 December 1998 on the
functioning of the internal market in relation to the free movement of goods among the Member
States. OJ L337/8 of 12.12.98; Article 2, quoted above. 1. Revise Treaty provisions on economic freedoms This bal-
ances the discrepancy between the power of employers benefiting from European trans-
national economic integration, and the relative weakness of a labour movement largely
confined to national boundaries in its collective bargaining and collective action. Finally, as proposed under short-term measures, the Treaties could explicitly provide
for an “anti-social dumping principle” against abuse of the exercise of economic free-
doms. 52 This could build on the precedent of the recognition of management and labour as institutional ac-
tors in the Maastricht Agreement’s provisions on social dialogue. States. OJ L337/8 of 12.12.98; Article 2, quoted above.
51 Thus reinforcing the ECJ’s jurisprudence establishing a general principle of substance that EC law
protects fundamental rights. 50 As in the “Monti” Regulation, Council Regulation (EC) No. 2679/98 of 7 December 1998 on the
functioning of the internal market in relation to the free movement of goods among the Member
States. OJ L337/8 of 12.12.98; Article 2, quoted above.
51 Thus reinforcing the ECJ’s jurisprudence establishing a general principle of substance that EC law
protects fundamental rights.
52 This could build on the precedent of the recognition of management and labour as institutional ac-
tors in the Maastricht Agreement’s provisions on social dialogue. a) Structure of the ECJ There have been reforms to the structure of the ECJ in the past.53 The decisions in
Viking, and particularly in Laval with its bizarre interpretation of the Posting Directive
and dramatic consequences for the entire Swedish system of industrial relations, make a
powerful case for establishment of a specialist tribunal: the chambre social of the Euro-
pean Court. 3. Revise the subsidiarity principle to exclude EC competence to regulate collective
action National laws on collective action by workers and trade unions reflect an equilibrium
carefully constructed over time. It is an area of law jealously guarded by Member
States. The principle of subsidiarity aims to preclude EC law intervening as EU inter-
vention could be destabilising. Transnational collective action raises difficult questions
in private international law: which national court has jurisdiction and the law of which
Member State applies. Different laws and different courts take different views regarding
the legality of cross-border collective action. The Services Directive rejected the “country of origin” principle to protect national
social models, in particular, as regards collective bargaining, collective agreements and
collective action: “the exercise of fundamental rights as recognised in the Member
States and Community law… the right to negotiate, conclude and enforce collective
agreements and to take industrial action in accordance with national law and prac-
tices…” (Article 1(7)). The principle of subsidiarity could be reworked to reflect explic-
itly the protection of collective action. 2. Revise the Treaty to prevent employers using economic freedoms against trade
unions taking collective action (horizontal direct effect) EC Treaty Articles 43 (freedom of establishment) and 49 (freedom of services) were
aimed at regulatory action by public authorities (vertical direct effect) or professional
associations regulating access to the labour market. This is not the case with trade un-
ions engaging in collective action in pursuance of a collective agreement which regu-
lates substantive terms and conditions of employment, not free movement. To apply horizontal direct effect to collective agreements means the ECJ will be
flooded with endless references from national courts asking whether a specific collec-
tive agreement in a particular Member State’s collective bargaining system possessed
the requisite regulatory effect. 50 As in the “Monti” Regulation, Council Regulation (EC) No. 2679/98 of 7 December 1998 on the
functioning of the internal market in relation to the free movement of goods among the Member
States. OJ L337/8 of 12.12.98; Article 2, quoted above. 50 As in the “Monti” Regulation, Council Regulation (EC) No. 2679/98 of 7 December 1998 on the
functioning of the internal market in relation to the free movement of goods among the Member
States. OJ L337/8 of 12.12.98; Article 2, quoted above. 51 Thus reinforcing the ECJ’s jurisprudence establishing a general principle of substance that EC law
protects fundamental rights. 51 Thus reinforcing the ECJ’s jurisprudence establishing a general principle of substance that EC law
protects fundamental rights. 52 This could build on the precedent of the recognition of management and labour as institutional ac-
tors in the Maastricht Agreement’s provisions on social dialogue. 52 This could build on the precedent of the recognition of management and labour as institutional ac-
tors in the Maastricht Agreement’s provisions on social dialogue. 84 10.577
– Scope of Action at the European Level Scope of Action at the European Level The Treaty should be revised to clarify that economic freedoms may not be invoked
against trade unions taking collective action. 3. Revise the subsidiarity principle to exclude EC competence to regulate collective
action 54 As in the “Monti” Regulation, quoted above. 53 The Single European Act 1986 created the European Court of First Instance. The Treaty of Nice
2000 allows for the creation of specialist tribunals; the first is the European Civil Service Tribunal. the “Monti” Regulation, quoted above. d) Composition of the ECJ The legislative institutions of the EU, Council and Parliament (and in the future, even
the Commission) reflect the relative weight of different Member States. The European
Court does not: each Member State appoints one judge. The latest enlargement means
there are 15 judges from the “old” Member States and 12 from the “new” Member
States.58 55 This was the effect of the decision in Albany, where the ECJ ruled that collective agreements fell
outside the competition provisions of the Treaty. 56 In European Social Policy: Between fragmentation and integration, (eds. Stephan Liebfried and
Paul Pierson, The Brookings Institution, Washington, 1995), the editors offer insights into the role
of the courts in EC labour law: (p. 26) "...joint-decision traps in the United States helped push na-
tional initiatives for social reform in a rights-based, court-led direction, with disappointing results... the relatively activist role of the European Court of Justice in social policy stems in part from a
similar desire of European federalists to avoid the daunting problems associated with joint decision-
making through the European Council". And pp. 36-37: "...the dilemmas of shared decision-making
in the EU lead to strategies of circumvention… the efforts of actors to escape the gridlock in social
policy-making is a central theme. For advocates of social integration, institutional constraints within
the Union have made court-led policy development an important path of social reform... [However]
multi-tiered systems make centralized policymaking difficult for a reason - to protect local interests
- and circumvention of these protections is likely to generate resentment... one aspect of the current
disquiet over the Union's 'democratic deficit'...". 57 This should be extended to allowing the social partners to take legal action against the Member
States or the other EU institutions for failing to observe Community law (e.g. against the Commis-
sion for failure to observe Treaty provisions on the social dialogue process). 57 This should be extended to allowing the social partners to take legal action against the Member
States or the other EU institutions for failing to observe Community law (e.g. against the Commis-
sion for failure to observe Treaty provisions on the social dialogue process). 58 The Viking and Laval cases were illustrations of the fact that the disparity in labour costs among
“new” and “old” Member States and the willingness of enterprises to exploit economic freedoms
has the potential of social dumping as between “old” and “new” Member States. c) Procedures of the ECJ The ETUC’s experience in Viking demonstrated that, although cases before the ECJ
may be of vital concern to trade unions, the rules of procedure before the Court make it
difficult for trade unions to make representations. The consequence is that the ECJ may
not be alerted to important issues. In particular, the rules of procedure should acknowl-
edge that the privileged status of the social partners as institutional actors in the EU
Treaty authorises them, like the Member States, to intervene before the ECJ.57 Brian Bercusson Brian Bercusson specifically as regards collective action, by curtailing the jurisdiction of the ECJ.55 Ar-
ticle 234 EC could be amended to limit the discretion of national courts to make pre-
liminary references to the ECJ on matters concerned with collective action.56 specifically as regards collective action, by curtailing the jurisdiction of the ECJ.55 Ar-
ticle 234 EC could be amended to limit the discretion of national courts to make pre-
liminary references to the ECJ on matters concerned with collective action.56 56 In European Social Policy: Between fragmentation and integration, (eds. Stephan Liebfried and
Paul Pierson, The Brookings Institution, Washington, 1995), the editors offer insights into the role
of the courts in EC labour law: (p. 26) "...joint-decision traps in the United States helped push na-
tional initiatives for social reform in a rights-based, court-led direction, with disappointing results...
the relatively activist role of the European Court of Justice in social policy stems in part from a
similar desire of European federalists to avoid the daunting problems associated with joint decision-
making through the European Council". And pp. 36-37: "...the dilemmas of shared decision-making
in the EU lead to strategies of circumvention… the efforts of actors to escape the gridlock in social
policy-making is a central theme. For advocates of social integration, institutional constraints within
the Union have made court-led policy development an important path of social reform... [However]
multi-tiered systems make centralized policymaking difficult for a reason - to protect local interests
- and circumvention of these protections is likely to generate resentment... one aspect of the current
disquiet over the Union's 'democratic deficit'...". 55 This was the effect of the decision in Albany, where the ECJ ruled that collective agreements fell
outside the competition provisions of the Treaty. b) Competences of the ECJ The ECJ (and the EU) should not have competence to override fundamental rights (to
collective action) protected in Member States.54 This could be reinforced, for example, 85 /10.5771/
– included 9 judges from the “old” Member States (Austria, Belgium, Denmark, Finland, Greece, Ire-
land, Luxembourg, Spain and the United Kingdom), and 4 judges from the “new” Member States
(Estonia, Latvia, Lithuania, Poland). There was no judge from France, Germany or Italy. The juge
rapporteur in Viking was from Luxembourg ; the juge rapporteur in Laval was from Estonia. Sub-
missions broadly supporting the employer in Viking were made to the ECJ by representatives of all
4 “new” Member States on the Court and the Czech Republic, Hungary, and the United Kingdom.
Submissions broadly supporting the trade unions were made by representatives of 5 of the “old”
Member States on the Court (Austria, Belgium, Denmark, Finland and Ireland), and France, Ger-
many, Italy, Norway and Sweden. b) ILO Conventions All Member States, as members are bound by the ILO Declaration of Principles of
1998, and have ratified ILO Conventions 87 and 98. Article 307 EC means that the ob-
ligations under these Conventions “shall not be affected by the provisions of this
Treaty”. This could be made explicit as regards specific Conventions of the ILO, as in-
terpreted by the ILO’s Freedom of Association Committee. ILO Convention 87 has
been interpreted to include the right to strike. Rights under international labour law are
not to be affected by the free movement provisions of the Treaty. Scope of Action at the European Level Scope of Action at the European Level a) The European Social Charter The Lisbon Treaty proposes that the EU ratifies the European Convention on Human
Rights. Article 136 EC already refers to the Community “having in mind” the European
Social Charter (ESC) of 1961. The Treaty should allow for ratification by the EU of the
ESC, which includes the right to strike in Article 6(4). 59 In Viking, the standard is whether the threat to jobs and conditions is “serious” and the action is
“suitable”; in Laval, the standard is that stipulated in the Posting Directive. d) Composition of the ECJ There was a clear
difference between the submissions to the ECJ of the “old” (except for the UK) and “new” Member
States. See B. Bercusson, “The Trade Union Movement and the European Union: Judgment Day”,
(2007) 13 European Law Journal (No. 3, May), pp. 279-308. The Court deciding Laval and Viking 86 10.5771
– Scope of Action at the European Level 60 B.A. Hepple (ed), The Making of Labour Law in Europe: A Comparative Study of Nine Countries
up to 1945, London, Mansell, 1986.
61 An example is the British experience, see K.D. Ewing (ed), The Right to Strike: From the Trade
Disputes Act 1906 to a Trade Union Freedom Bill 2006, Liverpool, The Institute of Employment
Rights, 2007. 1. National labour law National labour law in the EU Member States emerged to redress the imbalance of
power between employer and employee in national labour markets. It did this through
two principal mechanisms: national legislation protecting employment and working
conditions and national organisations of workers, trade unions, engaging in collective
bargaining and collective action.60 The labour law on national organisations of workers developed in the Member States
through successive stages of repression, recognition and promotion. Although courts in
the nineteenth century initially invoked legal doctrines protecting economic freedom to
repress collective action by trade unions “in restraint of trade”,61 legislative policy and
judicial doctrine eventually recognised and even promoted the rights of workers and
their organisations to collective bargaining and collective action. c) Interpreting “proportionality” in light of international labour law The criterion used by the ECJ to assess the lawfulness of collective action is “propor-
tionality”.59 This is not the standard used in international labour law, either by the
Committee on Social Rights interpreting Article 6(4) of the European Social Charter
(ESC) (which the Community is to be “having in mind” under Article 136 EC) or by the
Freedom of Association Committee interpreting ILO Convention No. 87, ratified by all
Member States (usually before accession to the EC, hence “shall not be affected” by the
Treaty (Article 307 EC)). The principles established under the ESC and ILO should be invoked to shape the
criterion of “proportionality” invoked by the ECJ. 59 In Viking, the standard is whether the threat to jobs and conditions is “serious” and the action is
“suitable”; in Laval, the standard is that stipulated in the Posting Directive. 87 /10.5771/9783
– Brian Bercusson VIII. Conclusion 63 I concluded my article of May 2007 in the European Law Journal on an earlier stage of this litiga-
tion by speculating that the future of the European trade union movement but also of the EU could
be at stake. Now that the decisions have come out, it may be necessary to add the future of the
European Court to this list... 3. The law on workers’ collective action One response to the shift in the economic balance of power resulting from the growth
of the transnational economy is workers’ traditional defence of collective action. As
stated in the first sentence of this paper, a crucial element in maintaining a balance of
economic power within Member States is the legal right to take collective action. Na-
tional labour laws include the right to collective action. Though legal systems differ, no
Member State outlaws collective action. Under the pressure of EU law, Member States adapted their law to the requirements
of free movement in the single market. The EU law of the common market transformed
national rules governing the free movement of goods, services, capital or workers. However, national laws have not yet adapted to trade unions’ response in the form of
collective action which impacts on the transnational economy.62 62 B. Bercusson, “Foreword”, to F. Dorssemont, T. Jespers, A. van Hoek (eds), Cross-Border Collec-
tive Actions in Europe: A Legal Challenge, Intersentia, Antwerp-Oxford, 2007. 2. The balance of power in the EU economy The transnational economy of the European Union, like the national economies of
the Member States, requires an economic balance of power between employers and
workers. In the Member States, this balance is achieved, in part, through the collective
action of trade unions and organisations of employers. The social partners at EU level
have not achieved this balance of power. EU law on free movement transforms the balance of economic power in the EU. The
freedom of enterprises to move throughout the single European market has shifted the
balance of economic power towards employers. This is particularly evident in the over-
whelming economic power of multinational enterprises, the magnitude of transnational
capital movements and the social dumping effects of global trade. The changing balance
of economic power, together with competition over labour standards, weakens European
economic integration and undermines support for the European political project. 88 10.577
– Scope of Action at the European Level Scope of Action at the European Level 4. Transnational collective action and EU law Globalisation means that collective action frequently has an impact beyond national
borders. National rules on collective action are inadequate to regulate transnational col-
lective action having an impact on free movement in the EU. The decisions of the Euro-
pean Court of Justice in Laval, Viking and Rüffert, although not repressing collective
action, indeed recognising it as a fundamental right, conspicuously failed to rise to the
challenge of accommodating labour law doctrine to the new balance of power in a
transnational European economy.63 Action on the European level is required to com-
pensate for this failure. 89 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb 783845219165, am 24.10.2024, 06:46:57
- https://www.nomos-elibrary.de/agb Scope of Action at the European and National Level from the
Perspective of the European Commission Armindo Silva I. The ECJ Rulings The rulings on Viking, Laval and Rüffert, and now the most recent one on the case
Commission vs Luxembourg, deal with the issue of how to balance the protection of
workers’ rights and working conditions with the safeguard of fundamental freedoms as
established by primary and secondary EU law. − The Viking case is about whether it is legitimate for an international trade union
to use collective action in order to force a ferry company to drop its plans to re-
flag a ship from Finland to Estonia. − The Laval case is about whether it is legitimate for a Swedish trade union to use
collective action to force a Latvian company to enter a collective agreement set-
ting work and pay conditions that go beyond the core mandatory rules established
in the Posting of Workers Directive: − The Rüffert case is about whether public procurement rules in a German Land
can impose to a Polish undertaking higher wages than those set by the federal
collective agreement that is universally applicable. − The Luxembourg case is about whether it is legitimate for a member State to im-
pose to foreign service providers employment conditions that go well beyond the
same core of mandatory rules on the grounds that they are necessary for public
order, as well as the obligation to have a domiciled representative or to issue an
ex ante declaration. The two first rulings recognize the right to take collective action as a fundamental
right which forms an integral part of the general principles of Community law. They
also confirm that the trade unions will continue to be able to take action to protect
workers’ interests and combat social dumping. However, the exercise of such right
should be done in compliance with the Treaty and the fundamental freedoms of estab-
lishment and of service provision. Collective trade union action does not fall outside the scope of the Treaty despite the
lack of competence of the Community to legislate on these matters, by virtue of Article
137.5. Member States must therefore comply with Community law when exercising
their competence in setting rules in the area of industrial relations. This confirms exist-
ing jurisprudence in the areas of taxation and social security, such as in Walrave and
Koch, Bosman, and Wouters et al. Armindo Silva − What difficulties does the Commission identify in the application of the POW
Directive 96/71 and how can we solve them? I. The ECJ Rulings
92
II. Consequences of the ECJ rulings
93
III. Difficulties with the application of the POW Directive
95 I. The ECJ Rulings
II. Consequences of the ECJ rulings
III. Difficulties with the application of the POW Directive I. The ECJ Rulings 93 95 III. Difficulties with the application of the POW Directive First, let me thank the Federal Ministry of Employment and Social Affairs for the
opportunity given to the Commission to express our views in such an important debate. I am afraid however that I shall not be able to comment on most points raised in Prof. Bercusson’s very challenging paper. He suggests a number of fundamental changes in
the Treaty, in the wake of the Irish referendum, as a remedy for the difficulties in con-
ciliating the economic freedoms with the social rights that the recent rulings of the ECJ
made more apparent. For the Commission, as it was clearly stated before the European
Council last week by President Barroso, the priority is to carry on the ratification of the
Lisbon Treaty as it stands, while opening a period of reflection on the reasons and con-
sequences of the Irish vote with the purpose of finding an institutional and political so-
lution. The contribution of the Irish government to this reflection is fundamental. In the meantime, the policy-making process should continue, on the basis of the ex-
isting Treaty, with a particular emphasis on those dossiers that are important for meet-
ing the social concerns of EU citizens, such as employment, social inclusion, anti-
discrimination, health, and education. The social package that the Commission intends
to adopt next Wednesday, 2nd July, under the heading of a renewed Social Agenda, shall
include legislative and non-legislative policy proposals in these areas and will make
therefore a major step towards reviving the confidence of citizens in the EU capacity to
deliver in those areas that are closest to the citizen. In my presentation, I shall focus on three questions: − How does the Commission consider the recent ECJ rulings on Viking, Laval,
Rüffert and Luxembourg? − How does the Commission consider the recent ECJ rulings on Viking, Laval,
Rüffert and Luxembourg? − What consequences do we attach to such rulings ? 91 I. The ECJ Rulings The way the Court dealt with the issue of the horizontal effect of EU primary law is
in our view relatively more innovative and will certainly deserve a far-reaching debate,
which will stress its legal as well as political consequences. In Viking and in Laval, Ar- 92 Scope of Action at the European and National Level from the Perspective of the European Commission ticles 43 and 49 of the Treaty were considered as not applying only to the actions of
public authorities, but also to any rules that aim at regulating employment conditions in
a collective manner, such as those established by the social partners in their role of
agents to which national authorities delegate the task to implement EU directives. The Court found therefore that the classical tests of necessity and proportionality
should be applied in both cases in order to determine whether the restriction imposed to
the freedom of establishment or the freedom to provide services was justified. However,
while in the Viking case, the Court considered that it was for the national Court to judge
on that matter, in the Laval case, it carried out directly such verification and found out
that the union action was not proportionate in relation to the objective of upholding the
protection of workers. Another innovative aspect in the Court rulings, also deserving wide debate, is the in-
terpretation in the Laval ruling, of Article 3.7 of the Posting of Workers Directive as
meaning that the scope of employment conditions guaranteed to posted workers should
be limited to the core of mandatory rules defined in Article 3.1. This question should be
discussed against the background of the twofold purpose of the Directive: to ensure a
level-playing field between domestic and foreign providers of services; and to offer
minimum protection to workers who are temporarily posted to a country without par-
ticipating permanently in its labour market. In the Rüffert ruling, the Court declared illegitimate a legislative measure that did not
fix a rate of pay in accordance with any of the procedures laid down in the POW Direc-
tive: whereas Germany has a system for declaring collective agreements universally
applicable and has used this faculty for the construction sector, the law in question ap-
plies only to public contracts and not to private ones, and enforces a collective agree-
ment which has not been declared generally applicable. I. The ECJ Rulings Finally, in the Luxembourg case, the Court recognized all the items of non-
conformity raised by the Commission. It confirmed that Member States should not uni-
laterally interpret the concept of public policy provisions used in Article 3.10 of the
POW Directive in order to apply to undertakings employment conditions on top of those
included in the hard core. Especially when some of such conditions are ruled by laws
whose verification falls upon the authorities of the country of origin. It also confirmed
that Member States should not impose control measures that are disproportionate to the
objective of protecting workers, in line with previous case law and the guidelines set out
by the Commission in 2006. II. Consequences of the ECJ rulings The rulings will affect most directly the countries involved: Sweden, Germany and
Luxembourg. They should bring their legislation in line with Article 49 of the Treaty The rulings will affect most directly the countries involved: Sweden, Germany and
Luxembourg. They should bring their legislation in line with Article 49 of the Treaty 93 Armindo Silva and the POW Directive. This, in our view, does not appear as an impossible task. The
rulings do not oblige to review the fundamental principles on which the industrial rela-
tions systems of these countries are based. Also, the rulings make it clear that the POW
directive is not intended to harmonise the employment rules, but rather to coordinate
them in order to establish a level-playing field. In particular, Articles 3.1 and 3.8 allow
for a multiple choice of possibilities to determine the way by which minimum rates of
pay are determined. However, it is clear that the rulings will have consequences that extend well beyond
these three countries. First, because other countries, where minimum wages are set nei-
ther by law nor by universally applicable collective agreements, may find themselves in
the same situation. It is the case of Denmark, where a consultative tripartite commission
already proposed an amendment to Danish legislation which aims at ensuring that the
POW directive could be fully implemented in Denmark while maintaining the specific
characteristics of the Danish model of industrial relations. Second, and more importantly, because significant sectors of the public opinion call
for measures to reverse what they see as the two major consequences of the rulings: to
subordinate the fundamental right to collective action by trade unions to economic con-
siderations and to facilitate social dumping. We have difficulty in seeing these rulings as reversing a long established tradition of
EU jurisprudence in favour of social rights. In the Laval case, the collective action was
considered as non-proportionate because the obstacle that it created to the free provi-
sion of services could not be justified by its goal which was to impose employment
conditions going beyond what the Directive considers as necessary to grant protection
to workers. In other cases, such as in Schmidberger and in Omega, the Court considered
that the protection of fundamental rights is a legitimate interest that justifies a restriction
of the obligations imposed by Treaty-based economic freedoms. Scope of Action at the European and National Level from the Perspective of the European Commission In the sec-
ond case (Luxembourg) , because it is for the national authorities of the home country to
control the respect of an obligation that is established by EU directive 91/533. Scope of Action at the European and National Level from the Perspective of the European Commission Scope of Action at the European and National Level from the Perspective of the European Commission The application to posted workers of the whole range of employment conditions in
force in the host country would create far more costs for the service provider, who
would have to become aware of all employment legislation in force in every EU country
in which he would like to do business. Would that be justified in terms of workers' pro-
tection? Would such obligation entail more benefits for workers who are temporarily
posted across borders? This is the key question around which the necessary debate
should turn. It is fair to say that, so far, the cases that have been examined by the Court
– the compulsory contributions for the national training funds (in Laval) or the obliga-
tion to have a written statement from the employer (in Luxembourg) - have not pro-
vided sufficiently convincing justifications. . In the first case (Laval), because the
posted workers would not have the possibility to benefit from such training. In the sec-
ond case (Luxembourg) , because it is for the national authorities of the home country to
control the respect of an obligation that is established by EU directive 91/533. The application to posted workers of the whole range of employment conditions in
force in the host country would create far more costs for the service provider, who
would have to become aware of all employment legislation in force in every EU country
in which he would like to do business. Would that be justified in terms of workers' pro-
tection? Would such obligation entail more benefits for workers who are temporarily
posted across borders? This is the key question around which the necessary debate
should turn. It is fair to say that, so far, the cases that have been examined by the Court
– the compulsory contributions for the national training funds (in Laval) or the obliga- – the compulsory contributions for the national training funds (in Laval) or the obliga-
tion to have a written statement from the employer (in Luxembourg) - have not pro-
vided sufficiently convincing justifications. . In the first case (Laval), because the
posted workers would not have the possibility to benefit from such training. II. Consequences of the ECJ rulings And the right to launch
a collective action to protect host country workers from social dumping is recognised by
case law, since Arblade, as an imperative reason of general interest that can justify a
restriction of economic freedoms. This is not deadwood. As to social dumping, I would like to underline that the POW Directive is the most
effective instrument in the EU legal framework to prevent social dumping. It is based on
the principle of primacy of the law and collective agreements in the host country as the
regulating framework for the employment conditions of posted workers, who can bene-
fit automatically from such conditions without having to bargain. However, such pri-
macy only applies to a core of mandatory rules defined in Article 3.1. What is the rea-
son for such limitation? The temporary nature of posted work. Posted workers are not
part of the host country labour market. They have not made the choice to establish
themselves and to follow a professional career there. He/she was posted by a decision of
his/her employer which is part of the material conditions ensuring the provision of a
service. 94 III. Difficulties with the application of the POW Directive The Commission recognizes that the application of the POW Directive has given rise
to a number of difficulties. For instance, − Abuses in the form of successive postings of the same worker, who, for that rea-
son would tend to have a quasi-permanent connection with a certain labour mar-
ket; − Abuses in the form of undertakings functioning as letter-box companies with the
sole purpose of posting workers to a certain destination, as a way to evade from
the application of national rules; − Insufficient respect of host country rules as regards wages and health and safety
conditions by foreign undertakings; − Non-respect of social security obligations in the home country; − Insufficient information of workers and companies about their rights and obliga-
tions in the context of posting; − Insufficient administrative cooperation between national authorities of home and
host countries; − Lack of effective cross-border enforcement mechanisms in case of unlawful be-
haviour by undertakings. These difficulties can be overcome by an efficient working of the labour inspector-
ates and other bodies responsible for a correct enforcement of the legislation, provided
their action is proportionate and non-discriminatory. Also important is a more effective
administrative cooperation between such authorities. These difficulties, as well as those created for some countries by the recent Court rul-
ings, can be handled in the context of the present POW Directive. Priority should be 95 Armindo Silva given to remedy shortcomings in the implementation and enforcement of the Directive. With this purpose the Commission adopted on 3 April a recommendation which aims at
enhancing administrative cooperation between Member States in order to ensure better
protection of posted workers’ rights and more effective cross-border enforcement. It sets
the basis for enhanced fight against disrespect of workers’ rights and undeclared work. We are satisfied that the EPSCO Council on 9 June has endorsed this recommenda-
tion, and on this basis we are ready to carry on with its practical consequences: the crea-
tion of an expert Committee on posting of workers, with the participation of social part-
ners, which will be responsible for the exchange of good practice, and within this set-
ting, a working group to start developing an electronic information exchange system. We are very much aware of the political and legal importance of the recent Court rul-
ings. III. Difficulties with the application of the POW Directive It is therefore important to give sequence to open debates such as this one, here in
Berlin. The Commission is planning a Forum in October involving major stakeholders,
such as member States, social partners and legal and economic experts, to debate key
issues about the new challenges raised by increased workers mobility for fundamental
social rights, including collective bargaining and collective action, and will continue to
address their concerns in a constructive and balanced way. 96 Diskussionsbericht Diskussionsleitung: Christoph Schumacher-Hildebrand Diskussionsleitung: Christoph Schumacher-Hildebrand Diskussionsbericht Silva erklärte, dass die Entsenderichtlinie bestimme, dass Löhne dann auf entsandte
Arbeitnehmer anwendbar seien, wenn sie gesetzlich oder durch Allgemeinverbindlich-
keitserklärung festgelegt seien. Dabei handle es sich nicht um den Vorrang der Rege-
lungen des Herkunftsstaates, sondern um diejenigen des Aufnahmestaates. Nun stelle
sich die Frage, was als Lohn verstanden werden könne, der durch Tarifverträge festge-
legt worden ist, die für allgemeinverbindlich erklärt werden können. In den meisten
Ländern bereite dies keine Schwierigkeiten, da die Löhne entweder gesetzlich festgelegt
seien oder auf Allgemeinverbindlichkeitserklärungen beruhen würden. Es führe nur in
einzelnen Mitgliedstaaten zu Problemen, die kein nationales System hätten, Tarifverträ-
ge für allgemeinverbindlich zu erklären. Daher gebe es in diesen Ländern Arbeitneh-
mer, deren Löhne nicht auf diese Weise geschützt seien und die geringere Löhne erhal-
ten könnten, als sie in den Tarifverträgen festgelegt seien. Wenn entsandten Arbeitneh-
mern Löhne gewährt würden, die in Tarifverträgen festgelegt seien, die nicht für allge-
meinverbindlich erklärt worden sind, bedeute dies, dass wir gezwungen wären, Löhne
zu bezahlen, die über den Löhnen anderer Wettbewerbern im gleichen Arbeitsmarkt
liegen. Schweden zähle sicherlich zu den Ländern, die in ihrer Gesetzgebung Raum lassen
sollten, um Regelungen anzunehmen, wie sie die Entsenderichtlinie verlange. Die
Richtlinie biete drei verschiedene Möglichkeiten, Mindestlöhne festzulegen. Die Laval-
Rechtsprechung werde von der Kommission so verstanden, dass zur Bestimmung der
Mindestlöhne keiner dieser Wege zwingend vorgeschrieben sei. Es sei eine Frage der
Sichtweise, wie nationale Regelungen an die Richtlinienvorgaben angepasst werden
könnten. Silva führte weiter aus, es reiche nicht aus, dass die Kommission eine Überarbeitung
der Tarifverträge vorschlage, sondern es sei notwendig, Hinweise darüber zu erhalten,
dass der Rat mit einer Überarbeitung der Richtlinie einverstanden wäre. Die Kommissi-
on kenne die Situation im Rat und die dortigen Diskussionen zum Thema Arbeitszeiten. Auch sei sie der Öffentlichkeit bekannt. Dazu werde er jetzt nichts sagen. Es sei zu be-
denken, dass hier Teilnehmer aus Luxemburg, Schweden und Deutschland anwesend
seien, was sehr nützlich sei, aber eben nur eine Seite der Streitfrage wiedergebe. g
g
Silva erklärte, dass er mit der jetzigen Situation im Rat zufriedener als vorher sei, un-
abhängig davon, was über die am 10. Juni 2008 erreichte Übereinstimmung zur Ar-
beitszeit gedacht werde. Es sei der Kommission gelungen, einen langwierigen Streit im
Rat zu beenden. Wenn nun ein neuer Konflikt eröffnet werde, habe er keinerlei Zweifel,
dass es sich um einen größeren Konflikt handeln werde. Vielleicht irre er sich auch. Diskussionsleitung: Christoph Schumacher-Hildebrand In der Diskussion richtete zunächst Andersson seine Fragen an Silva. Andersson er-
klärte, dass er vor Inkrafttreten der Dienstleistungsrichtlinie mit Vertretern der Kom-
mission über das Thema „Herkunftsland“ diskutiert habe. Die Kommission hätte ihre
Position vertreten, aber als Gesetzgeber hätten das Parlament und der Rat die Sache
entschieden. Anschließend stellte Andersson die Frage, wie gleiche Voraussetzungen für alle ge-
schaffen werden können. Er fragte, ob dies der Fall sei, wenn Unternehmen in einem
anderen Land tätig würden und eines von ihnen einen Tarifvertrag auf dem landesübli-
chen Niveau unterschreibe, das andere Unternehmen sich dagegen mit der Begründung
weigere, dass die Vereinbarung nicht auf dem Mindeststandard beruhe. Diejenigen, die
den Mindeststandard unterschrieben, würden gewinnen. Er stellte daher die Frage, wie
es sich auf den Arbeitsmarkt auswirken werde, wenn ein ausländisches Unternehmen
nach dem anderen zum Zuge komme, indem es einen Tarifvertrag unterschreibe, der nur
den Mindeststandard absichert. Von Zuhörerseite wurde zum Ausdruck gebracht, dass die Vertreter des EuGH zwar
sagten, dass sie den vorgegebenen rechtlichen Rahmen einhalten würden, jedoch wür-
den die Entscheidungen des EuGH am Ende auch politische Vorgaben setzen. Es rufe
daher Erstaunen hervor, dass die Kommission jetzt schon sage, sie sehe z.B. bei der
Entsenderichtlinie keine Notwendigkeit, etwas zu ändern, und ausführe, dass mit den
betreffenden Urteilen kein Sozialdumping betrieben werden könne. Diese Reaktion der
Kommission sei nicht nachvollziehbar, zumal Bercusson nachgewiesen habe, welche
Auswirkungen dies auf die nationalen Standards der jeweiligen Mitgliedstaaten habe. Bislang hätten die Mitgliedstaaten immer noch ihre Souveränität, diese werde jedoch
durch die Rechtsprechung des EuGH angegriffen. Auch von anderer Seite wurde der Position der Kommission mit Unverständnis be-
gegnet und deutlich gemacht, dass eine Debatte über die grundlegenden Probleme ge-
wünscht werde, die sich aus den vier EuGH-Urteilen ergeben würden. Dabei handle es
sich um das Spannungsfeld Grundfreiheiten, Grundrechte, öffentliche Ordnung, natio-
nale und gemeinschaftliche Grundordnung. Hinsichtlich der Lösung dieser Probleme,
die hier aufeinander treffen würden, sei die Kommission noch eine grundlegende Ant-
wort schuldig. Insoweit genüge es nicht und werde nicht zum Ziel führen, wenn die
Kommission dazu auffordere, die nationalen Gesetze zu ändern und die Kontrolle der
Arbeitsbedingungen effizienter zu gestalten. Notwendig sei eine grundlegende Debatte
auf politischer Ebene mit einer klaren Stellungnahme, was die Kommission nun unter-
nehme. 97 97 845219165, am 24.10.2024, 06:46:57
- https://www.nomos-elibrary.de/agb Gestaltungsspielräume aus deutscher Sicht Wolfgang Däubler Das Thema „Gestaltungsspielräume aus deutscher Sicht“ wäre früher ein sehr schö-
nes und populäres gewesen. Die EG-Richtlinien zum Arbeitsrecht enthalten typischer-
weise Günstigkeitsklauseln. Inhaltlich fügen sie dem nationalen Arbeitsrecht Gleich-
heitsrechte hinzu, z. B. die Antidiskriminierungsrichtlinien, aber nicht nur diese. Weiter
bringen sie neue Verfahrensmöglichkeiten wie die Chance, einen europäischen Be-
triebsrat zu bilden. Dies hatte zur Folge, dass man sich zwar über Einzelheiten oft un-
eins war; insgesamt aber stand der europäische Gesetzgeber, speziell der Europäische
Gerichtshof bei Arbeitnehmern und Gewerkschaften in hohem Ansehen. Wenn man
Kritik übte, riskierte man, als schlechter Mensch betrachtet zu werden. Das hat sich schlagartig geändert. Die Entscheidungen „Viking“ und „Laval“ haben
große Unsicherheit im Arbeitskampfrecht geschaffen. Wir hatten vor einiger Zeit in
Deutschland den Konflikt um Nokia, das seine Niederlassung von Bochum nach Rumä-
nien verlagerte. Die Beteiligten haben den Konflikt ohne Arbeitskampf gelöst, aber man
kann natürlich nachträglich die Frage stellen: Wäre es zulässig gewesen, wenn die Be-
legschaft erklärt hätte: „Wir streiken für die Erhaltung des Standorts Bochum“ und
wenn sie dies in die Tat umgesetzt hätte? Und man kann weiter fragen, ob ein Streik um
einen Tarifsozialplan eventuell eine Maßnahme gewesen wäre, die die Ausübung der
Niederlassungsfreiheit „weniger attraktiv“ gemacht hätte – der erreichte Tarifvertrag
wäre ja für den Arbeitgeber automatisch mit bestimmten Nachteilen verbunden gewe-
sen. Im französischen Recht gibt es z. B. eine Regel, wonach ein Unternehmen, das sich
aus einer strukturschwachen Gegend zurückzieht, für jeden wegfallenden Arbeitsplatz
eine Ausgleichsabgabe bezahlen muss. Wäre nicht vielleicht auch das eine nationale
Regelung, die die Ausübung der Niederlassungsfreiheit „weniger attraktiv“ macht? Ich
würde diese Fragen keineswegs bejahen, aber ich weise darauf hin, dass der Europäi-
sche Gerichtshof hier ein erhebliches Maß an Rechtsunsicherheit geschaffen hat. g
Noch mehr als diese Rechtsunsicherheit beunruhigt uns der Umgang mit den Grund-
rechten. Die Grundrechte sind nach den Urteilen „Viking“ und „Laval“ nur ein Legiti-
mationstitel für Eingriffe in Grundfreiheiten. Sie sind nicht – wie andere schon gesagt
haben – das Primäre. In beiden Entscheidungen heißt es sinngemäß, man müsse abwä-
gen zwischen der Versammlungsfreiheit, der Meinungsfreiheit und der Menschenwürde
auf der einen Seite und den Grundfreiheiten auf der anderen Seite. Mit diesen Aussagen
stellt sich in Deutschland eine spezielle Frage. Das Bundesverfassungsgericht hat auf
die Kontrolle von Gemeinschaftsrechtsakten am Maßstab der Grundrechte des Grund-
gesetzes nur verzichtet, „solange“ ein äquivalenter Grundrechtsschutz durch den Euro-
päischen Gerichtshof oder auf andere Weise gewährleistet ist. Diskussionsbericht Deshalb sei eine Diskussion so wichtig. Aus diesem Grund würde die Kommission im
Oktober 2008 Vertreter aus allen Mitgliedstaaten einladen, um zu hören, was etwa Po-
len, Lettland und Italien hierzu zu sagen hätten. Bevor die Kommission in dieser Sache
einen Vorschlag präsentiere, müsste abgeschätzt werden, ob es im Rat zu einer gewissen
Art der Übereinstimmung darüber kommen könne, weiter voran zu schreiten und das 98 Diskussionsbericht Thema politisch zu debattieren, wie es vom Repräsentanten Luxembourgs vorgeschla-
gen werde. Damit beschäftige sich die Kommission gegenwärtig. Andersson ergriff noch einmal das Wort und bat darum, für weitere Einzelheiten sei-
nen Bericht zu lesen. Darin habe er einige detaillierte Vorschläge ausgeführt, wie die
Entsenderichtlinie geändert werden könnte, um die Probleme zu umgehen, die die
Rechtsprechung des EuGH hervorgerufen habe. Er sei von Silvas Ausführungen zum
Kompromiss vom 10. Juni 2008 über die Arbeitszeitrichtlinie beeindruckt, der eine
deutliche Abkehr von den Entscheidungen des EuGH zur Arbeitszeitrichtlinie zum Ziel
habe. Offensichtlich habe die Kommission kein Problem damit, in manchen Bereichen
Änderungen vorzunehmen. In änderungsbedürftigen Bereichen sollte daher vielleicht
die Kommission die Führung übernehmen, um die Mitgliedstaaten zu einem Fortschritt
zu bewegen. Simone Gräfin von Hardenberg 99 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb 783845219165, am 24.10.2024, 06:46:57
- https://www.nomos-elibrary.de/agb https://doi.org/10.5771/97838
Open Access –
- Wolfgang Däubler Schutz durch deutsche Grundrechte hängt also davon ab, dass man einen gleichwertigen
Schutz auf der Ebene der Gemeinschaft besitzt. Bisher konnte man mit Recht den Ein-
druck gewinnen, dass bei allen Abweichungen im Detail die Gleichwertigkeit gewähr-
leistet war. Wenn man nunmehr die Grundrechte unter den Vorbehalt der Grundfreihei-
ten stellt, wenn man sie nur insoweit gelten lässt, als die Grundfreiheiten nicht übermä-
ßig beschränkt werden, also die Abwägung zugunsten der Grundrechte ausfällt, dann ist
das nicht mehr dieselbe Form von Grundrechtsschutz, die wir im Grundgesetz haben. Im deutschen Recht wäre es völlig undenkbar, zu sagen oder zu schreiben: „Der Schutz
der Menschenwürde ist zu gewährleisten, soweit dadurch nicht in unverhältnismäßiger
Weise in Marktfreiheiten eingegriffen wird.“ Das wäre eine völlig unverständliche, aus
dem allgemeinen Konsens herausfallende Aussage. Das Bundesverfassungsgericht wird
Gelegenheit haben, zu dieser Problematik Stellung zu nehmen. Es gibt heute schon ein
anhängiges Verfahren, in dem es um die Qualität des Grundrechtsschutzes durch den
Europäischen Gerichtshof geht. Möglicherweise werden weitere Verfahren dieser Art
hinzu kommen. Ich bin mir nicht sicher, ob sich die Große Kammer des EuGH, die die
beiden Entscheidungen „Viking“ und „Laval“ erlassen hat, dieser Problematik wirklich
bewusst war. Ich bin nun aber gefragt, was die Reaktionen auf nationaler Ebene sein könnten au-
ßer der Einleitung oder Fortführung von Verfahren vor dem Bundesverfassungsgericht. Die Rüffert-Entscheidung hat die Tariftreueregeln der deutschen Bundesländer für un-
vereinbar mit dem Gemeinschaftsrecht erklärt. Dies hat viele kritische Stellungnahmen
provoziert, doch will ich diese jetzt nicht aufgreifen. Handlungsmöglichkeiten bestehen
„noch“ – so muss man sagen – insoweit, als man die Mindestlöhne über den Sektor der
Bauwirtschaft hinaus auf andere Bereiche ausdehnen kann. Ein solcher Schritt wäre
rechtlich möglich und auch sinnvoll. Angesichts der deutschen Tarifpraxis wäre es in
hohem Maße vernünftig, insoweit nicht nur zentrale sondern auch dezentrale Kompe-
tenzen zu schaffen. Nicht nur der Bundesarbeitsminister sondern auch die einzelnen
Landesarbeitsminister sollten die Möglichkeit erhalten, Tarifverträge nach dem Arbeit-
nehmerentsendegesetz für allgemeinverbindlich zu erklären. Wir haben nun einmal ein
dominierendes Modell von Flächentarifen in einzelnen Teilen unseres Territoriums. Von daher wäre es nur konsequent, das Arbeitnehmerentsendegesetz insoweit zu än-
dern, dass eine Delegation der dort vorgesehenen Befugnisse auf Landesarbeitsminister
möglich oder auch geboten ist. Dies ist ein Vorschlag, den ich allerdings aus Zeitgrün-
den nicht näher begründen kann. Ich gehe davon aus, dass trotz der Entscheidung in Sachen Kommission gegen Lu-
xemburg eine solche Regelung noch möglich wäre. Gestaltungsspielräume aus deutscher Sicht Der Verzicht auf den 101 Wolfgang Däubler Wolfgang Däubler Wolfgang Däubler Allerdings muss man sich fragen,
wie eigentlich der Begriff „Mindestlohn“ zu bestimmen ist. Die Frage ist schon ange-
sprochen, aber im Grunde nicht eindeutig beantwortet worden. Die These eines Kolle-
gen, wonach es auch zehn Mindestlöhne sein könnten je nach Tätigkeit und Qualifikati-
on der betroffenen Menschen, aber ohne Berücksichtigung ihres Dienstalters, ihrer Jahre
der Betriebszugehörigkeit, findet meine Zustimmung, aber es gibt hier natürlich 102 Gestaltungsspielräume aus deutscher Sicht Schwierigkeiten, weil niemand beurteilen kann, wie sich der Europäische Gerichtshof
zu einer solchen Ausdehnung stellen würde. Wir finden in der Entscheidung Kommissi-
on gegen Luxemburg eine geradezu akribische Analyse aller potentiellen Einwände
gegen Einzelregelungen des luxemburgischen Entsendegesetzes. So hatte beispielsweise
Luxemburg in ganz harmloser Weise festgeschrieben, dass die Vorgaben der Nachweis-
richtlinie sowie der Richtlinien über Teilzeit und Befristung auch von den entsendenden
Unternehmen einzuhalten waren, was im Einzelfall überprüft werden konnte. Es gab
damit also eine doppelte Kontrolle – eine durch den Entsendestaat und eine durch den
Aufnahmestaat. Man sollte denken, dass das keine Einwände provoziert: Das Ziel der
Gemeinschaft, den Richtlinien möglichst viel Durchschlagskraft zu verschaffen, ist auf
diese Weise doch nur besser sichergestellt. Nein, meinte der Europäische Gerichtshof,
dies sei nicht „erforderlich“, das stelle einen unverhältnismäßigen Eingriff in die Dienst-
leistungsfreiheit dar. Damit ist eine Lösung präferiert, die der Dienstleistungsrichtlinie
in ihrer ursprünglichen Fassung zugrunde lag, die dann aber aufgrund politischer Ent-
scheidungen wieder rückgängig gemacht wurde. Was politisch nicht erreichbar war,
wird bestimmten Leuten vom Europäischen Gerichtshof geschenkt; dies hinterlässt ei-
nen wenig erfreulichen Beigeschmack. Gerade im Hinblick darauf muss man heute je-
den rechtspolitischen Vorschlag im Anwendungsbereich des EG-Rechts mit einem Fra-
gezeichen versehen; man kann nie garantieren, ob die gewählte Lösung auch Bestand
hat, wenn der Europäische Gerichtshof mit der Sache befasst werden sollte. Ein weiterer Vorschlag betrifft den Bereich Vergaberecht. Wäre es nicht sinnvoll,
sich nicht nur auf die Frage der Einhaltung von Tarifverträgen zu konzentrieren, son-
dern auch andere sozialpolitisch relevante Vergabekriterien heranzuziehen? Es gibt eine
interessante Entscheidung des Europäischen Gerichtshofs im Fall „Nord – Pas-de-
Calais“ aus dem Jahr 2000. Danach ist es zulässig, Aufträge nur an solche Firmen zu
erteilen, die bereit sind, eine gewisse Anzahl an Arbeitslosen einzustellen. Dies ist ein
sehr interessanter Ansatz, den man vielleicht auch bei uns aufgreifen könnte – das Krite-
rium würde natürlich in gleicher Weise für deutsche wie für ausländische Interessenten
an dem fraglichen Auftrag gelten. Wolfgang Däubler Auch letztere können ja 10 oder 15 oder 20 Prozent
an bisher Arbeitslosen beschäftigen. Als Vergabekriterium könnte man sich weiter eine
besondere Förderung von Auszubildenden vorstellen – nicht im Sinne des Berufsbil-
dungsgesetzes, das ja nur für Deutschland gilt, sondern in dem Sinn, dass jeder Interes-
sent bestimmte Ausbildungsleistungen erbringen muss, die ggf. auch in der Beschäfti-
gung von Berufsanfängern liegen können. Weiter könnte man daran denken, solche
Auftragnehmer gezielt zu fördern, die Angehörige von Problemgruppen beschäftigen:
Behinderte, ethnische Minderheiten und andere, die in der Gefahr sind, vom Arbeits-
markt ausgeschlossen zu werden. Auch dies ist eine Möglichkeit, die nach der bisheri-
gen Rechtsprechung wohl noch Bestand haben würde. Schließlich müsste man sich überlegen, ob es nicht sinnvoll wäre, dem Europäischen
Gerichtshof nach einiger Zeit die Möglichkeit zu geben, sich die ganze Sache noch ein-
mal zu überlegen. Wir sind Optimisten, was die Lernfähigkeit anderer Menschen an- 103 Wolfgang Däubler geht. Es wurde mit Recht der fehlende europäische Diskurs angesprochen. Diesen Man-
gel kann man in den nächsten Jahren beheben; man kann sehr viel mehr miteinander
reden, als man dies in der Vergangenheit getan hat. Dann könnte möglicherweise der
Vorrang der Grundrechte vor den Grundfreiheiten ein sehr viel wichtigerer Topos als in
der bisherigen Rechtsprechung des EuGH werden. Ein neues „Rüffert-Verfahren“ müss-
te meines Erachtens auch das ILO-Übereinkommen Nr. 94 berücksichtigen, wonach die
öffentliche Hand bei der Auftragsvergabe bestimmte Bedingungen stellen, insbesondere
auch die Einhaltung von Tarifverträgen verlangen kann. Das Übereinkommen Nr. 94 ist
zwar von allen Mitgliedstaaten unterzeichnet, aber nur von 10 Mitgliedstaaten ratifiziert
worden. Es stellt aber inhaltlich der Sache nach eine Ergänzung zum ILO-
Übereinkommen Nr. 87 über Koalitionsfreiheit und kollektive Maßnahmen dar. Es hat
diesem gegenüber flankierende Wirkung, weil es einen spezifischen Durchsetzungsme-
chanismus vorsieht. Das Übereinkommen 87 gehört seinerseits zu den Kernarbeitsnor-
men, auf die auch der Europäische Gerichtshof zurückgegriffen hat, als es in der Vi-
king-Entscheidung um die Streikgarantie ging. Das Übereinkommen 87 steht dort neben
der Europäischen Sozialcharta, der Gemeinschaftscharta von 1989 und der Grundrech-
techarta von Nizza. Unter diesen Umständen wäre es naheliegend, sich beim Thema
„Umsetzungsmechanismen von Tarifverträgen“ auch auf das Übereinkommen 94 zu
stützen. Als Ergänzung des Übereinkommens 87 ist dieses genauso ein „Rechtserkennt-
nismittel“ in der Terminologie des EuGH, das die Basis für die Entwicklung allgemei-
ner Rechtsgrundsätze des Gemeinschaftsrechts darstellt. Perspektive der Gewerkschaften Helga Nielebock I. Grundsätzliches
II. Vorschläge
1. Vorschläge für die europäische Ebene
2. Vorschläge für die nationale Ebene I. Grundsätzliches
II. Vorschläge
1. Vorschläge für die europäische Ebene
2. Vorschläge für die nationale Ebene 107 Wolfgang Däubler In einem weiteren Verfahren könnte man auch die Frage aufwerfen, weshalb man bei
der Warenverkehrsfreiheit nach der Keck-Rechtsprechung den Mitgliedstaaten das
Recht einräumt, die Ausübungsformen selbst zu bestimmen und ihnen so einen be-
trächtlichen Spielraum eröffnet. Warum gilt Vergleichbares nicht auch bei der Nieder-
lassungs- und der Dienstleistungsfreiheit? Weiter wäre die Bereichsausnahme für die
Tarifautonomie zu erörtern, die vom Europäischen Gerichtshof in der Albany-
Entscheidung bejaht wurde. Die neueste Rechtsprechung stellt die Loyalität der Arbeitnehmer in den Kernländern
der EU auf eine harte Probe. Es ist erstaunlich, dass der Europäische Gerichtshof dies
tut in einer Zeit, wo die Zukunft des europäischen Projekts sehr prekär geworden ist. Man hat dies zuletzt an der Volksabstimmung in Irland gesehen, man konnte es vorher
an den Referenden in Frankreich und den Niederlanden ablesen. In einer solchen Situa-
tion sollte man die Loyalität der EG-Bürger nicht weiter strapazieren. Deshalb ist dem
EuGH zu raten, wieder jene kluge Zurückhaltung zu üben, die seine Rechtsprechung in
der Vergangenheit ausgezeichnet hat. 104 I. Grundsätzliches Dem DGB ist ein soziales Europa wichtig, da nur ein solches Europa ein Zusammen-
leben in Frieden ermöglicht. Unverzichtbar sind soziale Rechte für die Beschäftigten. Sie schaffen mit ihrer Ar-
beit erst die Werte für einen wirtschaftlichen Erfolg. Wir treten für ein politisch gestaltetes soziales Europa ein, das nicht nur von Markt-
kräften bestimmt wird, und erwarten eine Respektierung der nationalen Grundrechte
wie der Gemeinschaftsgrundrechte gegenüber den Grundfreiheiten – zumindest in ei-
nem angemessenen Verhältnis – in Deutschland durch die sog. praktische Konkordanz
hergestellt. Zum Ausgleich widerstreitender Interessen, insbesondere mit wirtschaftlichen und
sozialen Rechten, sind also beide Seiten gleichwertig in die Waagschale zu werfen. Da-
für muss das betroffene Grundrecht – in diesem Fall jeweils die Tarifautonomie – ein-
mal in ihrer wichtigen Form der Durchsetzung, dem Streik, im anderen Fall als die übli-
che Entgeltkategorie, als dem Ergebnis der kollektiven Ausübung von Rechten, die der
Staat akzeptiert und im Wege der Selbsthilfe der Ausgestaltung der Tarifparteien über-
lässt, dieses Grundrecht muss in seiner ihm eigenen Gänze und Tragweite beachtet wer-
den. Dabei ist das Agieren (die Freiheit der Wahl der Mittel) und die Zielsetzung der Ak-
teure (Freiheit der Tarifziele) als ein zu schützendes Recht an sich anzusehen bei Beach-
tung der Waffengleichheit. Es kann nicht einfach, wie der EuGH dies tut, ein Grund-
recht als Rechtfertigungsgrund herangezogen werden. Es hat vielmehr einen eigenen 105 Helga Nielebock Schutzbereich. Und dieser muss eigenständig berücksichtigt werden. Zudem dürfen
nicht unter den zwingenden Allgemeininteressen die Arbeitnehmerinteressen aus einer
individualistischen Perspektive des Arbeitnehmerschutzes heraus betrachtet werden. Schutzbereich. Und dieser muss eigenständig berücksichtigt werden. Zudem dürfen
nicht unter den zwingenden Allgemeininteressen die Arbeitnehmerinteressen aus einer
individualistischen Perspektive des Arbeitnehmerschutzes heraus betrachtet werden. Wir unterstützen eine Politik der Angleichung durch soziale Mindeststandards auf
dem Weg des Fortschritts für alle in Europa und meinen, dass in einem sozialen Europa
dies auch in dessen Grundrechtecharta und Vertrag deutlicher als bislang und in Korrek-
tur der Rechtsprechung des EuGH zum Ausdruck gebracht werden muss. Deshalb ist es
angebracht, dies deutlich zu verstärken und im Primärrecht festzulegen, dass es jeden-
falls keinen Vorrang der Grundfreiheiten vor klar definierten sozialen Grundrechten des
Gemeinschaftsrechts und der Mitgliedstaaten geben kann. Dieser Weg muss beschritten werden. Es gibt zugleich auch Chancen, dass der
EuGH seine Rechtsprechung noch einmal, wenn die EU-Charta dann voll in Kraft ist,
überdenkt. Die Rechtsprechung des EuGH wurde vom DGB oft, wenn auch nicht immer geteilt,
jedenfalls aber akzeptiert ebenso wie die Institution selbst und die Reichweite ihrer Ent-
scheidungen. Wir haben aber auch erfahren, dass der EuGH Argumenten gegenüber
offen ist, manchmal auch unter Einschränkung seiner bisherigen Rechtsprechung und
leider auch zuungunsten des Arbeitnehmers. Perspektive der Gewerkschaften Ausländer zum Tragen kommen kann, sofern dies nicht diskriminierend geschieht und
auch weitergehende Regelungsbereiche, die im Arbeitsverhältnis eine Rolle spielen,
unter Berücksichtigung der Dienstleistungsfreiheit verlangt werden können, denn an-
sonsten wäre die Tarifautonomie empfindlich betroffen. Alle europäischen Richtlinien im Arbeitsrecht sind von allen Unternehmern überall
wo sie tätig sind in der EU einzuhalten und müssen auch überall überprüft werden kön-
nen. Der enge Begriff, wonach eine Begrenzung der Dienstleistungsfreiheit nur aus-
nahmsweise auf der Basis der Entsenderichtlinie durch eine Kontrolle im Aufnahme-
staat erfolge, muss ebenfalls angegangen werden. In beiden Staaten (Aufnahme- wie Herkunftsland) sollte die Einhaltung von europäi-
schem Recht je nach den unterschiedlichen Möglichkeiten und zu den unterschiedlichen
Zeiten der Anwesenheit von Arbeitnehmern und Unternehmensvertretern kontrolliert
werden dürfen. 1. Vorschläge für die europäische Ebene Unter Berücksichtigung der Vorgaben durch die Rechtsprechung des EuGH, also
quasi systemimmanent, müssen sich die Bundesregierung, die nationalen Abgeordneten
und die Mitglieder des Europäischen Parlaments in den EU-Gremien für folgende Punk-
te einsetzen: − Unsere in der Verfassung verbrieften Rechte müssen vertreten und im Rahmen
der eigenen Bindung an die Grundrechte für die Tarifautonomie eingetreten wer-
den und − für eine entsprechende klarstellende Verankerung der Gleichgewichtigkeit von
sozialen Schutzrechten und Grundfreiheiten, z.B. durch eine entsprechende Ver-
ankerung eines sozialen Fortschrittsprotokolls im Vertrag, muss – wie bereits
ausgeführt – eingetreten werden. Zudem muss in der Arbeitnehmerentsenderichtlinie klargestellt werden, dass die dor-
tigen Mindeststandards keine Höchststandards sind, somit eine verbesserte Anwendung
national (Günstigkeitsprinzip, tarifliche Verbesserung) auch mit Wirkung für EU- Zudem muss in der Arbeitnehmerentsenderichtlinie klargestellt werden, dass die dor-
tigen Mindeststandards keine Höchststandards sind, somit eine verbesserte Anwendung
national (Günstigkeitsprinzip, tarifliche Verbesserung) auch mit Wirkung für EU- 106 Perspektive der Gewerkschaften 1 EuGH v. 25.10.2001, Finalarte, Rs. C-49/98, Slg. 2001, I-7831. 2. Vorschläge für die nationale Ebene Ziel für das Vergaberecht ist es eine Legitimationsbasis für Tariftreueerklärungen zu
schaffen, die alle – und nicht nur die im erstreckten Mindestlohntarifvertrag vereinbar-
ten – Entgeltgruppen umfasst und die alle in- und ausländischen Arbeitgeber bindet. Dies wäre zum einen möglich durch eine erleichterte Allgemeinverbindlicherklärung
(AVE) bzw. Rechtsverordnung (RVO) auf der Basis des AEntG (ggf. allein) zu diesem
Zweck. Damit wäre den Anforderungen der Entsenderichtlinie Genüge getan. Der Bund könnte diese Legitimation auch auf die Länder übertragen, z. B. im lau-
fenden Gesetzgebungsverfahren zum Vergaberecht, oder in der AEntG-Novelle. Der
Ansatz des BMAS im neuen Entwurf auch regionale Tarifverträge vorzusehen, war also
richtig und sollte unter diesem Aspekt erneut berücksichtigt werden. Andere soziale Kriterien wie Vorhandensein einer Interessenvertretung, Quote bei
Ausbildung und Einstellung Arbeitsloser wären möglich, weil öffentliches Interesse an
diesen Maßnahmen gegeben ist. Tariftreue ist auch im öffentlichen Interesse, da die
Unterbietung im Wettbewerb über Löhne und daraus resultierende Arbeitslosigkeit ver-
hindert werden sollen, wie auch die sozialen Sicherungssysteme gesichert werden sol-
len. Die bereits durch AVE oder RVO nach dem AEntG erstreckten Tarifverträge können
in die Ländervergabegesetze und das Bundesrecht übernommen werden. Und damit
auch bestehende Überwachungs- und Sanktionsmöglichkeiten weiterhin genutzt wer-
den. Die einfache Unanwendbarkeitserklärung einiger Länder ist ohne Not erfolgt und
muss korrigiert werden. Für das Problem der öffentlichen Aufträge gilt: die Differenzierung ist ein Ausdruck
des verkürzten Ansatzes des EuGH in diesem Zusammenhang nach dem jeweils zwin- 107 I. EuGH und Arbeitsmarkt In vielen Bereichen hat der EuGH mit seinen Entscheidungen tief in die Gestaltungs-
kompetenz des nationalen Gesetzgebers eingegriffen und damit Richtlinienumsetzungen
ad absurdum geführt. Aus jüngster Zeit sei an dieser Stelle nur erinnert an die miss-
glückten Entscheidungen in der Rechtssache Mangold, der Rechtssache Pfeiffer und der
Rechtssache Jaeger. g
Der EuGH hat gerade mit seiner Entscheidung in der Rechtssache Mangold Beschäf-
tigungschancen vertan. Allenfalls insoweit ließe sich die Behauptung rechtfertigen, „das
soziale Europa finde nicht statt“. Eine solche Aussage ist aber geradezu absurd unter
dem Gesichtspunkt, der EuGH schränke Arbeitnehmerrechte ein. Helga Nielebock genden Mindestschutz zu fragen, also Ausdruck, dass er nicht nach der Erforderlichkeit
des Eingriffs sondern der des Schutzes fragt. In der EuGH-Entscheidung „Finalarte“
war hingegen ein höherer tariflicher Urlaubsanspruch gegenüber gesetzlich festgelegtem
möglich.1 genden Mindestschutz zu fragen, also Ausdruck, dass er nicht nach der Erforderlichkeit
des Eingriffs sondern der des Schutzes fragt. In der EuGH-Entscheidung „Finalarte“
war hingegen ein höherer tariflicher Urlaubsanspruch gegenüber gesetzlich festgelegtem
möglich.1 108 I. EuGH und Arbeitsmarkt
II. EuGH und Tarifrecht
III. Entsendung in der EuGH-Rechtsprechung
IV. Arbeitskampf mit grenzüberschreitendem Charakter
V. Zusammenfassung I. EuGH und Arbeitsmarkt
II. EuGH und Tarifrecht
III. Entsendung in der EuGH-Rechtsprechung
IV. Arbeitskampf mit grenzüberschreitendem Charakter
V. Zusammenfassung Perspektive der Arbeitgeberverbände Roland Wolf I. EuGH und Arbeitsmarkt
109
II. EuGH und Tarifrecht
109
III. Entsendung in der EuGH-Rechtsprechung
110
IV. Arbeitskampf mit grenzüberschreitendem Charakter
110
V. Zusammenfassung
111 I. EuGH und Arbeitsmarkt
II. EuGH und Tarifrecht
III. Entsendung in der EuGH-Rechtsprechung
IV. Arbeitskampf mit grenzüberschreitendem Charakter
V. Zusammenfassung III. Entsendung in der EuGH-Rechtsprechung Bei den Entscheidungen Luxemburg, Rüffert und Laval geht es darum, wie ein Mit-
gliedsstaat der Europäischen Union in die Dienstleistungsfreiheit von Unternehmen aus
anderen Mitgliedsstaaten eingreifen darf. Zu Recht betont der EuGH, dass dies grund-
sätzlich nur zulässig ist, soweit Primär- oder Sekundärrecht einen solchen Eingriff zu-
lassen. Beim Einsatz entsandter Arbeitnehmer ist ein solcher Eingriff durch das Sekun-
därrecht vorgesehen, nämlich durch die Entsenderichtlinie. Der Europäische Gerichtshof arbeitet in den drei vorbezeichneten Entscheidungen
den Eingriff in die Dienstleistungsfreiheit am Kanon der Entsenderichtlinie ab. Dies ist den Eingriff in die Dienstleistungsfreiheit am Kanon der Entsenderichtlinie ab. Dies ist
– ob man die Entsenderichtlinie für richtig befindet oder nicht – nachvollziehbar. Mit-
gliedsstaaten dürfen danach nicht aus protektionistischen Erwägungen unter dem
Deckmantel des Arbeitnehmerschutzes eigenständige Regelungen erfinden, die den Ein-
satz von entsendeten Arbeitnehmern entweder unzulässig oder aber doch wirtschaftlich
sinnlos machen. II. EuGH und Tarifrecht Wenn man die Diskussion auf die jüngsten vier Entscheidungen zum Tarifrecht im
weiteren Sinne fokussiert (wobei Vergaberecht nicht mit Tarifrecht gleichzusetzen ist),
ist festzustellen, dass der EuGH maßvoll widerstreitende Interessen zum Ausgleich ge-
bracht hat. Anders, als es auf den ersten Blick scheinen mag, muss man dabei die Ent-
scheidungen Luxemburg, Rüffert und Laval auf der einen und Viking auf der anderen
Seite betrachten. 109 Roland Wolf IV. Arbeitskampf mit grenzüberschreitendem Charakter In der Rechtssache Viking handelt es sich demgegenüber um eine Entscheidung zum
Arbeitskampfrecht. Schon aus diesem Grunde muss gefragt werden, ob die Entschei-
dung überhaupt vor dem Hintergrund von Artikel 137 Abs. 5 EG-Vertrag hätte ergehen
dürfen. Wenn aber der EuGH die Niederlassungsfreiheit durch einen Arbeitskampf be-
rührt sieht, der nach nationalen Vorschriften zulässig ist, muss er dieses nationale Recht
in Einklang bringen mit der Grundfreiheit (hier der Niederlassungsfreiheit) des Europa-
rechts. Der EuGH postuliert sehr klar, dass der bei Viking relevante Boykott als Arbeits-
kampfmaßnahme der finnischen Gewerkschaft – verstärkt durch die internationale See-
arbeitergewerkschaft – nur das mildeste Mittel sein darf, um in Grundfreiheiten ein-
zugreifen. Dabei ist der Arbeitskampf nicht gerechtfertigt als eigenständiges Recht der
Gewerkschaft, sondern er wird gerechtfertigt im Verhältnis von Niederlassungsfreiheit
zum Schutz der Arbeitnehmerrechte. Anders gewendet: Wo es einen ausreichenden
Schutz der Arbeitnehmerrechte auf andere Weise gibt, darf kein Arbeitskampf stattfin-
den. Der nationale Gesetzgeber ist daher aufgefordert klarzustellen, dass Arbeitskämpfe
um Regelungsmaterien unzulässig sind, die dem Betriebsrat abschließend vorbehalten
sind – so in den §§ 111 ff. BetrVG. 110 Scope of Action from a Scandinavian (Nordic) Angle Niklas Bruun 113
114
116 I. Laval and Viking
II. Rüffert
III. Conclusion: ILO 94 and the Rüffert case 116 My understanding is that I am expected to discuss the new case-law from the ECJ
(Laval C-341/05, Viking C-438/05 and Rüffert C-346/06) and the problems they pose
for the labour market system in the Scandinavian countries. I take it for granted that the main content of these cases are known by the audience, I
therefore go straight on and discuss what we should do about this and what problems we
face from a Scandinavian point of view and even on a more general level. V. Zusammenfassung Aus den Entscheidungen Luxemburg, Rüffert und Laval ergibt sich für den deut-
schen Gesetzgeber keinerlei Handlungsbedarf. Die Entscheidungen machen noch ein-
mal deutlich, wie behutsam mit dem Instrument des Mindestlohns umgegangen werden
muss. Aus der Entscheidung Viking ergibt sich Handlungsbedarf, wenn das Bundesar-
beitsgericht weiter die Grundfreiheiten des europäischen Einigungswerks zu wenig be-
rücksichtigen und Tarifsozialpläne und Streiks um diese weiter für zulässig erachten
sollte. 111 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb 783845219165, am 24.10.2024, 06:46:57
- https://www.nomos-elibrary.de/agb https://doi.org/10.5771/97838
Open Access –
- 1 Paragraph 3(1) of the Landesvergabegesetz, headed ‘Declaration that the collective agreement will
be complied with’ states:
“Contracts for building services shall be awarded only to undertakings which, when lodging a ten-
der, undertake in writing to pay their employees, when performing those services, at least the remu-
neration prescribed by the collective agreement at the place where those services are performed and
at the time prescribed by the collective agreement. For the purposes of the first sentence, the term Niklas Bruun tion. Finland has a well established system for making collective agreements binding
“erga omnes”, which makes the situation regarding the implementation of the Posting of
Workers Directive (96/71/EC) less problematic for Finland. tion. Finland has a well established system for making collective agreements binding
“erga omnes”, which makes the situation regarding the implementation of the Posting of
Workers Directive (96/71/EC) less problematic for Finland. For both Denmark and Sweden the national Acts on Posting of workers must be
amended as least regarding minimum wages. There is in the author´s view no need to
change the system fundamentally, but the question is how the new legislation can build
upon the national branch level collective agreements (covering a vast majority of the
workers in the sector) and at the same time fulfil the EC requirements concerning cov-
erage, non discrimination and transparency? If the social parties cannot solve the issue
of minimum wages in collective agreements, there is in the end no other solution than
legislated minimum wages. The necessary legislative amendments are in a preparatory stage both in Denmark
and Sweden. In both countries tripartite bodies have been appointed to prepare for
amendments in legislation and collective agreements. In Denmark this tripartite body
presented its proposals on 19 June 2008 and the responsible Minister Claus Hjort
Fredriksen has declared that the Danish government will take action accordingly. The
tripartite body suggests an amendment to the Danish Act on posting of workers which
explicitly would state that industrial action can be undertaken against foreign service
providers in the same way as action can be taken against Danish ones. An explicit new
condition for such industrial action is that the representative national collective agree-
ment applicable in the branch contains clear stipulations on the minimum wages that a
foreign service provider has to pay. This again creates a responsibility for the social
partners to amend the collective agreements accordingly. In Sweden the preparation of legal measures to be undertaken is still in a preparatory
phase and it is not self-evident what solutions will be proposed. I. Laval and Viking These judgments deal with a peace-obligation for transnational industrial action that
has an impact on one of the four EC freedoms (free movement for goods, labour, ser-
vices and capital). The judgments do have a clear impact on Scandinavian law. This impact is partly
common for all the Scandinavian or even Nordic countries. The impact of the Viking-
judgment restricting collective action in situations of crossborder establishment is
clearly similar for all Nordic countries and will certainly have implications on how trade
unions and their international organisations take use of industrial action in such situa-
tions. The Laval-judgment again will clearly bring about changes to the law in force in
these countries. In this respect there are however also several different features in the
situation of the respective states: Sweden has a special situation with its unique Lex
Britannia that has to be dealt with since the ECJ declared that it is not compatible with
EC law. Finland, Denmark and Norway have ratified ILO Convention 94 on Social
Clauses in Public Contracts (public procurement). Sweden has not ratified that Conven- 113 Niklas Bruun II. Rüffert In the Rüffert case a company, Objekt und Bauregie, was awarded a public contract. The company employed as a subcontractor another undertaking established in Poland. In summer 2004 this undertaking came under suspicion of having employed workers on
the building site at a wage below that provided for in the ‘Buildings and public works’
collective agreement.1 Following investigation, Land Niedersachsen terminated the con- 114 0 5
– Scope of Action from a Scandinavian (Nordic) Angle tract with Objekt und Bauregie based on the fact, inter alia, that Objekt und Bauregie
had failed to fulfil its contractual obligation to comply with the wage provision in a col-
lective agreement which it was bound to by law. A penalty notice was issued against the
person primarily responsible at the undertaking established in Poland, accusing him of
paying 53 workers engaged on the building site only 46.57% of the prescribed mini-
mum wage. The issue went at first instance to the regional court in Hannover and then to the hig-
her regional court, which stayed the proceedings and referred the following question to
the European Court of Justice (ECJ): “Does it amount to an unjustified restriction on the freedom to provide services under
the EC Treaty if a public contracting authority is required by statute to award contracts
for building services only to undertakings which, when lodging a tender, undertake in
writing to pay their employees, when performing those services, at least the remunerati-
on prescribed by the collective agreement in force at the place where those services are
performed?” The ECJ read the Posting of Workers’ Directive 96/71/EC2 in the light of Article 49
EC. The ECJ held that the German federal legislation did not satisfy the conditions re-
garding minimum wages in the host country which are binding on a service provider as
regards payment of posted workers. According to the court, the legislation did not dec-
lare collective agreements generally applicable, and applied only to a part of the sector
(public contracts), although in fact it did also apply to the main part of the private sec-
tor. Nor did the legislation satisfy the criteria laid down in Article 3(8) of the Directive. The ECJ gave a very restrictive interpretation of Article 3(7) of the Directive, which
states that paragraphs 1 to 6 shall not prevent application of terms and conditions of
employment which are more favourable to workers. 3 Case C-341/05, Laval un Partneri Ltd v Svenska Byggnadsarbetareförbundet, Svenska Byggnadsar-
betareförbundet, avd. 1, Svenska Elektrikerförbundet, Opinion of Advocate General Paolo Mengoz-
zi, 23 May 2007, ECJ decision, 18 December 2007. ‘services’ means services provided by the principal contractor and by subcontractors. The first sen-
tence shall also apply to the award of transport services in local public transport.” 2 Directive 96/71/EC concerning the posting of workers in the framework of the provision of services.
OJ 1996, L18/1. ‘services’ means services provided by the principal contractor and by subcontractors. The first sen-
tence shall also apply to the award of transport services in local public transport.”
2 Directive 96/71/EC concerning the posting of workers in the framework of the provision of services.
OJ 1996, L18/1.
3 Case C-341/05, Laval un Partneri Ltd v Svenska Byggnadsarbetareförbundet, Svenska Byggnadsar-
betareförbundet, avd. 1, Svenska Elektrikerförbundet, Opinion of Advocate General Paolo Mengoz-
zi, 23 May 2007, ECJ decision, 18 December 2007. Niklas Bruun Land Niedersachsen. The Rüffert judgment is, in my view, the first where the ECJ no
longer requires that competition be on equal terms between national and foreign service
providers. Rather, the Court takes the position that foreign service providers are to be
allowed a competitive advantage by paying lower wages, contrary to what is provided
by the federal law referring to the wage level in the sectoral collective agreement. II. Rüffert In sum, the Court came to the conc-
lusion that a Member State is not entitled to impose such legislation on contractors. Di-
rective 96/71, interpreted in the light of Article 49 EC, precludes a Member State from
adopting legislation such as that in force in Land Niedersachsen. The ECJ judgment is in many ways problematic from a legal point of view. Here I do
not present any general critique of the conclusions in the Rüffert case and only discuss
some issues related to ILO 94. It has, however, to be stated that in Rüffert, contrary to
what was argued to be the case in the Laval judgment3, the wage level was transparent
and easy to ascertain in advance. It is also clear that the legislation is valid on national
level and binds German companies that take part in a public procurement procedure in ‘services’ means services provided by the principal contractor and by subcontractors. The first sen-
tence shall also apply to the award of transport services in local public transport.” 2 Directive 96/71/EC concerning the posting of workers in the framework of the provision of services. OJ 1996, L18/1. 2 Directive 96/71/EC concerning the posting of workers in the framework of the provision of services. OJ 1996, L18/1. 115 Niklas Bruun III. Conclusion: ILO 94 and the Rüffert case The federal legislation in Land Niedersachsen complies precisely with the obligation
imposed by ILO Convention 94 on those states that have ratified it. Article 2 of Con-
vention 94 states: “Contracts to which this Convention applies shall include clauses ensuring to the
workers concerned wages (including allowances), hours of work and other conditions of
labour which are not less favourable than those established for work of the same charac-
ter in the trade or industry concerned where the work is carried on – (a) by collective
agreement…”. Germany has not ratified Convention ILO 94. However, the Convention has been ra-
tified by a considerable number of Member States: Austria (1951), Belgium (1952),
Denmark (1955), Finland (1951), France (1951), The Netherlands (1952), Italy (1952),
Spain (1971) and the UK (1950, though it was denounced by the UK in 1982). Among
the new Member States, at least Bulgaria (1955) and Cyprus (1960) have ratified the
Convention. In light of this, the ECJ’s interpretation of the Posting Directive in the Rüffert case is
remarkable. When both adopting and implementing the Posting Directive, the Commis-
sion declared that the Directive was fully consistent with ILO Convention 94. The posi-
tion after Rüffert is that the legal situation will vary among Member States. Those
Member States that have ratified ILO 94 before being bound by the Treaty can, accor-
ding to Article 307 EC, still apply ILO 94, at least during a lengthy transitory period if it
is to be denounced. From a Scandinavian angle one may ask whether this means that
Finland and Denmark are in a different position than Sweden, because the latter has not
ratified the Convention. The conflict between the Rüffert judgment and ILO 94 is clear but of limited scope. It does not apply to public procurement exclusively within a Member State (where there
is no cross-border posting of workers); it does not apply when posting is of workers
from a non-EU Member State; and it does not apply when cross-border work is perfor-
med in the context of public procurement in forms which do not involve posting of
workers. View of the Swedish Trade Union Movement Claes-Mikael Jonsson First of all, thank you for giving the Swedish Trade Union movement the opportunity
to comment on recent developments in Community Law here today. I share most of the views expressed by professor Bruun, but also much of what pro-
fessors Lyon-Caen, Rebhahn and Bercusson said this morning, in particular concerning
the problems caused by the new interpretation of the Posting Directive and the new re-
strictions on collective actions introduced on Community level by the European Court
of Justice. It is our belief that the ECJ, through its recent case law, has planted a time-bomb un-
der the European Union. The fire will start when workers see their wages undercut by
posted workers. It might even be so that the fire has already started. The “no” in the
referendum in Ireland could be interpreted in such a way. Community law cannot only
be interpreted narrowly through contradictory legal texts. Political and social realities
must always be a factor. The EU was at a crossroad six months ago. The ECJ could have chosen another path. The Court had alternative routes, which the Advocate Generals showed in all four cases. The road chosen was neither “the only one” nor “necessary”. Free movement without
responsibility was not the answer to all the questions ECJ had been asked. The EU is founded upon compromises. One important compromise, if not the most
important one, is the balance between two conflicting views – EU as an instrument for
companies to compete on an international arena with free movement as the primary tool
– and the other view – EU as an instrument for Trade Unions and Governments to cre-
ate a countervailing force to companies and capital which acts on a global scene. These
two perspectives could also be described as EU’s economic and social dimensions. A sound balance between the social and economic dimensions is crucial for the EU. Jaques Delors struck a compromise with the European Trade Union movement when he
relaunched the Internal Market in the late 80’s. The European Trade Union movement
promised not to oppose the Internal Market and got an “Espace social” in return. This
“gentlemen’s agreement” was simple – no internal market without a social dimension. This agreement has been revoked by the ECJ through its recent case law. The social
dimension has been severely undermined. Swedish Trade Unions fought for equal treatment in Laval. III. Conclusion: ILO 94 and the Rüffert case The starting point for regulation of public procurement in the European Union has
been that the Member States should be free to stipulate national standards, including for 116 Scope of Action from a Scandinavian (Nordic) Angle wage regulation, provided there is equal treatment and no discrimination whatsoever is
allowed in respect of foreign serviceproviders. This starting point is fully compatible
with the ILO 94. The Rüffert case has created confusion, uncertainty and the risk of different rules ap-
plying in Member States. The conclusion is that the Posting Directive 96/71/EC, as in-
terpreted by the ECJ in the light of Article 49, must be clarified in order to be in harmo-
ny with ILO Convention 94. This could quite easily be done by inserting a clause into the Directive clearly stating
that either the public sector (procurement) can be regarded as a separate sector within
the meaning of the Directive (Article 3(8)) or that wage standards linked to or clearly
defined in collective agreements in a specific sector, at national or federal level, can be
considered as part of the minimum standard that foreign service providers are obliged to
apply under condition that this is a general requirement under the EC Public Procure-
ment regime. It is important that the European Union Member States defend the freedom of all
Member States to continue to adhere to ILO Convention 94 and apply the principles on
equal treatment of employers that is the fundament of this instrument. 117 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
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- View of the Swedish Trade Union Movement But instead of “equal pay
for equal work” we got “minimum pay for equal work”. We expected double demands
to be forbidden, and had already started our work to prevent this. We knew that our sys-
tem had some transparency problems, but we had also initiated a process to improve the
situation. But the ECJ did much more than necessary in the Laval case. Carefully devel- 119 Claes-Mikael Jonsson oped balances in national industrial relations systems have been distorted. One should
keep in mind that the EU has 27 different labour market models. They all reflect differ-
ent balances of power between Capital and Labour. The ECJ will become largely un-
popular as it moves delicately balanced power between the social partners in the Mem-
bers States. Let me also in this context comment on the German employers’ representative who
claimed that Laval went bankrupt. This claim is not true. It was their letter box com-
pany, Baltic Bygg, without employees or assets, their “door-opener” to undercut condi-
tions on the Swedish labour market that went bankrupt. Laval is still doing business in
Latvia. Let me also, as a comment to the German employers’ representative concerning
Lex Britannia, state that this law is not remarkable or protectionist in any way, it is not
more than a functional equivalent to a mechanism which declares collective agreements
generally binding. An industrial relations system, such as the Swedish, based on the
autonomy of the social partners must have such a mechanism. All recent case law from the ECJ can be analysed both from a narrower national per-
spective and a more overarching European perspective. For example the horizontal di-
rect effect for trade union activities which was introduced in the Viking and Laval
cases. The ECJ could have given, as for Competition law, a larger scope of autonomy to
the social partners in line with the Albany judgement. The ECJ does not appear to trust Member States, as the recent Luxemburg case illus-
trates well. Neither can regional authorities be trusted by the ECJ, what the rationale of
the Rüffert case seems to be. The ECJ has also redefined the Posting of Workers direc-
tive into a maximum Directive. And the Court also struck an illusory balance between
free movement and fundamental rights, which in practise subordinates fundamental
rights to free movement. However, I will not have time to discuss this further now. View of the Swedish Trade Union Movement I will stick to the Swedish perspective, but one should keep in mind, that even though
many consequences can be solved on national basis, structural general problems on
European level remain – if equal treatment of workers, the rationale under article 39,
should be restored also for posted workers. The new industrial relations system con-
structed by the ECJ on Community level, which restricts the right to collective action in
the Member States should also be abolished. The recommendation by Professor Bercus-
son, to use the Keck-option should be seriously considered by the ECJ. If not, The
Court will have to deal with a potential flood of cases where political, economical and
social tensions are very high. The EU runs a great risk of getting even more unpopular. This would be ironic in a time when EU is needed more than ever to face challenges on
a global scale. The Swedish autonomous collective bargaining model consists of strong social part-
ners, who regulate the labour market more or less independently of the State. This is in
contrast with the so called continental models where the State have a more primary role
in the regulation of the labour market. It appears as if the ECJ measures the Swedish (or
Scandinavian) autonomous labour market models through a lens shaped by a continental 120 View of the Swedish Trade Union Movement view. This has created a number of problems in Sweden. Let me name some, beside the
fact that the right to industrial action has been restricted by Community law. Collective wage formation on the Swedish labour market might be severely dis-
turbed. Decentralized and local bargaining will probably become difficult. Many sectors
and branches do not have minimum wages, and where such are to find, they are seldom
in use due to local and flexible collective bargaining. It is ironic that the rationale of the
ECJ, a naïve concept of minimum wages, is most likely to create a less flexible and
more rigid wage formation, as there will be a pressure upwards on minimum wages in
the years to come, this while the legislative institutions in Brussels cherish the Scandi-
navian flexicurity models. Posted workers, which constitute a minor part of the labour
market, could thus affect the whole system for wage formation in Sweden. Another problem is to restore collective agreements as the primary regulatory in-
strument also for posted workers. View of the Swedish Trade Union Movement We don’t want the state to interfere with the labour
market. No matter if it is the Swedish or the European State. The social partners should
be the principal actors when it comes to the regulation of relations between workers and
their employers. The social dimension of the EU has been severely damaged. Political action is
needed. A first step must be to open the Posting of Workers directive for revision. It
needs a broader legal base, a pluralistic view on national labour market systems and to
re-establish its character as a minimum Directive. Secondly, Heads of States and Prime
Ministers need to adopt the Social Progress Clause which the ETUC have proposed. Action is needed urgently. A Europe built solely on free movement, where Member States are prevented from
protecting its own and foreign workers have no future. The EU is needed and it is too
important to be jeopardized through bad judgements. Future challenges must be met
with both a social and economic dimension. 121 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
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- View of the Confederation of Swedish Enterprise Lars Gellner I. The Rüffert ruling
II. The Laval ruling 123
124 123 124 I. The Rüffert ruling Professor Bruun thinks the ECJ’s interpretation of the Posting Directive in the Rüf-
fert case is astonishing. The reason is that, according to the ECJ’s interpretation, the
Directive is in conflict with ILO Convention No 94. I cannot understand this reaction. A possible conflict between EU legislation and
agreements that Member States have made is not an unanticipated situation. It is fore-
seen in the Treaty, article 307. And that article establishes the fact that a Member State
that has produced an agreement in conflict with the Treaty must make efforts to remove
elements in conflict with the Treaty. The 97th ILO-session in Geneva in June this year was – among other things – about
the issue of integrating labour clauses into public procurement contracts. According to
the provisional record from the session the basic assumption by the Committee of Ex-
perts seemed to be that competition on the basis of labour costs is socially unhealthy
and should always be avoided. When we talk about public procurement contracts it is important to evaluate whether
or not competition eliminates corruption, increases productivity and transparency and
procures goods and services at the best value for money or best quality for the price. In my opinion these are important factors to be considered. In my opinion these are important factors to be considered. There is a number of concerns related to the Convention. There are about 60 coun-
tries that have ratified the Convention, but only one quarter of them are actually apply-
ing it. The Committee of Experts in the ILO-session has noted that Member States of
ILO are unwilling to take the necessary action to implement the Convention. One con-
clusion is that the principle that the State should act as a model employer by offering the 123 Lars Gellner most advantageous conditions to workers paid indirectly through public funds does not
seem to be popular. Furthermore the prevailing view of governments is that workers employed under
procurement contracts are not in need of special protection over and above national la-
bour and employment laws. The idea of including labour clauses in public contracts is
not widely accepted among Member States of ILO. Against this background perhaps the Rüffert ruling is not very alarming. II. The Laval ruling In Sweden the ruling in the Laval case has led to a widespread debate regarding trade
union rights. From trade unions and some politicians on the left wing there have been
claims to change the Swedish legislation in order to guarantee that the system with col-
lective agreements can be used against foreign EU-employers. Efforts are even made by
trade unions to postpone Sweden’s approval of the Lisbon Treaty due to the conse-
quences of the Laval ruling. Maybe a solution including collective agreement is good. The alternative is, roughly
speaking, minimum pay determined by the legislator, directly or indirectly. The Con-
federation of Swedish Enterprise has not yet made a choice what solution to promote. However, there is one legal problem with the collective agreement-solution. Accord-
ing to Swedish labour law there is no guarantee, in my opinion, that the foreign employ-
ees will get the pay regulated by the agreement. They are not members of the Swedish
trade union that is part of the collective agreement and their employment contracts will
not be filled out by a collective agreement at the workplace - because there is no such
agreement at the workplace. As the Rüffert ruling establishes, it is not enough if a Member State in the national
Posting of Workers legislation only refers to a collective agreement for setting the pay. It is, in other words, not allowed to in blank delegate the establishing of pay to the so-
cial partners at the national level. This is not a legislation in accordance with article 3.1,
first paragraph. There are drawbacks also with the legislator-solution as well. I do admit that letting a
question of setting wages be solved by politicians is not the common way in Sweden. It
must however be considered that we do not have a tradition of regulating wages for for-
eign employees. Let me finish by declaring that Sweden and other states have joined the EU because
they think that a membership best promotes economic development and increased wel-
fare. The freedom to provide services is one of the fundamental principles of the Com-
munity and therefore one of great importance for employers all over Europe. It would
have been remarkable if ECJ should not have defended that principle. 124 Diskussionsbericht Diskussionsleitung: Horstpeter Kreppel Diskussionsleitung: Horstpeter Kreppel Ausgangspunkt der Diskussion bildete die mehrfach geäußerte Kritik an der EuGH-
Rechtsprechung, die ökonomischen Grundfreiheiten zu stark zulasten der Grundrechte
zu betonen und die Grundrechte lediglich als Rechtfertigungsgrund zu berücksichtigen. Dieser Kritik wurde entgegen gehalten, dass der EuGH die Grundrechte nur aus prozes-
sualen Gründen im Rahmen der Verhältnismäßigkeit prüfe, diese dadurch jedoch nicht
als Rechtsfertigungsgründe materiell degradiere. Nach anderer Ansicht habe in den Ent-
scheidungen Viking und Laval prozessual keine Notwendigkeit bestanden, die Grund-
rechte lediglich als Ausnahmetatbestand zu behandeln. Sie seien daher in diesen beiden
Urteilen in völlig vermeidbarer Weise relativiert worden. Als positives Gegenbeispiel
wurde erneut das Schmidberger-Urteil hervorgehoben, in dem der EuGH ausdrücklich
gesagt habe, dass es uneinschränkbare und einschränkbare Grundrechte gebe und eine
Abwägung nur in Bezug auf einschränkbare Grundrechte vorgenommen werde. Dieser
Ansatz sei korrekt und daher weiterzuverfolgen. Es wurde grundsätzlich davon ausgegangen, dass die Grundrechte und die Grund-
freiheiten auf gleicher Stufe stehen, denn beide seien im Primärrecht verankert und
würden vom EuGH anerkannt werden. Uneinigkeit bestand jedoch über ihre konkrete
Ausgestaltung. Eine Seite stufte die Grundfreiheiten nicht lediglich als ökonomische
Rechte, sondern als grundrechtsgleiche oder grundrechtsähnliche Rechte ein, da sie das
Freizügigkeitsrecht und das Verbot der Diskriminierung beinhalten würden und diese
beiden Rechtsgüter auch nach deutschem Verfassungsrecht Grundrechte seien. Von anderer Seite wurde vorgebracht, dass der EuGH die Grundfreiheiten zu Un-
recht grundrechtsgleich behandle. Die Grundfreiheiten seien lediglich Regelungen, die
dazu dienen würden, dass Europa ökonomisch zusammenwächst, d.h. sie seien darauf
begrenzt, Protektionismus zu verhindern und daher restriktiv auszulegen. Dagegen
müssten die verfassungsrechtlich garantierten Grundrechte so ausgestaltet werden, dass
sie sich möglichst weitgehend entfalten können. Darüber hinaus wurde auf die unterschiedliche Durchschlagskraft abgestellt. Die
Durchschlagskraft der Grundfreiheiten sei nach der EuGH-Rechtsprechung eine univer-
selle, da sie auch unter Privatleuten uneingeschränkt gelten würden; eine vergleichbare
Drittwirkung gebe es bei den Grundrechten nicht. Das Verhältnis der Durchschlagskraft
sei allerdings eine noch zu klärende Frage. Zur Dienstleistungsfreiheit wurde angemerkt, dass ein Mittelweg zwischen dem
Recht des Herkunftsstaates und dem Recht des Aufnahmestaates gefunden werden müs-
se, denn die Dienstleistungsfreiheit betreffe Arbeitnehmer, die lediglich vorübergehend
im Aufnahmestaat tätig seien, d.h. auch noch dem Recht ihres Herkunftsstaates unter- 125 Diskussionsbericht worfen seien. In ihrem Herkunftsland würden regelmäßig Renten- und Krankenversi-
cherungsbeiträge für sie entrichtet. Wenn sie vollständig dem Recht des Aufnahmestaats
unterstellt würden, würde man den im Vertrag angelegten Unterschied zwischen Nie-
derlassungsfreiheit und Dienstleistungsfreiheit vermischen. Simone Gräfin von Hardenberg Diskussionsleitung: Horstpeter Kreppel Dann gäbe es ein Problem
mit dem Primärrecht, das die Verfassung der Gemeinschaft darstelle. Zum Verhältnis Europäische Gemeinschaft und Soziales wurde vorgebracht, dass die
Sozialstandards in Europa nach Gründung der Europäischen Gemeinschaft gestiegen
seien, da Länder mit einem hohen Sozialstandard (wie z.B. Frankreich) zur Supranatio-
nalisierung bereit gewesen seien. Auch die Löhne seien gestiegen und würden weiter
steigen. So wären z.B. bei den drei baltischen Staaten Erhöhungen des durchschnittli-
chen Lohnniveaus um über 20 % zu verzeichnen, bei anderen Staaten über 10 %. In
Deutschland sei es nicht zum Rückschritt gekommen. Daher wäre es nicht gerechtfer-
tigt, den EuGH so hinzustellen, als ob er den Raubtierkapitalismus unterstütze, denn –
wie bereits in den Vorträgen angeklungen – seien auch die Arbeitnehmer und die Ge-
werkschaften vielfach doch recht zufrieden mit seiner Rechtsprechung. So habe der
EuGH z.B. den Bereitschaftsdienst in die Höchstarbeitszeit eingeschlossen und Raum-
pfleger/-innen im Fall eines Betriebsübergangs in ihren Arbeitsverhältnissen geschützt. p
g
g
g
g
Vor dem Hintergrund des veränderten Gleichgewichts zwischen Grundfreiheiten und
nationalen Grundrechten wurde die Frage gestellt, ob nun Europa oder die einzelnen
Mitgliedstaaten für ein Auspendeln dieser veränderten Gewichte zuständig seien. Wenn
ein Arbeitnehmer aus einem Mitgliedstaat mit einem niedrigen Sozialstandard in einen
Mitgliedstaat mit einem höheren Sozialstandard entsandt werde, dann sei für ihn das
Minimum gleichzeitig das Maximum, denn er könne in seinem Herkunftsland nicht auf
bessere Arbeitsbedingungen zurückgreifen. Daher stelle sich die Frage, ob es sich dabei
um eine Problematik handele, die national oder auf europäischer Ebene gelöst werden
müsse. Es bestand einheitlich der Wunsch nach einer Verbesserung der gegenwärtigen Si-
tuation. Es wurde u.a. vorgeschlagen, die Entsenderichtlinie zu ändern, auch wenn eine
Änderung nicht leicht durchzusetzen sei. Daher lag die Hoffnung vorrangig auf einer
Änderung der EuGH-Rechtsprechung, indem sich der EuGH in Zukunft selbst Grenzen
setzt und auf weitere Eingriffe in nationale Kompetenzen verzichtet. 126 Scholz: Wir haben zu dieser Veranstaltung mit so vielen Experten eingeladen, um zu lernen. Diese Veranstaltung ist getragen von der Sorge, dass eine Verschiebung der Sozialbe-
ziehungen zu Lasten der Arbeitnehmer und Arbeitgeber stattfindet und Dinge nicht so
funktionieren, wie wir uns das in unseren nationalen Staaten vorstellen. Ein Blick auf
die abstrakten rechtlichen Regelungen reicht dazu sicherlich nicht aus. Als in Deutsch-
land das Bürgerliche Gesetzbuch geschrieben wurde, kam der Arbeitsvertrag darin nicht
vor. Gesetze, die damals geschaffen wurden, haben sich allmählich im Rahmen von
rechtlichen Bewegungen und Demokratisierungsprozessen weiterentwickelt. Die Frage
ist, ob wir bei der Notwendigkeit der wirtschaftlichen Integration Mittel und Wege ge-
funden haben, die selbstverständlichen Rechte der Arbeitnehmer und Arbeitgeber zum
Ausdruck zu bringen. Wenn die politischen Institutionen in Europa mit dem erreichten
Stand im Bereich Arbeitsbeziehungen nicht Schritt halten können, dann kann es auf
Dauer zu einer Legitimationskrise kommen. I. Podiumsdiskussion Möller: Ein wichtiger Aspekt, den wir vertiefen sollten, ist die politische Dimension. Wie
kann man politisch auf die Auswirkungen der Urteile reagieren, die sich auf das Ar-
beitsrecht der Mitgliedstaaten niedergeschlagen haben? Es wurde gesagt, den Entschei-
dungen ist die Tendenz zu entnehmen, die unternehmerische Freiheit im Binnenmarkt
zu betonen und gleichzeitig den sozialpolitischen Spielraum der Mitgliedsstaaten einzu-
schränken, ohne dabei eine Kompensation auf politischer Ebene vorzusehen. Herr Mi-
nister Scholz, verschiebt der Europäische Gerichtshof tatsächlich die Balance zwischen
Dienstleistungsfreiheit und sozialen Schutzrechten der Arbeitnehmer? Podiums- und Plenumsdiskussion Moderation: Karl-Dieter Möller I. Podiumsdiskussion Möller: Wie aber kann es gelingen, den die Mitgliedstaaten verbindenden politischen Willen
in Gesamteuropa so zum Ausdruck zu bringen, wie es innerhalb der einzelnen Mitglied-
staaten im Wege von politisch-demokratischen Prozessen geschieht? Wie reagiert die
deutsche Politik, wenn sie merkt, dass dies auf Europaebene nicht gelingt, sondern
vielmehr der EuGH seinen eigenen Weg geht, der von Deutschland nicht geteilt wird? Es wurde vorhin der Vorschlag gemacht, die Entsenderichtlinie zu ändern. Gibt es hier- 127 Podiums- und Plenumsdiskussion zu Überlegungen auf deutscher Seite und Gespräche mit ihren Kolleginnen und Kolle-
gen auf europäischer Ebene? Möller: Das war eine schöne politische Antwort. Herr Hoffmann, die Frage an Sie, vorhin
wurde gesagt, egal, welche Lösung es gibt, es wird immer Probleme geben. Teilen Sie
diese Ansicht? Scholz: Wir haben das Gefühl, dass wir etwas tun müssen, denn das, was dem Willen der
demokratisch Gewählten und dem Willen der deutschen Bevölkerung entspricht, kann
nicht so umgesetzt werden, wie wir uns das gedacht haben. Wie wir darauf konkret rea-
gieren sollen und können, das wollen wir gerade auch durch diese Veranstaltung und
ihre Auswertung lernen. Insofern kann ich während der Veranstaltung noch kein Ergeb-
nis verkünden. Wir haben diese Veranstaltung auch durchgeführt, um unseren Hand-
lungsrahmen besser einschätzen zu können und um eine europäische Diskussion über
die Handlungsnotwendigkeiten und das Erreichbare zu entfachen. Möller: Hoffmann: Sicher, es wird immer Probleme geben, die Frage ist, wie wir uns dieser Probleme
annehmen und welche Lösungen dazu zur Verfügung gestellt werden. Wir haben heute
viel aus juristischer Perspektive erfahren, jedoch sollte die politische Diskussion, aber
auch die politischen Prozesse und der politische Wille mehr in den Vordergrund gerückt
werden, damit mehr bei den Bürgerinnen und Bürgern ankommt. Wir brauchen eine
politische Debatte nicht nur im Lichte der jüngsten EuGH-Rechtsprechung, sondern
müssen auch die Frage klären, welches Europa wir in der Zukunft anstreben. Welchen
Stellenwert haben wirtschaftliche Freiheiten gegenüber sozialen Grundrechten? Das
irische Referendum hat gezeigt, dass hier noch viel Diskussionsbedarf besteht. Wir als
Europäischer Gewerkschaftsbund haben den Verfassungsvertrag – also auch den Re-
formvertrag – unterstützt, aber nicht deshalb, weil er der beste Vertrag ist, sondern weil
die Grundrechtscharta damit rechtverbindlich wäre und die Rechtsprechung des EuGH
dann möglicherweise etwas anders ausgefallen wäre. Möller: Herr Levits, was nehmen Sie aus der heutigen Diskussion mit? Wird die Kritik an der
EuGH-Rechtsprechung von den Richtern ernst genommen oder fühlen sich die Richter
davon nicht angesprochen? Levits: Der Richter spricht durch seine Urteile, auch wenn bei Urteilen, die wie bei uns kol-
lektiv verfasst werden, die einzelne Richtermeinung nicht erkennbar wird. Jedes Ge-
richt, insbesondere die höchsten Gerichte, müssen sich der Kritik und der Diskussion
stellen. Das gilt auch für den EuGH, der diese Kritik keineswegs als eine Art Majestäts-
beleidigung auffasst, sondern den intellektuellen Dialog innerhalb einer interessierten 128 Podiums- und Plenumsdiskussion juristisch-politischen Öffentlichkeit schätzt. Insoweit hat Bundesverfassungsrichter Di
Fabio die maßgebliche Öffentlichkeit treffend formuliert. Dialoge, Kritiken und Diskus-
sionen in den einzelnen Mitgliedstaaten sind für das Gericht und für mich persönlich
sehr wichtig, damit das Gericht nicht im „luftleeren Raum“ agiert. Ich kann nicht sagen,
ob und welche Auswirkungen damit verbunden sein werden, sondern nur, dass jedes
Argument für den Richter von Belang ist, denn ein Teil seines Berufes besteht darin,
Argumente anzuhören und zu verarbeiten. Möller: Das ist möglicherweise aus Ihrer Sicht, Herr Andersson, etwas anders. Es wurde be-
reits angesprochen, dass Sie einen Bericht vorgelegt haben, in dem Sie das Europäische
Parlament auffordern, in dieser Sache tätig zu werden. Vielleicht könnten sie uns ein
paar Stichworte daraus nennen und mitteilen, was das Europäische Parlament aus Ihrer
Sicht nach diesen Urteilen unternehmen sollte. Teilen Sie die Einschätzung von Herrn
Levits, dass es in den 27 Mitgliedstaaten diese Ausgewogenheit gibt? Möller: Kurze Nachfrage, Herr Levits: Kommt die Kritik nur aus Deutschland oder kommt
sie auch aus anderen Ländern? Gibt es so etwas wie eine Presseschau beim Europäi-
schen Gerichtshof, aus der sich diese öffentlichen Kritiken oder Meinungen erkennen
lassen? Levits: Levits: Ja, es gibt eine wöchentliche Pressezusammenstellung über die Reaktionen zu unse-
ren Urteilen. Die Kritiken und Debatten der besprochenen Urteile waren sehr unter-
schiedlich und zeigten – ganz grob gesagt – ein eher ausgewogenes Bild, d.h. in einigen
der 27 Mitgliedstaaten wurden diese Urteile eher kritisiert, in anderen wiederum sehr
positiv aufgenommen. Verschiedene Staaten und auch einzelne soziale Gruppen inner-
halb eines Mitgliedstaates vertraten unterschiedliche Interessen. Möller: Möller: Podiums- und Plenumsdiskussion Lebensbedingungen, d.h. um ein gutes Sozialsystem, um ein angemessenes Einkommen
und darum, einen Job zu haben usw. Die gesamte Politik dreht sich um diese Fragen. Wenn wir als Politiker diese Bedürfnisse vergessen, werden uns die Menschen den Rü-
cken kehren. Wir haben eine breite Diskussion darüber, wie wir mit der Globalisierung
umgehen. Was aber unternimmt die Politik? Es geht nicht darum, ein Einkommen auf
indischem oder chinesischem Niveau zu haben. Wir sollten durch Forschung und Ent-
wicklung, durch Investitionen in menschliche Arbeitskraft und durch angemessene Ar-
beit, gutes Einkommen und durch gute Arbeitsbedingungen in den Wettbewerb treten. Da haben wir die Flexibilität, wir sprechen von „flexicurity“. Das dänische System ist
nicht das einzige, das wir ansehen können, aber es ist ein sehr flexibles System mit einer
großen Verantwortung für die Sozialpartner und ihre Verhandlungen. Die Rechtspre-
chung geht insoweit in eine völlig andere Richtung. Das beunruhigt mich und ich hoffe,
es beunruhigt auch das Europäische Parlament. Die Kommission scheint es nicht zu
beunruhigen, aber es sollte sie beunruhigen, denn es geht um die Zukunft Europas und
um seine Akzeptanz unter den Bürgerinnen und Bürgern. Als Gesetzgeber müssen wir
reagieren. Wir müssen das Gleichgewicht zwischen Dienstleistungsfreiheit und nationa-
len Rechten wie Tarifverhandlungen und betrieblichen Wahlen finden. Die Lösung sehe
ich im sozialen Fortschritt. Ich bin nicht ganz sicher, das erkennen zu können, da wir im
Bereich des Sekundärrechts die Dienstleistungsrichtlinie, Entsenderichtlinie und die
Monty Regelung haben. Es wurde gesagt, dass diese nicht in die nationalen Systeme der
Tarifverhandlungen und in die Arbeitnehmer- und Arbeitgeberrechte eingreifen sollten,
aber das scheint nicht auszureichen. Und warum nicht? Der Grund liegt vielleicht darin,
dass es sich hier um Sekundärrecht handelt und die Dienstleistungsfreiheit zum Primär-
recht zählt. Dagegen müssen wir etwas tun. Das ist die eine Sache. Die andere Sache, die ich in meinem Bericht behandelt habe, betrifft die Mindest-
standards, die für bestimmte Gruppen verpflichtend sind. Es sollte nicht nur für einzelne
Nationalitäten zur allgemeinen Regel werden. Die Gleichbehandlung ist wichtiger. Wenn ein polnischer, ein deutscher, ein schwedischer oder ein dänischer Arbeitnehmer
die gleiche Arbeit im gleichen Arbeitsmarkt verrichten, dann müssen sie auch gleich
behandelt werden. Das sollte die Hauptrolle spielen. Wir haben viele Mindeststandards
in der europäischen Gesetzgebung, so z.B. im Bereich Umwelt, Gesundheit und Sicher-
heit. Man kann überall noch bessere Bedingungen einführen, als sie im Herkunftsland
vorhanden sind. Andersson: Das Gericht hat sein Urteil gefällt. Es hat seine Funktion in der Europäischen Ge-
meinschaft. Nun ist es Aufgabe der Kommission, des Parlaments und des Rates, zu
handeln. Wir sind der Gesetzgeber und wir müssen die Verantwortung übernehmen. Nicht zu handeln, bedeutet zuzustimmen. Bevor ich auf meinen Bericht – den Entwurf – eingehe, müssen wir diese Beurtei-
lung in einem breiten politischen Zusammenhang sehen. Zum Beispiel, wie sehen dies
die Bürger und Bürgerinnen heute? Irland ist nicht das einzige Beispiel. Wenn wir im
Mai nach Schweden kommen, findet dort eine Abstimmung über den Lissaboner Ver-
trag statt. Es ist mit einer Ablehnung von 60 bis 70 % zu rechnen, die vor allem auf die
Laval-Entscheidung zurückzuführen ist. Warum? Weil es um das Gleichgewicht geht. Und was ist unser Thema in Europa? Geht es nur um den offenen Binnenmarkt, den
Binnenmarkt für Unternehmen? Nein, es geht in erster Linie um Menschen und ihre 129 Podiums- und Plenumsdiskussion Koskelo: Ich glaube nicht, dass in Finnland eine Abstimmung über den Lissaboner Vertrag
derart negativ ausgefallen wäre und das sicherlich nicht wegen den Entscheidungen
Laval, Viking und Rüffert. Ihre zweite Frage hat sich bei uns noch nicht konkret gestellt. Wenn wir uns z.B. die
heutigen Vorträge in Erinnerung rufen, dann sehen wir, dass Arbeitskampfmaßnahmen
und Streik als Grundrechte in vielen nationalen Rechtssystemen – so auch in Finnland –
anerkannt sind. Selbst wenn sie mit dem Grundrecht auf Koalitionsfreiheit verbunden
sind, handelt es sich nicht um ein absolutes Recht. Die verfassungsmäßige Auslegung
durch den Gesetzgeber sagt sehr deutlich, dass der genaue Inhalt des Rechts auf Ar-
beitskampfmaßnahmen und die Ausübung dieses Rechts dem Gesetz unterworfen sind. Es handelt sich daher nicht um ein schrankenloses Recht wie es bei einigen Grundrech-
ten der Fall ist. Die Europäische Menschenrechtskonvention enthält einige Rechte, die
schrankenlos gelten, aber einige der zentralen Grundrechte der Konvention sind
Schranken unterworfen. Eine Schranke sind z.B. die Rechte Dritter. Der entscheidende Punkt, der heute mehrfach angesprochen wurde, besteht darin,
wie wir mit dem Gleichgewicht umgehen. Dieser Punkt betrifft die nationale Ebene
ebenso wie die Gemeinschaftsebene. Nach der bisherigen Diskussion habe ich den Ein-
druck, dass der Gerichtshof etwas zu hart und unangemessen für Dinge getadelt wird,
die nicht wirklich Probleme des Gerichts sind. Das Problem ist ein anderes. Es besteht
darin, dass wir innerhalb der Europäischen Union, innerhalb des Binnenmarktes, sehr
viele verschiedene Sozialstandards haben. Daher ist die Wahrung des Gleichgewichts
schwieriger geworden. Wir müssen die Prinzipien des Binnenmarktes mit dem Wunsch
nach einem guten Sozialstandard für jedermann in Einklang bringen. Möller: Podiums- und Plenumsdiskussion Denken Sie darüber nach, was passieren würde, wenn diese Bedingun-
gen in allen anderen Bereichen eingeführt würden. Das ist das Problem. Der letzte Punkt: Wir haben verschiedene Modelle von Tarifverhandlungen, Gesetz-
gebung, Arbeitsmarktpolitiken etc. Wir können diese nicht alle harmonisieren, aber sie
sollten gleich behandelt werden, wenn die Art von Regelungen eingeführt wird, wie wir
sie in der Entsenderichtlinie haben. Das ist der wesentliche Inhalt meines Berichts. Möller: Möller: Herzlichen Dank. Frau Koskelo, würden oder hätten in Ihrem Land aufgrund der La-
val, Rüffert oder Viking-Rechtsprechung auch 60 bis 70 % gegen den Lissaboner Ver- 130 Podiums- und Plenumsdiskussion trag gestimmt? Gibt es in Ihrem Land einen Konflikt zwischen nationalem Verfassungs-
recht und europäischem Recht? Koskelo: Koskelo: Koskelo: Sicherlich können wir eine solche Möglichkeit nicht ausschließen, aber – wie ich be-
reits gesagt habe – ist es bei uns aktuell noch kein Thema. Die eigentlichen Probleme
liegen nach meiner Ansicht im Umgang mit dem Gleichgewicht. Dann kann es zu dem
Problem kommen, dass wir anders mit diesem Gleichgewicht umgehen als der EuGH. Dieses Problem existiert bislang nur theoretisch. Möller: Herr Dreesen, was halten Sie von den heute angesprochenen Entscheidungen, die ja
doch sehr unterschiedlich aufgenommen werden? Halten Sie diese Entscheidungen für
reinen Protektionismus, wie Herr Wolf andeutete? Dreesen: Ich teile die Meinung von Herrn Wolf. Es ist aber möglich, eine lange und fruchtbare
Verbindung zum Binnenmarkt zu haben. Wie andere Teilnehmer möchte auch ich Fra-
gen stellen. Ist es fair, dass eine in London ansässige Gewerkschaft Arbeitsbedingungen
für Arbeitnehmer aus Estland regelt, die auf einer finnischen Fähre arbeiten? Ist es fair,
dass die Europäische Union ein finnisches Unternehmen daran hindern kann, nach Est-
land zu gehen? Ist es fair, dass schwedische Arbeitnehmer lettische Arbeitnehmer daran
hindern können, eine Arbeit in Schweden zu finden? Möglicherweise ist ihr Lohn in
Schweden höher als in Lettland. Ist es fair, dass öffentliche Arbeitgeber mehr bezahlen
als der Markt fordert? Ist es fair gegenüber deutschen Arbeitnehmern? Ist es fair gegen-
über dem Steuerzahler? Ich bin der Ansicht, dass es unter der Prämisse der Fairness sehr
schwierig ist, das Gleichgewicht zwischen den verschiedenen Prinzipien herzustellen. Möller: Sie haben die sozialen Unterschiede in Europa angesprochen. Meine Frage zielt auf
die deutschen Verhältnisse. Ich glaube, Professor Däubler hat bereits gesagt, dass das
deutsche Bundesverfassungsgericht sich vorbehält, selbst zu entscheiden, wenn Grund-
rechte der Deutschen durch Europäisches Recht tangiert werden. Bisher hat das Bun-
desverfassungsgericht immer gesagt, der Europäische Gerichtshof hat diese Aufgabe in
unserem Sinne übernommen. Unter Umständen kann es aber zu einem Konflikt zwi-
schen dem Europäischen Gerichtshof und dem Bundesverfassungsgericht kommen. Mir
ist in letzter Zeit aufgefallen, dass zahlreiche Delegierte von Verfassungsgerichten aus
neuen Mitgliedstaaten unser Bundesverfassungsgericht besuchen, um sich über seine
Arbeitsweise und Rechtsprechung zu informieren. Gibt es eine vergleichbare Tendenz
auch bei Ihnen in Finnland? 131 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
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– Möller: Kommen wir zu Herrn Combrexelle. Die Ratspräsidentschaft läuft auf Sie zu. Wer-
den die Franzosen auch an die Fairness, an die deutschen und auch an die französischen
Steuerzahler denken? Wird man an die Stärkung der sozialen Rechte denken? Combrexelle: Combrexelle: Sicher wird es ein Überdenken der Gerichtsurteile geben. Ich würde einfach fragen,
was war der Auftrag des Binnenmarktes? Es geht darum, zu klären, wie der Binnen-
markt mit der Idee einer qualitativen Beschäftigung vereinbar ist. Und bei dieser Frage
gibt es offene Punkte. Jeder ist für seine Antwort verantwortlich, die er geben möchte. Der Gerichtshof und die Richter haben große Freiheiten geschaffen. Und nun geht es
um die sehr sensiblen Fragen des Arbeitsrechts. Mein Gefühl ist, dass die Rechtspre-
chung des Gerichtshofs zu Fragen des Arbeitsrechts noch nicht völlig unausgewogen
ist. Die heutigen Debatten haben jedenfalls gezeigt, dass es noch Bedarf an Gesprächen
zwischen den EuGH-Richtern, den nationalen Richtern und den Gerichten gibt. Es ist
wichtig, dass man in diesen Dialogen eine klare Vorstellung über die richterliche Ant-
wort gewinnt zu der Frage der Vereinbarkeit des Binnenmarktes mit den sozialen As-
pekten, mit dem Beschäftigungsschutz und mit der Sicherung qualitativer Arbeitsplätze. 132 Podiums- und Plenumsdiskussion Die Antwort darf nicht einseitig juristisch ausfallen, sondern sie muss auch politisch
sein. Es wurde heute bei den vielen Debatten deutlich, dass das Thema des kollektiven
Arbeitsrechts einen untergeordneten Rang einnimmt. Sie wissen, Frankreich war schon
immer sehr mit der Frage des Kollektivvertragsrechts verbunden. Ich sage voraus, dass
die französische Präsidentschaft ebenso fühlt und natürlich auch die Minister, die sehr
aufmerksam die Positionen der Mitgliedstaaten verfolgen. Dies gilt besonders für dieje-
nigen Staaten, die von dieser Rechtsprechung unmittelbar betroffen sind. Ich glaube,
dass die Sorge Frankreichs dazu beigetragen hat, zuzuhören, zu sehen und einen Befund
über die Konsequenzen der Rechtsprechung des Gerichtshofs für die verschiedenen na-
tionalen Gesetzgeber zu verfassen. Diese Fragen werden auf der Tagesordnung der
nächsten Sitzung des Ministerrats stehen, so dass man einen klaren Bericht darüber hat
und erkennen kann, was man gemacht hat. Noch einen letzten Punkt möchte ich erwähnen und die Gelegenheit nutzen, da so
viele universitäre Rechtsgelehrte anwesend sind. Ich habe wahrgenommen, dass von
ihnen die Aspekte des kollektiven Arbeitsrechts verteidigt werden. Die Universität ist
der Ort, an dem man sich mit dem Gleichschritt im kollektiven Arbeitsrecht beschäftigt. Gleichzeitig versuchen die Mitgliedsstaaten und die Kommission, das Arbeitsrecht wei-
ter zu entwickeln für den Fall, dass man sich auf die „Flexicurity“ berufen kann. Ich bin
der Meinung, das Gefühl der Verantwortung sollte auch die Universitäten erfassen, da-
mit sie ihren Einsatz zur Weiterentwicklung des Arbeitsrechts im Schoße der europäi-
schen Union verstehen und auch verstehen, was „Flexicurity“ bedeutet. Combrexelle: Wenn man des-
halb in gewisser Weise von der Qualität der Beschäftigung spricht, dann in Verbindung
mit den Urteilen Laval und Viking, aber auch in Verbindung mit „Flexicurity“. Möller: Möller: Vielen Dank für Ihre Ausführungen. Herr Minister Scholz, ich möchte Sie doch noch
nicht aus der Verantwortung entlassen und stelle meine Frage daher ein weiteres Mal. Ist man in Ihrem Ministerium nicht schon einen Schritt weiter als nur zu sagen, wir tra-
gen Verantwortung? Stichwort Mindestlöhne: Gibt es Überlegungen, die gegenwärtige
politische Auseinandersetzung auch auf die europäische Ebene zu übertragen? Wie se-
hen die Pläne des Ministeriums für die nächsten Jahre, sagen wir mal bis 2009 aus? Möller: Dann darf ich aber doch noch eine Frage zum Rüffert-Urteil nachschieben. Bei dieser
Entscheidung haben die deutsche, die belgische, die dänische und die französische Re-
gierung sowie Irland, Zypern, Österreich, Finnland, Norwegen und Polen Erklärungen
abgegeben. Bis auf Polen haben alle gesagt, dass eine Tarifklausel möglich ist. Haben
diese Erklärungen die EuGH-Richter überhaupt nicht beeindruckt? Levits: Ich habe bereits erwähnt, dass im Urteil nicht alle Erwägungen in Erscheinung treten. Mehr kann ich als Richter dazu leider nicht sagen. Scholz: Scholz: In Deutschland sind die Entscheidungen getroffen. Wir haben uns in der Regierung
darauf verständigt, dass wir in Deutschland mehr Mindestlöhne einführen und dass wir
das auf der Basis zweier schon vorhandener Gesetze tun. Das erste Gesetz betrifft das
deutsche Entsendegesetz, das wir weiter entwickeln werden und das auch schon bisher
die Grundlage für mehrere Tarifbereiche bildet, in denen wir Mindestlöhne festsetzen. Dies gilt im Wesentlichen für den Baubereich, die Gebäudereiniger und die Briefdienst-
leistungen. Wir werden das in diesem Jahr noch gesetzgeberisch für weitere Branchen
ergänzen, die sich bei uns gemeldet haben und mit denen wir uns politisch verständigt
haben. Voraussetzung soll sein, dass mehr als 50 % der Arbeitnehmer bei tarifgebunde- 133 Podiums- und Plenumsdiskussion nen Arbeitgebern beschäftigt werden. Darüber hinaus existiert das Mindestarbeitsbedin-
gungsgesetz von 1952. In diesem Gesetz wollen wir Mindestregelungen festsetzen für
Beschäftigungsbereiche, in denen die Beschäftigten durch keine Gewerkschaft und kei-
nen Arbeitgeberverband vor Ausbeutung beschützt werden können. Diese Vorhaben
bewegen sich im Rahmen des Europäischen Rechts. Selbstverständlich gelten die Rege-
lungen für In- und Ausländer gleichermaßen. Für Europa planen wir keine eigenständige Mindestlohninitiative, da es sich hierbei
um nationales Recht handelt und die Lohnniveaus in den einzelnen Ländern sehr unter-
schiedlich sind, so dass eine gemeinsame Regelung noch lange nicht möglich sein wird. Wir vergleichen uns zwar gerne mit den Vereinigten Staaten von Amerika, in denen es
einen gesetzlichen Mindestlohn von $ 7,20 gibt, aber das für Europa allgemein vorzu-
schreiben, würde sicherlich manchen Mitgliedstaat noch überfordern. Von daher steht
das nicht auf der Tagesordnung. Ein Satz noch zur Verknüpfung der europäischen und der nationalen Debatte. Wenn
wir das Rüffert-Urteil bisher richtig verstanden haben, dann bedeutet es, dass allge-
meinverbindliche Tarifverträge als Grundlage für Entscheidungen bei der Vergabe von
Aufträgen genommen werden können. Das beinhaltet in jedem Fall die Zulässigkeit von
allgemein gesetzlichen Mindestlöhnen, so dass es eine Lösung gibt, die der nationale
Gesetzgeber, wenn er sich dazu durchringen kann, in der eigenen Hand hat. Das macht
uns zwar nicht weniger skeptisch gegenüber der ergangenen Entscheidung, aber es ist
jedenfalls ein Argument, das wir auch ernst nehmen müssen und das wir auch ernst
nehmen. Möller: Herr Levits, können Sie die Interpretation durch Minister Scholz bestätigen Levits: Ich bitte um Verständnis, dass ich die Urteile nicht interpretiere. Podiums- und Plenumsdiskussion Podiums- und Plenumsdiskussion Dreesen: Aus dänischer Sicht bin ich der Auffassung, dass wir eine Lösung gefunden haben. Die europäische Wirtschaft wird es in der hier diskutierten Weise wohl nicht unterstüt-
zen. Wir werden abwarten, was passiert. Möller: Möller: Herr Minister Scholz, es gibt verschiedene Möglichkeiten auf Europa, auf den EuGH
einzuwirken. Deshalb eine Frage zur Wahl der Richter. In Deutschland erfolgt die Wahl
nach Parteiproporz. Nach sechs Jahren kommt der nächste Richter oder die nächste
Richterin, obwohl in so kurzer Zeit noch nicht einmal eine richtige Einarbeitung mög-
lich ist. Daher habe ich den Eindruck, dass deutsche Richter nie Einfluss auf die Recht-
sprechung des Europäischen Gerichtshofs ausüben können. In der kurzen Zeit können
sie nicht einmal Kammerpräsident werden. Bei anderen Ländern, wie etwa Spanien und
Griechenland, sieht es anders aus. Warum verwendet die deutsche Regierung, die deut-
sche Politik so wenig Mühe auf die Auswahl deutscher Richter am Europäischen Ge-
richtshof und warum endet das so häufig im Parteienstreit? Scholz: Möller: Möller: Dafür haben wir Verständnis. Herr Dreesen, Minister Scholz hat angedeutet, dass wir
die Entsenderichtlinie verbessern müssen. Das hat auch die Diskussion ergeben. Wie
stehen die Arbeitgeber in Europa dazu? 134 Scholz: Scholz: Erstens, ich habe auch gehört, dass es diese Kritik gibt. Zweitens, das Auswahlver-
fahren ist seriös und findet ordnungsgemäß statt. Und drittens, dass Parteien sich an den
Diskussionen beteiligen, ist kein Parteienstreit, sondern Demokratie. Möller: Frau Koskelo, was halten Sie von der jetzigen Wahl der Richter und Richterinnen
zum Europäischen Gerichtshof? Koskelo: Koskelo: Jeder Mitgliedstaat entscheidet selbst, in welchem Verfahren er seine Richter wählt. In Finnland hatten wir eine Diskussion darüber. Es wurde vorgeschlagen, das Auswahl-
verfahren breiter und transparenter zu gestalten. Die Regierung hat darauf noch nicht
reagiert. Aber ich möchte betonen, dass jeder EuGH-Richter seine Loyalität nicht den
Mitgliedstaaten, nicht einem einzigen Staat und auch nicht seinem Mitgliedstaat schul-
det, sondern nur den europäischen Verträgen. Wie Richter Levits bereits angedeutet hat,
ist der gesamteuropäische Blick entscheidend. Wenn wir uns die Rechtsprechung des
EuGH anschauen, dann wird leicht erkennbar, dass sie sehr durch die vorangegangene
Rechtsprechung geprägt ist. Das hat auch unsere Diskussion ergeben. Die Rechtspre-
chung steht nicht für eine radikale Periode des EuGH, sondern ist vielmehr als eine kon-
tinuierliche Entwicklung zu verstehen. Am Anfang standen die Grundfreiheiten, die
wirtschaftlichen Freiheiten. Die wichtigste Entwicklung in der Rechtsprechung des
EuGH ist in erster Linie die Tatsache, dass soziale Aspekte, die soziale Dimension und
Grundrechte eine immer wichtigere Rolle spielen. Die heutigen Konsequenzen dieser
kontinuierlichen Entwicklung sind auf eine veränderte Wirklichkeit zurückzuführen. 135 Podiums- und Plenumsdiskussion Darüber hinaus muss das Gericht Entscheidungen treffen, obwohl Europa letztlich in
den Händen der Mitgliedstaaten liegt. In der Hand des EuGH liegen sie nur dann,
wenn die Mitgliedstaaten die Vorgaben nicht eingehalten haben Darüber hinaus muss das Gericht Entscheidungen treffen, obwohl Europa letztlich in
den Händen der Mitgliedstaaten liegt. In der Hand des EuGH liegen sie nur dann, g
g
g
wenn die Mitgliedstaaten die Vorgaben nicht eingehalten haben. Möller: Da der europäische Gerichtshof auch bei dieser Diskussion im Mittelpunkt steht,
möchte ich Ihnen, Herr Levits, als EuGH-Richter, das Schlusswort geben. Zuvor möch-
te ich aber gerne noch die etwas saloppe Frage anschließen: Warum sind Urteile des
Europäischen Gerichtshof eigentlich nur zu verstehen, wenn man vorher die Schlussan-
träge der Generalanwälte liest? Levits: Die Generalanwälte haben ihre eigene Sicht der Dinge und sind insoweit wissen-
schaftlich und rechtlich viel freier. Sie können verschiedene Aspekte in ihre Überlegun-
gen einbeziehen und sich auch auf Literaturmeinungen stützen. Das Gericht kann sich
dagegen nicht auf Literatur stützen, denn welche sollen wir zitieren? II. Plenumsdiskussion Im Rahmen der nun folgenden Plenumsdiskussion wurde an Herrn Levits die Frage
gestellt, ob es tatsächlich nur Zufall sei bzw. von der prozessualen Situation abhänge,
dass die Grundrechte als Rechtfertigungsgrund herangezogen würden. Es könne doch
auch damit zusammenhängen, dass der EuGH als Gerichtshof der Europäischen Ge-
meinschaft von den Marktfreiheiten ausgehe und dann entscheide, inwieweit der einzel-
ne Staat die Grundfreiheiten beschneiden könne. Levits erklärte, dass dies in der Tat von der vorgelegten Fragestellung abhänge und
daher von Fall zu Fall unterschiedlich sei. Abstrakt gedacht, stünden die Grundrechte
grundsätzlich auf der gleichen Stufe wie die Grundfreiheiten. Die Grundrechte seien
zwar nicht geschrieben, aber im Rahmen allgemeiner Rechtsgrundsätze anerkannt wor-
den. Das habe der Gerichtshof auch mehrfach betont. Es wäre allerdings viel besser
gewesen, wenn die Charta in Kraft gewesen wäre. Nach Ablehnung der Verfassung ha-
be sich der Gerichtshof mit der Zitierung der Grundrechtscharta bewusst zurückgehal-
ten, um nicht in den laufenden politischen Prozess einzugreifen. Kurz vor dem irischen
Referendum habe man Mut gefasst und sie in einem Fall beiläufig zitiert, wohl in der
Erwartung, dass der Lissaboner Vertrag zustande komme. Wenn die Fragestellung es
erfordere, müsse der EuGH seine Pflicht tun. Die Tendenz gehe aber stets dahin, die
Sache zurück an die nationalen Gerichte zu verweisen. Von Zuhörerseite wurde angemerkt, dass die Politik auf das Gemeinschaftsrecht auf
der europäischen Ebene nicht in derselben Weise einwirken könne wie im nationalen
Bereich, wenn sie das Recht ändere. Wenn der EuGH die Ansicht vertrete, dass das
Gemeinschaftsrecht, das Primärrecht nun gemäß „a“ und nicht mehr gemäß „b“ zu beur-
teilen sei, dann müssten alle 27 Mitgliedstaaten zustimmen, um wieder von „a“ zu „b“
zurückzukehren. Auf nationaler Ebene genüge dagegen die einfache Mehrheit und im
nationalen Verfassungsrecht eine 2/3-Mehrheit. Das sei ein ganz großer Unterschied. Daher wurde die Frage gestellt, ob nicht dieser Unterschied dazu führen sollte, dass der
EuGH bei der Auslegung des Rechts zurückhalternder als nationale Gerichte vorgeht. Seine Rechtsprechung erwecke aber den Eindruck, dass er großzügiger und vorantrei-
bender als nationale Gerichte mit dem geschriebenen Recht umgehe. Levits war der Ansicht, dass dieser interessante Gesichtspunkt – die europäische Ge-
setzgebung sei viel schwieriger zu ändern – genauer diskutiert werden sollte, denn das
juristische Gerichtsverständnis auf Europaebene sollte ein anderes sein als das eines
nationalen Verfassungsgerichts. In der Literatur sei diese Frage noch nicht genügend
diskutiert worden. Scholz: Die deutschen
Richter würden die deutsche Literatur zitieren, aber die griechische Literatur ist viel-
leicht ganz anderes. Daher ist die Institution der Generalanwälte wichtig, um die
Rechtssache umfassend beleuchten zu können. Das Gericht folgt häufig den General-
anwälten, manchmal aber auch nicht, denn es ist in seiner Entscheidung frei. Ich möchte noch auf die Frage eingehen, wie man den Gerichtshof beeinflussen
kann. Durch den Richter ist es sicherlich nicht möglich, denn er ist kein Agent des Mit-
gliedstaates, sondern er ist dem Gesamteuropäischen, der Union verpflichtet und nicht
dem Mitgliedstaat. Den Gerichtshof kann man vielmehr – wie mein Sitznachbar schon
richtig angemerkt hat – durch eine entsprechende Gesetzgebungsinitiative beeinflussen,
da das Gericht im Rahmen der juristischen Methodik an die Gemeinschaftsgesetzge-
bung gebunden ist. Das Recht liegt in den Händen der Politik, in den Händen der Mit-
gliedstaaten, die das Recht setzen. Dies kann geschehen, wenn sich die Politik z.B. in
Folge dieser Diskussion aktiv wird und auf europäischer Ebene eine Gesetzgebungsini-
tiative einbringt, um das Anliegen, das hier vorgetragen wurde, in Recht umzusetzen. Dann wird auch das Gericht diesem Recht gemäß der juristischen Methode Beachtung
schenken. Dieser politische Weg über die Gesetzgebung ist die richtige Art, den Ge-
richtshof zu beeinflussen. Möller: Herzlichen Dank. Eine klare Aufforderung an die Politik, die Sie zum Schluss for-
muliert haben. Meine Damen und Herren, ich möchte Ihnen nun Gelegenheit geben,
Fragen an das Podium zu richten. 136 Podiums- und Plenumsdiskussion Podiums- und Plenumsdiskussion II. Plenumsdiskussion Letztlich würde es bedeuten, dass der Gerichtshof politischer urteilen
sollte und dagegen würden wieder andere ihre Stimmen erheben. Scholz war ebenfalls der Ansicht, dass die Entscheidungsprozesse der europäischen
Institutionen mühsamer seien als auf nationaler Ebene. Daraus müssten Konsequenzen
gezogen werden, weil es sonst zu riesigen Legitimationskrisen kommen könne. Er habe 137 Podiums- und Plenumsdiskussion schon seit langem folgenden Eindruck: Würde man die Bevölkerung Europas jeweils
national fragen, ob sie der Meinung sind, dass Europa bedeute, dass in ihrem eigenen
Land soziale Rechte abgebaut werden könnten, würde es 27mal ein „Ja“ geben. Dies
gelte auch für Länder, die aus der Perspektive eines deutschen Ministers den Eindruck
erweckten, dass sie nur von Europa profitieren können. Dennoch existiere dieses
Selbstverständnis mehrheitlich. Die sehr unterschiedlichen Arbeitsrechtskulturen, Sozi-
albeziehungskulturen würden für etwas besonders Gutes gehalten. Daher werde davon
ausgegangen, es könne nur schlechter werden. Gleichzeitig sei der institutionelle Pro-
zess besonders mühselig. Es sei ähnlich wie beim Bundesrat. Ein Ministerpräsident
werde nicht wegen seines Abstimmungsverhaltens in den Bundesrat entsandt, sondern
für das, was er als Ministerpräsident in der Tätigkeit als Regierungschef eines Landes
vorschlage. So ähnlich sei es auch auf der deutschen Gesamtebene. Es gebe keine euro-
päische Öffentlichkeit, die wirklich durchdringe. Es gebe keinen Diskussionsprozess,
der bei den Bürgern das Gefühl hinterlasse, zu wissen, was getan werden könne, wenn
man der Rechtsprechung des EuGH entgegenwirken möchte. Keiner könne genau sa-
gen, was zu tun sei und wie lange es dauern werde. Dies müsse das Gericht bedenken,
damit es mit seinen Entscheidungen keine Legitimationskrisen auslöse. Solche Legiti-
mationskrisen würden ansonsten bewirken, dass die Unvollständigkeit des demokrati-
schen Willensprozesses auf europäischer Ebene, seine Kompliziertheit, Langsamkeit
und die vielfältigen Verästelungen dazu führten, dass der Gesetzgeber in vielen Fällen
ausfalle, in denen der Bürger erwarte, dass er eine Regelung darüber treffe, was die
Bürger als Mindestbestand erwarteten und was sie vom EuGH abgesichert wissen wol-
len. Der EuGH solle keine Politik betreiben und die europäische Integration Europas
nicht nach eigenem Gusto herstellen, aber er müsse sich der möglichen Schwierigkeiten
bewusst sein. Das sei vielleicht ein Ergebnis der Diskussion. Und wenn Levits zum
Ausdruck bringe, dass dies ein interessanter Gedanke sei, dann sei dies Grund zur Freu-
de. An Hoffmann und Scholz wurde eine Frage zur Bewertung des sogenannten Kom-
promisses zum Arbeitszeitrecht gestellt. Dort stelle sich die Situation nämlich umge-
kehrt dar. Der EuGH habe mit seiner Entscheidung eine neue Rechtssetzung bewirkt. II. Plenumsdiskussion Wie ist der jetzige Tatbestand zu bewerten? Scholz erläuterte, dass der EuGH eine Richtlinie anders als der Richtliniengeber aus-
gelegt habe. Der EuGH habe die Meinung vertreten, dass Bereitschaftszeit im deutschen
Sprachgebrauch Arbeitszeit sei, die bezahlt werden müsse. Einige Mitgliedstaaten (wie
z.B. die Bundesrepublik Deutschland sowie Spanien und Polen) hätten dies umgesetzt. Alle Mitgliedstaaten seien davon bedroht, dass die Kommission sie im Hinblick auf die
Rechtsprechung des EuGH verklagt und sie auffordert, diese Richtlinie so einzuhalten,
wie sie der EuGH interpretiert hat. In diesem Fall sei zu erwarten, dass die Mitgliedstaa-
ten unterliegen. Daher gebe es eine Mehrheit im Rat dafür, die Richtlinie abzuändern. Insoweit habe der demokratische Willensbildungsprozess funktioniert, auch wenn mög-
licherweise falsch herum. Es stehe also fest, dass es im Europäischen Rat eine Mehrheit 138 Podiums- und Plenumsdiskussion dafür geben werde, die Richtlinie so zu ändern, dass jedenfalls ein Teil der Bereit-
schaftszeit in den Mitgliedstaaten als unbezahlte Arbeitszeit bewertet werden könne. Aus der Sicht eines deutschen Arbeitsministers bedeute dies, dass jedenfalls sicherzu-
stellen sei, dass der Fortschritt, der hierzulande erreicht worden sei, festgehalten werden
könne. Deshalb habe er sich als Minister bei dem Kompromiss in Luxemburg bemüht,
eine Regelung zu finden, die sagt, dass dies durch Tarifverträge geregelt werden könne. Dies sei gelungen. Daher seien Tarifverträge weiter zulässig, die die deutschen Gewerk-
schaften mit den Arbeitgebern (z.B. im Krankenhausbereich) geschlossen hätten. Solan-
ge er Arbeitsminister sei, werde es keine Gesetzgebung geben, die in Deutschland hinter
den erreichten Stand zurückfalle. Bereitschaftszeit werde in Deutschland bezahlte Ar-
beitszeit bleiben. Damit dies durchgesetzt werden könne, sei die Richtlinie notwendig,
so wie sie jetzt geplant sei. Bei der Mehrheit der Mitgliedstaaten stehe jetzt schon fest,
dass sie sich von der Rechtsprechung des Europäischen Gerichtshofs distanzierten, in-
dem sie erlaubten, was dieser verboten habe. Hoffmann erläuterte, dass dies für Deutschland der Fall sein möge, für Europa sei
diese Arbeitszeitrichtlinie eine Katastrophe. Er zeigte sich empört darüber, dass die
Mitgliedstaaten nicht aufgefordert werden, das bestehende europäische Arbeitszeitge-
setz umzusetzen, wenn der Europäische Gerichtshof aus Sicht des Gewerkschaftsbundes
eine richtige und progressive Rechtsprechung vornehme. Das Arbeitszeitgesetz werde
nicht an die Rechtsprechung angepasst, sondern versucht, eine andere Rechtsgrundlage
zu schaffen. Wenn dieses umgekehrte Vorgehen öffentlich diskutiert würde, würden die
Bürger und Bürgerinnen Europa nicht verstehen. Wenn Arbeiter bei Ford am Band auf
einmal die Bänder für drei Stunden still legten, dann handle es sich um inaktive Ar-
beitszeit, die nicht bezahlt werde. Simone Gräfin von Hardenberg * Professor Brian Bercusson verstarb am 17. August 2008. II. Plenumsdiskussion Das könne nicht im Sinne des Gesetzgebers gewesen
sein. Die Lösung sei für Europa ungeeignet. Dieser Weg habe auch wenig mit Demo-
kratie zu tun. Daher hoffe er, über das Europäische Parlament mehr Öffentlichkeit
schaffen zu können, möglicherweise auch für Korrekturen, die dann dem Arbeitszeitge-
setz von seinem Ursprung her gerecht werden. Andersson stellte klar, dass im Europäischen Parlament über die Arbeitszeit disku-
tiert werden wird. Die Kommission habe auf die geänderte Rechtsprechung zur Arbeits-
zeit reagiert. Wenn jetzt keine Reaktion erfolge, dann würde dies bedeuten, dass es ak-
zeptiert werde. Man müsse reagieren und hätte letztlich schon viel früher reagieren
müssen. In seinem Schlusswort betonte Scholz, dass Europa eine europäische und auch eine
juristische Öffentlichkeit brauche. Die Probleme sollten erörtert werden, wie sie sind,
wie sie sich bewegen und wie sie sich weiterentwickeln sollen. Die Referate, Diskussi-
onen und die Äußerungen des Publikums hätten wesentlich zu einer weiteren Mei-
nungsbildung beigetragen, aus denen nun Schlüsse zu ziehen seien. 139 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb 783845219165, am 24.10.2024, 06:46:57
- https://www.nomos-elibrary.de/agb Autorenverzeichnis ANDERSSON, Jan, Mitglied des Europäischen Parlaments, Sozialdemokratische Frak-
tion, Stuvaregatan 11, SE-252 67 Helsingborg, Schweden BERCUSSON†*, Brian, Professor, Chair in European Social and Labour Law at King’s
College London, Strand, London WC2R 2LS, England, Vereinigtes Königreich BRUUN, Niklas, Professor in Private Law, University of Helsinki, Director IPRUniver-
sity Center, P.O. Box 479, 00100 Helsinki, Finnland DÄUBLER, Wolfgang, Prof. Dr., Professor für Deutsches und Europäisches Arbeits-
recht, Bürgerliches Recht und Wirtschaftsrecht an der Universität Bremen, Fachbe-
reich Rechtswissenschaft, 28353 Bremen GELLNER, Lars, Head of Labour Law at the Confederation of Swedish Enterprise,
Storgatan 19, SE-114 82 Stockholm, Schweden JONSSON, Claes-Mikael, Legal adviser at the Swedish Trade Union Confederation
(LO-Sweden), Landesorganisationen i Sverige, 105 53 Stockholm KOCH, Ulrich, Richter am Bundesarbeitsgericht, Hugo-Preuß-Platz 1, 99084 Erfurt KOKOTT, Juliane, Prof. Dr., Gerichtshof der Europäischen Gemeinschaften, Cabinet
Kokott, Th.M. 2214, L-2925 Luxembourg, Luxemburg LEVITS, Egils, Dr.h.c., Dipl.Pol., Richter am Europäischen Gerichtshof, 47 Rue Nico-
las Martha, L-2133 Luxembourg, Luxemburg LYON-CAEN, Antoine, Prof. Dr. à l’Université Paris Ouest, Directeur d’études à
l’EHESS – Nanterre La Défense Institut International pour les Etudes Comparati-
ves, 4, rue de la Michodière, 75002 Paris, Frankreich 141 NIELEBOCK, Helga, Bereichsleiterin, Deutscher Gewerkschaftsbund, Henriette-Herz-
Platz 2, 10178 Berlin REBHAHN, Robert, Univ. Prof. Dr., Professor für Arbeits- und Sozialrecht an der Uni-
versität Wien, Schenkenstraße 8-10, 1010 Wien, Österreich SILVA, Armindo, EMPL/F – Social dialogue, social rights, working conditions, adap-
tion to change DG Employment, Social Affairs and Equal Opportunities, European
Commission, B-1049 Bruxelles, Belgien WOLF, Roland, Geschäftsführer Arbeitsrecht, Bundesvereinigung der Deutschen Ar-
beitgeberverbände, Breite Straße 29, 10178 Berlin 142 ANDERSSON, Jan, siehe Autorenverzeichnis ANDERSSON, Jan, siehe Autorenverzeichnis BECKER, Ulrich, Prof. Dr., LL.M. (EHI), Direktor des Max-Planck-Instituts für aus-
ländisches und internationales Sozialrecht, Honorarprofessor an der Juristischen
Fakultät der Ludwig-Maximilians-Universität München, Amalienstr. 33, 80799
München BERCUSSON, Brian, siehe Autorenverzeichnis BERCUSSON, Brian, siehe Autorenverzeichnis COMBREXELLE, Jean-Denis, Directeur des Relations du Travail, Ministère de
l’Emploi, du Travail et de la Cohésion sociale, 39-43, quai André Citroën, 75902
Paris cedex 15, Frankreich DREESEN, Fleming, Businesseurope und Direktor für Arbeitsrecht bei der Vereinigung
dänischer Arbeitgeber, Dansk Arbeijdsgiverforening, Vester Voldgrade 113, 1790
København V, Dänemark HOFFMANN, Reiner, stellvertretender Generalsekretär des DGB, Deutscher Gewerk-
schaftsbund, Henriette-Herz-Platz 2, 10178 Berlin KOBERSKI, Wolfgang, Ministerialdirigent, Leiter der Abteilung Europäische Union,
Europäische Beschäftigungs- und Sozialpolitik, Bundesministerium für Arbeit und
Soziales, Wilhelmstr. 49, 10117 Berlin KOSKELO, Pauliine, Präsidentin des Obersten Gerichts Finnlands, PL 301, 00171 Hel-
sinki, Finnland KREPPEL, Horstpeter, Richter am Gericht für den öffentlichen Dienst der Europäi-
schen Union, L-2925 Luxembourg, Luxemburg 143 LEVITS, Egils, siehe Autorenverzeichnis
LYON-CAEN, Antoine, siehe Autorenverzeichnis
MÖLLER, Karl-Dieter, Leiter der ARD-Fernsehredaktion Recht und Justiz, SWR Fern-
sehen, Redaktion ARD-Ratgeber Recht, 76037 Karlsruhe
PEUTL, Christa, Dr., Bundeskanzleramt, Ballhausplatz 2, 1014 Wien, Österreich
REBHAHN, Robert, siehe Autorenverzeichnis
SILVA, Armindo, siehe Autorenverzeichnis
SCHOLZ, Olaf, Bundesminister für Arbeit und Soziales, Bundesministerium für Arbeit
und Soziales, Wilhelmstr. 49, 10117 Berlin
SCHUMACHER-HILDEBRANDT, Christoph, Ministerialdirigent, Leiter der Unterab-
teilung Europäische Union, Europäische Beschäftigungs- und Sozialpolitik, Bun-
desministerium für Arbeit und Soziales, Wilhelmstr. 49, 10117 Berlin LEVITS, Egils, siehe Autorenverzeichnis REBHAHN, Robert, siehe Autorenverzeichnis SCHOLZ, Olaf, Bundesminister für Arbeit und Soziales, Bundesministerium für Arbeit
und Soziales, Wilhelmstr. 49, 10117 Berlin SCHOLZ, Olaf, Bundesminister für Arbeit und Soziales, Bundesministerium für Arbeit
und Soziales, Wilhelmstr. 49, 10117 Berlin SCHUMACHER-HILDEBRANDT, Christoph, Ministerialdirigent, Leiter der Unterab-
teilung Europäische Union, Europäische Beschäftigungs- und Sozialpolitik, Bun-
desministerium für Arbeit und Soziales, Wilhelmstr. 49, 10117 Berlin SCHUMACHER-HILDEBRANDT, Christoph, Ministerialdirigent, Leiter der Unterab-
teilung Europäische Union, Europäische Beschäftigungs- und Sozialpolitik, Bun-
desministerium für Arbeit und Soziales, Wilhelmstr. 49, 10117 Berlin SCHUMACHER-HILDEBRANDT, Christoph, Ministerialdirigent, Leiter der Unterab-
teilung Europäische Union, Europäische Beschäftigungs- und Sozialpolitik, Bun-
desministerium für Arbeit und Soziales, Wilhelmstr. 49, 10117 Berlin 144 1 ABl. C 364 vom 18.12.2000, S. 1.
2 ABl. L 18 vom 21.1.1997, S. 1.
3 ABl. L 134 vom 30.4.2004, S. 114. Entschließung des Europäischen Parlaments
vom 22. Oktober 2008 zu den
Herausforderungen für Tarifverträge in der EU
(2008/2085(INI)) Das Europäische Parlament , 1 ABl. C 364 vom 18.12.2000, S. 1. 3 ABl. L 134 vom 30.4.2004, S. 114. 2 ABl. L 18 vom 21.1.1997, S. 1. Das Europäische Parlament , Hinweis auf Artikel 2, insbesondere Spiegelstrich 1, und Artikel 3 Buch- − unter Hinweis auf Artikel 2, insbesondere Spiegelstrich 1, und Artikel 3 Buch-
stabe j des Vertrags über die Europäische Union, − unter Hinweis auf Artikel 2, insbesondere Spiegelstrich 1, und Artikel 3 Buch
stabe j des Vertrags über die Europäische Union, − unter Hinweis auf die Artikel 136, 137, 138, 139 und 140 des EG-Vertrags, − unter Hinweis auf die Artikel 12, 39 und 49 des EG-Vertrags, − unter Hinweis auf den Vertrag über die Europäische Union in der Fassung des
Vertrags von Lissabon vom 13. Dezember 2007, insbesondere seinen Artikel 3, Hinweis auf den Vertrag über die Europäische Union in der Fassung des
ags von Lissabon vom 13. Dezember 2007, insbesondere seinen Artikel 3, − unter Hinweis auf Artikel 152 des Vertrags über die Arbeitsweise der Europäi-
schen Union, in dem die Bedeutung des sozialen Dialogs und der Tarifverhand-
lungen für die Entwicklung anerkannt wird, − unter Hinweis auf Artikel 27, 28 und 34 der Charta der Grundrechte der Europäi-
schen Union1, − unter Hinweis auf die Europäische Konvention zum Schutze der Menschenrechte
und Grundfreiheiten, insbesondere ihren Artikel 11, − unter Hinweis auf die Europäische Sozialcharta, insbesondere ihre Artikel 5, 6
und 19, − unter Hinweis auf das Europäische Übereinkommen über die Rechtsstellung der
Wanderarbeitnehmer, − unter Hinweis auf die Richtlinie 96/71/EG des Europäischen Parlaments und des
Rates vom 16. Dezember 1996 über die Entsendung von Arbeitnehmern im
Rahmen der Erbringung von Dienstleistungen2 (Entsenderichtlinie), − unter Hinweis auf den Bericht der Dienststellen der Kommission über die Durch-
führung der Richtlinie 96/71/EG (SEK(2006)0439) (Dienstleistungsbericht), − unter Hinweis auf die Richtlinie 2004/18/EG des Europäischen Parlaments und
des Rates vom 31. März 2004 über die Koordinierung der Verfahren zur Vergabe
öffentlicher Bauaufträge, Lieferaufträge und Dienstleistungsaufträge3 (Vergabe-
richtlinie), 145 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb Entschließung des Europäischen Parlaments − unter Hinweis auf die "Monti-Klausel" in der Verordnung (EG) Nr. 2679/98 des
Rates vom 7. Dezember 1998 über das Funktionieren des Binnenmarktes im Zu-
sammenhang mit dem freien Warenverkehr zwischen den Mitgliedstaaten4, − unter Hinweis auf die "Monti-Klausel" in der Verordnung (EG) Nr. 2679/98 des
Rates vom 7. 4 ABl. L 337 vom 12.12.1998, S. 8. 17 ABl. C 313 E vom 20.12.2006, S. 452.
18 ABl. C 92 E vom 16.4.2004, S. 404.
19 ABl. C 102 E vom 24.4.2008, S. 321.
20 Angenommene Texte, P6_TA(2007)0574. Das Europäische Parlament , Oktober 2006 zur Anwendung
der Richtlinie 96/71/EG über die Entsendung von Arbeitnehmern17, − unter Hinweis auf seine Entschließung vom 26. Oktober 2006 zur Anwendung
der Richtlinie 96/71/EG über die Entsendung von Arbeitnehmern17, − unter Hinweis auf seine Entschließung vom 15. Januar 2004 zur Durchführung
der Richtlinie 96/71/EG in den Mitgliedstaaten18, − unter Hinweis auf seine Entschließung vom 15. Januar 2004 zur Durchführung
der Richtlinie 96/71/EG in den Mitgliedstaaten18, − unter Hinweis auf seine Entschließung vom 15. Januar 2004 zur Durchführung
der Richtlinie 96/71/EG in den Mitgliedstaaten18, − unter Hinweis auf seine Entschließung vom 23. Mai 2007 zur Förderung einer
menschenwürdigen Arbeit für alle19, − unter Hinweis auf seine Entschließung vom 23. Mai 2007 zur Förderung einer
menschenwürdigen Arbeit für alle19, − unter Hinweis auf die vom Europäischen Rat auf seiner Tagung vom
12./13. Dezember 2007 angenommenen gemeinsamen Grundsätze beim Flexicu-
rity-Ansatz sowie seine Entschließung vom 29. November 2007 zu gemeinsamen
Grundsätzen für den Flexicurity-Ansatz20, − gestützt auf Artikel 45 seiner Geschäftsordnung, − in Kenntnis des Berichts des Ausschusses für Beschäftigung und soziale Angele-
genheiten sowie der Stellungnahmen des Rechtsausschusses und des Ausschusses
für Binnenmarkt und Verbraucherschutz (A6-0370/2008), A. in der Erwägung, dass der EG-Vertrag die in der Charta der Grundrechte der Eu-
ropäischen Union, in den Verfassungen der Mitgliedstaaten und in diversen internatio-
nalen Verträgen und Übereinkommen verankerten Grundrechte als wichtigste Bezugs-
werte des Gemeinschaftsrechts und der in der Gemeinschaft üblichen Praxis anerkennt, B. in der Erwägung, dass der EG-Vertrag eine Reihe einschlägiger Grundsätze fest-
legt; in der Erwägung, dass zu den wichtigsten Zielen der Gemeinschaft ein Binnen-
markt gehört, der durch die Beseitigung der Hindernisse für den freien Waren-, Perso-
nen-, Dienstleistungs- und Kapitalverkehr zwischen den Mitgliedstaaten gekennzeichnet
ist und eine soziale Dimension hat, C. in der Erwägung, dass einer dieser Grundsätze darin besteht, die verfassungsmä-
ßigen Grundrechte der Bürger anzuerkennen, wozu das Recht, Gewerkschaften zu bil-
den, das Streikrecht und das Recht, Tarifverträge auszuhandeln, gehören, D. in der Erwägung, dass die Freizügigkeit der Arbeitnehmer, die Niederlassungs-
freiheit und der freie Dienstleistungsverkehr fundamentale Prinzipien des Binnenmark-
tes sind, E. in der Erwägung, dass gemäß Artikel 39 des EG-Vertrags die Freizügigkeit der
Arbeitnehmer die Abschaffung jeder auf der Staatsangehörigkeit beruhenden Diskrimi-
nierung der Arbeitnehmer in den Mitgliedstaaten in Bezug auf Beschäftigung, Entloh-
nung und sonstige Arbeits- und Beschäftigungsbedingungen mit sich bringt, F. Das Europäische Parlament , Dezember 1998 über das Funktionieren des Binnenmarktes im Zu-
sammenhang mit dem freien Warenverkehr zwischen den Mitgliedstaaten4, − unter Hinweis auf die Richtlinie 2006/123/EG des Europäischen Parlaments und
des Rates vom 12. Dezember 2006 über Dienstleistungen im Binnenmarkt5, − unter Hinweis auf das Urteil des Gerichtshofs der Europäischen Gemeinschaften
(EuGH) vom 27. März 1990 in der Rechtssache C-113/89, Rush Portuguesa Lda
gegen Office National d'Immigration6, − unter Hinweis auf die Urteile des EuGH vom 9. August 1994 in der Rechtssache
C-43/93, Vander Elst7, 23. November 1999 in den verbundenen Rechtssachen C-
369/96 und C-376/96, Arblade8, 25. Oktober 2001 in den verbundenen Rechtssa-
chen C-49/98, C-50/98, C-52/98, C-54/98, C-68/98 und C-71/98, Finalarte9, 7. Februar 2002 in der Rechtssache C-279/00, Kommission/Italien10, 12. Oktober
2004 in der Rechtssache C-60/03, Wolff & Müller GmbH11, 21. Oktober 2004 in
der Rechtssache C-445/03, Kommission/Luxemburg12, und 19. Januar 2006 in
der Rechtssache C-244/04, Kommission/Deutschland13, − unter Hinweis auf das Urteil des EuGH vom 11. Dezember 2007 in der Rechtssa-
che C-438/05, International Transport Workers' Federation and Finnish Seamen's
Union/Viking Line ABP14 (Rechtssache Viking), − unter Hinweis auf das Urteil des EuGH vom 18. Dezember 2007 in der Rechtssa-
che C-341/05, Laval un Partneri Ltd15, − unter Hinweis auf das Urteil des EuGH vom 3. April 2008 in der Rechtssache C-
346/06, Rüffert16, − unter Hinweis auf die folgenden Übereinkommen der Internationalen Arbeitsor-
ganisation (ILO): Übereinkommen Nr. 94 Arbeitsklauseln (Öffentliche Verträge),
Nr. 87 Vereinigungsfreiheit und Schutz des Vereinigungsrechtes, Nr. 98 Vereini-
gungsrecht und Recht zu Kollektivverhandlungen, Nr. 117 Grundlegende Ziele
und Normen der Sozialpolitik, insbesondere Teil IV, Nr. 154 Tarifverhandlun-
gen, 4 ABl. L 337 vom 12.12.1998, S. 8. 5 ABl. L 376 vom 27.12.2006, S. 36. 6 Slg. 1990, I-1417. 7 Slg. 1994, I-3803. 8 Slg. 1999, I-8453. 9 Slg. 2001, I-7831. 10 Slg. 2002, I-1425. 11 Slg. 2004, I-9553. 12 Slg. 2004, I-10191. 13 Slg. 2006, I-885. 14 Slg. 2007, I-10779. 15 Slg. 2007, I-11767. 16 Noch nicht in der Sammlung der Rechtsprechung veröffentlicht. 10 Slg. 2002, I-1425. 11 Slg. 2004, I-9553. 12 Slg. 2004, I-10191. 13 Slg. 2006, I-885. 14 Slg. 2007, I-10779. 15 Slg. 2007, I-11767. 16 Noch nicht in der Sammlung der Rechtsprechung veröffentlicht. 146 zu den Herausforderungen für Tarifverträge in der EU (2008/2085(INI)) − unter Hinweis auf seine Entschließung vom 26. Oktober 2006 zur Anwendung
der Richtlinie 96/71/EG über die Entsendung von Arbeitnehmern17, − unter Hinweis auf seine Entschließung vom 26. Das Europäische Parlament , in der Erwägung, dass der EG-Vertrag Beschränkungen der Grundfreiheiten zu-
lässt, sofern sie einen mit dem Vertrag vereinbaren legitimen Zweck verfolgen, aus
zwingenden Gründen des Allgemeininteresses gerechtfertigt sind, zur Erreichung der 147 https://doi.org/10.5771/97
Open Access – https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb Entschließung des Europäischen Parlaments verfolgten Zwecke geeignet sind und nicht über das zur Erreichung dieser Ziele Erfor-
derliche hinausgehen; in der Erwägung, dass parallel dazu Artikel 52 der Charta der
Grundrechte der Europäischen Union vorsieht, dass Einschränkungen bei der Ausübung
der in dieser Charta anerkannten Rechte und Freiheiten unter Wahrung des Grundsatzes
der Verhältnismäßigkeit nur dann erfolgen dürfen, wenn sie notwendig sind und den
von der Union anerkannten dem Gemeinwohl dienenden Zielsetzungen oder den Erfor-
dernissen des Schutzes der Rechte und Freiheiten anderer tatsächlich entsprechen, G. in der Erwägung, dass der EuGH das Recht auf Durchführung einer kollektiven
Maßnahme als Grundrecht anerkennt, das damit fester Bestandteil der allgemeinen
Grundsätze des Gemeinschaftsrechts ist; in der Erwägung, dass dieses Recht mit der
Ratifizierung des Vertrags von Lissabon auch in den Verträgen verankert werden wird, H. in der Erwägung, dass die Kommission mehrfach hervorgehoben hat, welche Be-
deutung die bestehenden nationalen Rahmenregelungen für Beschäftigung und Tarif-
verhandlungen für den Schutz der Arbeitnehmerrechte haben, I. in der Erwägung, dass der Bericht der Kommission "Arbeitsbeziehungen in Europa
2006" zeigt, dass hoch entwickelte Tarifverhandlungen einen positiven Einfluss auf die
soziale Eingliederung haben können, J. in der Erwägung, dass die Gemeinschaft und die Mitgliedstaaten gemäß Artikel
136 des EG-Vertrags (...) "folgende Ziele verfolgen: (...) die Verbesserung der Lebens-
und Arbeitsbedingungen, um dadurch auf dem Wege des Fortschritts ihre Angleichung
zu ermöglichen", und in der Erwägung, dass zur Erreichung dieses Ziels die Kommissi-
on gemäß Artikel 140 des EG-Vertrags die Zusammenarbeit zwischen den Mitgliedstaa-
ten im Bereich der Sozialpolitik fördert, insbesondere auf dem Gebiet des Koalitions-
rechts und der Kollektivverhandlungen zwischen Arbeitgebern und Arbeitnehmern, K. in der Erwägung, dass gemäß der Präambel der Entsenderichtlinie Bedingungen
eines freien und fairen Wettbewerbs sowie Maßnahmen, die die Wahrung der Rechte
der Arbeitnehmer entsprechend dem Rechtsrahmen für die Beschäftigung im jeweiligen
Land und die Arbeitsbeziehungen in den Mitgliedstaaten garantieren, Voraussetzung für
eine Förderung des länderübergreifenden Dienstleistungsverkehrs sind, L. 845219165, am 24.10.2024, 06:46:57
https://www.nomos-elibrary.de/agb Das Europäische Parlament , in der Erwägung, dass die Entsenderichtlinie in Erwägung 12 unmissverständlich
besagt, dass "das Gemeinschaftsrecht (...) die Mitgliedstaaten nicht daran (hindert), ihre
[nationalen] Gesetze oder die von den Sozialpartnern abgeschlossenen Tarifverträge auf
sämtliche Personen anzuwenden, die - auch nur vorübergehend - in ihrem Hoheitsgebiet
beschäftigt werden, selbst wenn ihr Arbeitgeber in einem anderen Mitgliedstaat ansässig
ist", und dass "das Gemeinschaftsrecht (…) es den Mitgliedstaaten nicht (verbietet), die
Einhaltung dieser Bestimmungen mit angemessenen Mitteln sicherzustellen", M. in der Erwägung, dass das Ziel der Entsenderichtlinie – ein fairer Wettbewerb
sowie Maßnahmen, die die Wahrung der Rechte der Arbeitnehmer garantieren – in einer
Ära, in der sich die länderübergreifende Erbringung von Dienstleistungen ausweitet,
wichtig ist für den Schutz der betroffenen Arbeitnehmer, und zwar unter Einhaltung des 148 zu den Herausforderungen für Tarifverträge in der EU (2008/2085(INI)) Beschäftigungsrechts und der Arbeitsbeziehungen in den Mitgliedstaaten, sofern damit
nicht gegen die gemeinschaftlichen Rechtsvorschriften verstoßen wird, N. in der Erwägung, dass gemäß der Entsenderichtlinie die Gesetze der Mitgliedstaa-
ten einen Kern zwingender Bestimmungen über ein Mindestmaß an Schutz für die ent-
sandten Arbeitnehmer festlegen, das im Gastland zu gewährleisten ist, der aber nicht der
Anwendung von Arbeitsbedingungen, die für die Arbeitnehmer günstiger sind, entge-
genstehen darf, O. in der Erwägung, dass gemäß Artikel 3 Absatz 8 der Entsenderichtlinie die Richt-
linie entweder durch Rechts- oder Verwaltungsvorschriften oder durch Tarifverträge
umgesetzt werden kann, die für allgemein verbindlich erklärt wurden oder die allgemein
verbindlich sind für alle vergleichbaren Unternehmen in dem betreffenden Industrie-
zweig oder die von den auf nationaler Ebene repräsentativsten Organisationen der Ta-
rifvertragsparteien geschlossen und auf dem gesamten nationalen Hoheitsgebiet ange-
wandt werden; der EuGH bestätigt ebenfalls, dass die Mitgliedstaaten, da die Entsende-
richtlinie nicht darauf abzielt, die Systeme zur Festlegung der Arbeits- und Beschäfti-
gungsbedingungen zu harmonisieren, ihre Freiheit behalten, auf nationaler Ebene ein
System zu wählen, das die genannte Richtlinie nicht ausdrücklich vorsieht, P. Das Europäische Parlament , in der Erwägung, dass die Kernbestimmungen in Artikel 3 Absatz 1 der Entsende-
richtlinie aus international zwingenden Bestimmungen bestehen, die die Mitgliedstaaten
gemeinsam beschlossen haben; mit der Feststellung, dass die Vorschriften im Bereich
der öffentlichen Ordnung in Artikel 3 Absatz 10 ebenfalls aus international zwingenden
Bestimmungen bestehen, dass sie aber insofern einen Rahmen darstellen, als die Mit-
gliedstaaten über einen gewissen Ermessensspielraum verfügen, wie sie sie in ihrem
nationalen Recht festlegen; ferner mit der Feststellung, dass Artikel 3 Absatz 10 für die
Mitgliedstaaten insofern von Bedeutung ist, als dass er die Berücksichtigung verschie-
dener arbeitsmarktpolitischer, sozialpolitischer und anderer Belange einschließlich des
Schutzes der Arbeitnehmer unter Beachtung des Grundsatzes der Gleichbehandlung
ermöglicht, Q. in der Erwägung, dass die Mobilität der Arbeitnehmer in hohem Maße zu Be-
schäftigung, Wohlstand und EU-Integration beigetragen und den Bürgern neue Mög-
lichkeiten zum Erwerb von Wissen und Erfahrung sowie zur Erreichung eines besseren
Lebensstandards geboten hat, R. in der Erwägung, dass Artikel 28 der Charta der Grundrechte der Europäischen
Union das Recht auf Kollektivverhandlungen und Kollektivmaßnahmen kodifiziert, S. in der Erwägung, dass die Entsenderichtlinie mehr als einer Million Arbeitnehmer
die Möglichkeit zur Arbeit im Ausland unter sicheren Bedingungen ohne Probleme oder
Konflikte gegeben hat, T. in der Erwägung, dass die einheitliche Anwendung und Durchsetzung der Be-
stimmungen der Entsenderichtlinie von entscheidender Bedeutung für die Erreichung
ihrer Ziele ist, insbesondere für die Einhaltung von in den Mitgliedstaaten bestehenden
Regelungen betreffend die Tarifverträge, 149 Entschließung des Europäischen Parlaments Entschließung des Europäischen Parlaments U. in der Erwägung, dass es in Artikel 3 Absatz 1 Buchstabe a der Dienstleistungs-
richtlinie ausdrücklich heißt, dass sie die Entsenderichtlinie nicht ersetzen soll und diese
unberührt lässt, V. in der Erwägung, dass für den freien Warenverkehr die folgende Klausel (bekannt
als "Monti-Klausel") in die Verordnung (EG) Nr. 2679/98 aufgenommen wurde - Arti-
kel 2: "Diese Verordnung darf nicht so ausgelegt werden, dass sie in irgendeiner Weise
die Ausübung der in den Mitgliedstaaten anerkannten Grundrechte, einschließlich des
Rechts oder der Freiheit zum Streik, beeinträchtigt. Diese Rechte können auch das
Recht oder die Freiheit zu anderen Handlungen einschließen, die in den Mitgliedstaaten
durch die spezifischen Systeme zur Regelung der Beziehungen zwischen Arbeitneh-
mern und Arbeitgebern abgedeckt werden", W. in der Erwägung, dass Artikel 1 Absatz 7 der Dienstleistungsrichtlinie festlegt:
"Diese Richtlinie berührt nicht die Ausübung der in den Mitgliedstaaten und durch das
Gemeinschaftsrecht anerkannten Grundrechte. Sie berührt auch nicht das Recht, gemäß
nationalem Recht und nationalen Praktiken unter Wahrung des Gemeinschaftsrechts
Tarifverträge auszuhandeln, abzuschließen und durchzusetzen sowie Arbeitskampfmaß-
nahmen zu ergreifen", X. in der Erwägung, dass der Europäische Rat Grundsätze für die Schaffung von Ar-
beitsmarktmodellen festgelegt hat, die zusätzlich zu einem hohen Maß an Sicherheit
auch einen hohen Grad von Flexibilität aufweisen (bekannt als "Flexicurity-Modell"); in
der Erwägung, dass anerkannt wird, dass ein wichtiger Aspekt eines erfolgreichen Fle-
xicurity-Modells starke Sozialpartner mit beträchtlichem Spielraum für Tarifverhand-
lungen sind, Y. in der Erwägung, dass es in der Zuständigkeit des EuGH liegt, das Gemein-
schaftsrecht unter Berücksichtigung der Grundrechte und -freiheiten auszulegen und die
Wahrung des Rechts bei der Auslegung und Anwendung des EG-Vertrags zu sichern, Z. in der Erwägung, dass es Aufgabe nationaler Gerichte ist, im Einzelfall zu prüfen,
ob die Kriterien betreffend die Beschränkung von Grundfreiheiten und deren Verein-
barkeit mit dem Gemeinschaftsrecht erfüllt sind, AA. in der Erwägung, dass das Recht auf Durchführung einer kollektiven Maßnahme
und auf Abschluss von Tarifverträgen ein Grundrecht ist, das fester Bestandteil der all-
gemeinen Grundsätze des Gemeinschaftsrechts ist; in der Erwägung, dass sich der
EuGH in diesem Zusammenhang nicht auf eine vom Parlament (als Mitgesetzgeber)
nicht angenommene Erklärung des Rates und der Kommission vom 24. September 1996
stützen sollte, die die Auslegung der Begriffe "Vorschriften im Bereich der öffentlichen
Ordnung" und "für die politische Ordnung entscheidende nationale Vorschriften" nur
auf in der Gesetzgebung festgelegte verbindliche Vorschriften beschränken würde, g
g
g
g
AB. Entschließung des Europäischen Parlaments in der Erwägung, dass das Urteil des EuGH vom 21. September 1999 in der
Rechtssache Albany International BV21 auf dem Gebiet des Wettbewerbsrechts den AB. in der Erwägung, dass das Urteil des EuGH vom 21. September 1999 in der
Rechtssache Albany International BV21 auf dem Gebiet des Wettbewerbsrechts den 21 Slg. 1999, I-5751. 150 /10.5771/9
– – zu den Herausforderungen für Tarifverträge in der EU (2008/2085(INI)) Gewerkschaften in Bezug auf Arbeitsmarktfragen einen beträchtlichen Ermessensspiel-
raum eingeräumt hat, AC. in der Erwägung, dass festgestellt worden ist, dass es beim EuGH und zwischen
dem Gerichtshof und seinen Generalanwälten in mehreren Rechtssachen betreffend die
Entsenderichtlinie, insbesondere in den genannten Rechtssachen Laval und Rüffert,
unterschiedliche Ansichten und Auslegungen gab; in der Erwägung, dass angesichts
solcher unterschiedlichen Ansichten und Auslegungen möglicherweise eine Klarstel-
lung betreffend das Gleichgewicht zwischen Grundrechten und Grundfreiheiten erfor-
derlich ist, 1. unterstreicht, dass die Dienstleistungsfreiheit ein Grundpfeiler des europäischen
Einigungswerkes ist; ist jedoch der Ansicht, dass dies einerseits gegen die in den Ver-
trägen dargelegten Grundrechte und sozialen Ziele und andererseits gegen das Recht der
öffentlichen und sozialen Partner, Nichtdiskriminierung, Gleichbehandlung und Verbes-
serung der Lebens- und Arbeitsbedingungen sicherzustellen, abgewogen werden sollte;
erinnert daran, dass das Recht auf Kollektivverhandlungen und das Recht auf Kollek-
tivmaßnahmen Grundrechte sind, die in der Charta der Grundrechte der Europäischen
Union anerkannt werden, und dass die Gleichbehandlung ein Grundprinzip der Europäi-
schen Union darstellt; 2. ist der Meinung, dass jeder EU-Bürger das Recht haben sollte, überall in der Euro-
päischen Union zu arbeiten, und dabei ein Recht auf Gleichbehandlung haben sollte;
bedauert daher, dass dieses Recht nicht einheitlich in der gesamten EU angewandt wird;
ist der Auffassung, dass die noch bestehenden Übergangsbestimmungen einer genauen
Überprüfung durch die Kommission unterzogen werden sollten, um festzustellen, ob sie
wirklich notwendig sind, um Verzerrungen auf den nationalen Arbeitsmärkten zu ver-
hindern, und dass sie, wo dies nicht der Fall ist, so rasch wie möglich abgeschafft wer-
den sollten; 2. Entschließung des Europäischen Parlaments ist der Meinung, dass jeder EU-Bürger das Recht haben sollte, überall in der Euro-
päischen Union zu arbeiten, und dabei ein Recht auf Gleichbehandlung haben sollte;
bedauert daher, dass dieses Recht nicht einheitlich in der gesamten EU angewandt wird;
ist der Auffassung, dass die noch bestehenden Übergangsbestimmungen einer genauen
Überprüfung durch die Kommission unterzogen werden sollten, um festzustellen, ob sie
wirklich notwendig sind, um Verzerrungen auf den nationalen Arbeitsmärkten zu ver-
hindern, und dass sie, wo dies nicht der Fall ist, so rasch wie möglich abgeschafft wer-
den sollten; 3. betont, dass die Dienstleistungsfreiheit weder dem Grundrecht der Sozialpartner,
den sozialen Dialog voranzutreiben und Arbeitskampfmaßnahmen zu ergreifen, wider-
spricht noch über diesem steht, zumal dies in mehreren Mitgliedstaaten ein verfas-
sungsmäßiges Recht ist; betont, dass die Monti-Klausel die verfassungsmäßigen Grund-
rechte im Rahmen des Binnenmarktes schützen wollte; erinnert gleichzeitig daran, dass
die Freizügigkeit der Arbeitnehmer eine der vier Freiheiten des Binnenmarktes ist; 4. begrüßt den Vertrag von Lissabon und die Tatsache, dass die Charta der Grund-
rechte der Europäischen Union rechtsverbindlich werden soll; stellt fest, dass dies das
Recht der Gewerkschaften, Tarifverträge auf den geeigneten Ebenen auszuhandeln und
abzuschließen sowie bei Interessenkonflikten kollektive Maßnahmen (z. B. Streik) zur
Verteidigung ihrer Interessen zu ergreifen, einschließen würde; 5. betont, dass die Dienstleistungsfreiheit nicht über den in der Charta der Grund-
rechte der Europäischen Union dargelegten Grundrechten steht und insbesondere nicht
über dem Recht der Gewerkschaften, Arbeitskampfmaßnahmen zu ergreifen, zumal dies 151 Entschließung des Europäischen Parlaments in mehreren Mitgliedstaaten ein verfassungsmäßiges Recht ist; betont daher, dass die
EuGH-Urteile in den Rechtssachen Rüffert, Laval und Viking zeigen, dass unbedingt
geklärt werden muss, dass wirtschaftliche Freiheiten, wie sie in den Verträgen verankert
sind, so ausgelegt werden sollten, dass sie nicht die Wahrnehmung grundlegender Sozi-
alrechte beeinträchtigen, wie sie in den Mitgliedstaaten und vom Gemeinschaftsrecht
anerkannt werden, einschließlich des Rechts, Tarifverträge auszuhandeln, abzuschließen
und durchzusetzen und kollektive Maßnahmen zu ergreifen, und nicht gegen die Auto-
nomie der Sozialpartner verstoßen, wenn diese Grundrechte in Verfolgung der sozialen
Interessen und des Schutzes der Arbeitnehmer ausgeübt werden; 6. betont, dass die Entsenderichtlinie den staatlichen Behörden und den Sozialpart-
nern erlaubt, für die Arbeitnehmer günstigere Beschäftigungs- und Arbeitsbedingungen
entsprechend den unterschiedlichen Traditionen in den Mitgliedstaaten festzulegen; 7. Entschließung des Europäischen Parlaments verweist darauf, dass Erwägung 22 der Entsenderichtlinie feststellt, dass die Richt-
linie das Recht der Mitgliedstaaten in Bezug auf kollektive Maßnahmen zur Verteidi-
gung beruflicher Interessen, das durch Artikel 137 Absatz 5 des EG-Vertrags bestätigt
wird, unberührt lässt; 8. betont deshalb, dass Gleichbehandlung und gleicher Lohn für gleiche Arbeit am
gleichen Arbeitsplatz, wie dies die Artikel 39 und 12 des EG-Vertrags vorsehen, ge-
währleistet und verstärkt werden müssen; ist der Auffassung, dass im Rahmen der
Dienstleistungsfreiheit oder der Niederlassungsfreiheit die Staatsangehörigkeit des Ar-
beitgebers bzw. der Arbeitnehmer oder der entsandten Arbeitnehmer Ungleichheiten in
Bezug auf Arbeitsbedingungen, Entgelt oder die Wahrnehmung von Grundrechten wie
des Streikrechts nicht rechtfertigen darf; 9. unterstreicht, dass es wichtig ist, negative Folgen für Arbeitsmarktmodelle zu ver-
hindern, die bereits ein hohes Maß an Flexibilität auf dem Arbeitsmarkt mit einem ho-
hen Maß an Sicherheit verbinden können, und diesen Ansatz weiter voranzutreiben; Allgemeine Auswirkungen Allgemeine Auswirkungen 10. weist darauf hin, dass die horizontale Wirkung bestimmter Vorschriften des EG-
Vertrags von der Erfüllung konkreter Bedingungen abhängt, so u. a. davon, dass diese
Vorschriften einer natürlichen Person, die ein Interesse an der Einhaltung der betreffen-
den Verpflichtungen hat, Rechte verleihen; äußert seine Besorgnis darüber, dass die
horizontale Wirkung von Artikel 43 des EG-Vertrags unter den speziellen Umständen
der jüngsten Urteile des EuGH klar nachgewiesen wurde, und ist der Ansicht, dass die
Zahl der Verfahren beim Gerichtshof infolgedessen zunehmen könnte; 11. begrüßt, dass entsprechend den Prinzipien und Traditionen der Europäischen
Union viele Mitgliedstaaten in Zusammenarbeit mit den Sozialpartnern hohe Standards
bei den Arbeitsbedingungen eingeführt haben, die das Wohlergehen aller Arbeitnehmer
verbessern und das Wirtschaftswachstum sowie die Wettbewerbsfähigkeit steigern; 12. ist der Ansicht, dass die mit der Entsenderichtlinie und der Dienstleistungsrichtli-
nie verfolgte Absicht des Gesetzgebers nicht mit Auslegungen vereinbar ist, die als Auf-
forderung zu unlauterem Wettbewerb zwischen Unternehmen verstanden werden könn- 152 /10.5771
– zu den Herausforderungen für Tarifverträge in der EU (2008/2085(INI)) ten; stellt fest, dass Unternehmen, die Tarifverträge unterzeichnen und einhalten, im
Wettbewerb mit Unternehmen, die dies nicht tun, benachteiligt sein könnten; g
13. ist der Auffassung, dass die korrekte Anwendung und Durchsetzung der Bestim-
mungen der Entsenderichtlinie wesentlich ist, um das Erreichen ihrer Ziele zu gewähr-
leisten, d.h. die Erleichterung der Erbringung von Dienstleistungen bei gleichzeitiger
Gewährleistung eines angemessenen Schutzes der Arbeitnehmer und die uneinge-
schränkte Einhaltung bestehender Tarifverträge in den Mitgliedstaaten, in die Arbeit-
nehmer im Rahmen dieser Richtlinie entsandt werden; 14. Entschließung des Europäischen Parlaments ist ferner der Ansicht, dass die Freiheit, grenzüberschreitende Dienstleistungen
im Binnenmarkt anzubieten, weiter dadurch gestärkt wird, dass sichergestellt ist, dass
für inländische und ausländische Dienstleister am Ort der Erbringung der Dienstleistung
ähnliche Wirtschafts- und Arbeitsmarktbedingungen herrschen; 15. spricht sich für eine Förderung der Wettbewerbsfähigkeit mit Hilfe von Wissen
und Innovation aus, so wie dies die Lissabon-Strategie vorsieht; 15. spricht sich für eine Förderung der Wettbewerbsfähigkeit mit Hilfe von Wissen
und Innovation aus, so wie dies die Lissabon-Strategie vorsieht; 16. äußert Bedenken an der Einführung eines Grundsatzes der Verhältnismäßigkeit
bei Maßnahmen gegen Unternehmen, die sich auf das Niederlassungsrecht oder das
Recht auf Erbringung grenzüberschreitender Dienstleistungen berufen, um bewusst die
Arbeitsbedingungen zu unterlaufen; ist der Ansicht, dass das Recht auf Arbeitskampf-
maßnahmen zur Aufrechterhaltung der Gleichbehandlung und zur Sicherung men-
schenwürdiger Arbeitsbedingungen nicht in Frage gestellt werden darf; 16. äußert Bedenken an der Einführung eines Grundsatzes der Verhältnismäßigkeit
bei Maßnahmen gegen Unternehmen, die sich auf das Niederlassungsrecht oder das
Recht auf Erbringung grenzüberschreitender Dienstleistungen berufen, um bewusst die
Arbeitsbedingungen zu unterlaufen; ist der Ansicht, dass das Recht auf Arbeitskampf-
maßnahmen zur Aufrechterhaltung der Gleichbehandlung und zur Sicherung men-
schenwürdiger Arbeitsbedingungen nicht in Frage gestellt werden darf; 17. betont, dass die wirtschaftlichen Freiheiten in der EU nicht so ausgelegt werden
dürfen, dass die Unternehmen das Recht hätten, nationalen sozial- und arbeitsrechtli-
chen Bestimmungen und Verfahren auszuweichen oder diese zu umgehen oder einen
unlauteren Wettbewerb bei Löhnen und Arbeitsbedingungen durchzusetzen; ist daher
der Auffassung, dass länderübergreifende Maßnahmen von Unternehmen, die die Be-
schäftigungsbedingungen im Gastland möglicherweise unterlaufen, verhältnismäßig
sein müssen und nicht automatisch mit den Bestimmungen des EG-Vertrags z. B. über
den freien Dienstleistungsverkehr oder die Niederlassungsfreiheit gerechtfertigt werden
können; 17. betont, dass die wirtschaftlichen Freiheiten in der EU nicht so ausgelegt werden
dürfen, dass die Unternehmen das Recht hätten, nationalen sozial- und arbeitsrechtli-
chen Bestimmungen und Verfahren auszuweichen oder diese zu umgehen oder einen
unlauteren Wettbewerb bei Löhnen und Arbeitsbedingungen durchzusetzen; ist daher
der Auffassung, dass länderübergreifende Maßnahmen von Unternehmen, die die Be-
schäftigungsbedingungen im Gastland möglicherweise unterlaufen, verhältnismäßig
sein müssen und nicht automatisch mit den Bestimmungen des EG-Vertrags z. B. über
den freien Dienstleistungsverkehr oder die Niederlassungsfreiheit gerechtfertigt werden
können; 18. betont, dass das Gemeinschaftsrecht den Grundsatz der Nichtdiskriminierung be-
achten muss; betont ferner, dass der gemeinschaftliche Gesetzgeber gewährleisten muss,
dass keine Hürden für Tarifverträge aufgebaut werden, u.a. Entschließung des Europäischen Parlaments 20. stellt fest, dass aufgrund der sozialen Aspekte, auf die in Artikel 26 und 27 der
Vergaberichtlinie Bezug genommen wird, die Mitgliedstaaten faire Wettbewerbsbedin-
gungen schaffen können, indem sie Arbeits- und Beschäftigungsbedingungen festlegen,
die über die zwingenden Bestimmungen für ein Mindestmaß an Schutz hinausgehen; 20. stellt fest, dass aufgrund der sozialen Aspekte, auf die in Artikel 26 und 27 der
Vergaberichtlinie Bezug genommen wird, die Mitgliedstaaten faire Wettbewerbsbedin-
gungen schaffen können, indem sie Arbeits- und Beschäftigungsbedingungen festlegen,
die über die zwingenden Bestimmungen für ein Mindestmaß an Schutz hinausgehen; 21. ist der Auffassung, dass die eingeschränkte Rechtsgrundlage der Freizügigkeit in
der Entsenderichtlinie möglicherweise dazu führt, dass die Entsenderichtlinie als aus-
drückliche Aufforderung zu unlauterem Wettbewerb in Bezug auf Löhne und Arbeits-
bedingungen verstanden wird; vertritt daher die Auffassung, dass die Rechtsgrundlage
der Entsenderichtlinie dahingehend ausgeweitet werden könnte, dass sie auch auf die
Freizügigkeit der Arbeitnehmer Bezug nimmt; 22. betont, dass die jetzige Situation im Ergebnis dazu führen könnte, dass Arbeit-
nehmer in Gastländern in einen Niedriglohnwettbewerb gezwungen werden; ist daher
der Auffassung, dass in allen Mitgliedstaaten für eine konsequente Umsetzung der Ent-
senderichtlinie gesorgt werden muss; 23. erinnert daran, dass neun Mitgliedstaaten das ILO-Übereinkommen Nr. 94 über
Arbeitsklauseln (Öffentliche Verträge) ratifiziert haben; bedauert, dass auch in der
Rechtsprechung das ILO-Übereinkommen Nr. 94 nicht angemessen beachtet wird, und
ist beunruhigt darüber, dass die Anwendung dieses Übereinkommens in den betreffen-
den Mitgliedstaaten im Widerspruch zur Anwendung der Entsenderichtlinie stehen
könnte; fordert die Kommission auf, diesen Sachverhalt dringend zu klären und auch
weiterhin die Ratifizierung dieses Übereinkommens zu fördern, um die Einführung von
Sozialklauseln in vergaberechtlichen Vorschriften, die ihrerseits ein Ziel der Vergabe-
richtlinie ist, weiter voranzutreiben; 24. stellt fest, dass nicht anerkannt worden ist, dass gemäß den ILO-Übereinkommen
Nr. 87 und 98 Einschränkungen des Rechts auf Arbeitskampfmaßnahmen und von
Grundrechten nur aus Gründen der Gesundheit, der öffentlichen Ordnung und anderer
ähnlicher Faktoren gerechtfertigt werden können; g
g
;
Forderungen 25. fordert alle Mitgliedstaaten zur ordnungsgemäßen Durchsetzung der Entsende-
richtlinie auf; betont ferner, dass die Arbeitsmarktvorschriften und die Vorschriften
betreffend Verhandlungen und Tarifverträge in die Zuständigkeit der Mitgliedstaaten
und der Sozialpartner fallen; weist diesbezüglich darauf hin, dass es Aufgabe der Mit-
gliedstaaten ist, gemäß dem Subsidiaritätsprinzip die Präventiv-, Kontroll- und Sankti-
onsmaßnahmen in vollem Maße auszuschöpfen und zu verbessern; 26. Entschließung des Europäischen Parlaments für solche, mit denen der
Grundsatz des gleichen Entgelts für gleiche Arbeit für alle Arbeitnehmer am Arbeits-
platz unabhängig von ihrer Staatsangehörigkeit bzw. der ihres Arbeitgebers am Ort der
Erbringung der Dienstleistung umgesetzt wird, oder für Arbeitskampfmaßnahmen zur
Unterstützung eines solchen Vertrags entsprechend den einzelstaatlichen Gesetzen oder
Praktiken; 19. räumt ein, dass die Urteile des EuGH in den Rechtssachen Laval, Rüffert und
Luxemburg für große Beunruhigung hinsichtlich der Auslegung der Richtlinien für eine
Mindestharmonisierung gesorgt haben; 19. räumt ein, dass die Urteile des EuGH in den Rechtssachen Laval, Rüffert und
Luxemburg für große Beunruhigung hinsichtlich der Auslegung der Richtlinien für eine
Mindestharmonisierung gesorgt haben; 153 Entschließung des Europäischen Parlaments Entschließung des Europäischen Parlaments ist der Auffassung, dass die geltenden gemeinschaftlichen Rechtsvorschriften
sowohl Schlupflöcher als auch Unstimmigkeiten aufweisen und sich daher für Ausle-
gungen der Entsenderichtlinie angeboten haben, die nicht in der Absicht des gemein-
schaftlichen Gesetzgebers lagen, der ein faires Gleichgewicht zwischen der Dienstleis-
tungsfreiheit und dem Schutz der Arbeitnehmerrechte anstrebte; fordert die Kommissi-
on auf, die notwendigen Legislativvorschläge auszuarbeiten, die dazu beitragen würden,
widersprüchlichen Auslegungen künftig vorzubeugen; 154 zu den Herausforderungen für Tarifverträge in der EU (2008/2085(INI)) 27. begrüßt deshalb die Erklärung der Kommission vom 3. April 2008, in der sie sich
nicht nur dazu verpflichtet hat, auch weiterhin einen auf niedrigen Sozialstandards be-
ruhenden Wettbewerb zu bekämpfen, sondern auch betont hat, dass die Dienstleistungs-
freiheit nicht im Widerspruch zum Grundrecht auf Streik und Mitgliedschaft in einer
Gewerkschaft steht und in keiner Weise über diesem Grundrecht steht; spricht sich für
die unverzügliche Umsetzung der Schlussfolgerungen des Rates vom 9. Juni 2008 aus,
um Mängel bei der Umsetzung zu beheben, weitere problematische Situationen und
Missbrauch zu verhindern und das angestrebte Klima gegenseitigen Vertrauens zu
schaffen; fordert die Kommission und die Mitgliedstaaten auf, eine engere Zusammen-
arbeit zwischen den Mitgliedstaaten, den nationalen Behörden und der Kommission mit
Blick auf die Überwachung und den Austausch bewährter Verfahren zu fördern; ist der
Auffassung, dass dies ein wirksames Mittel wäre, um Missbrauch zu bekämpfen; 28.stellt fest, dass es wichtig ist, dass die Regeln auf dem EU-Arbeitsmarkt transpa-
rent und für alle gleichwertig sind, dass es aber aufgrund der unterschiedlichen politi-
schen Traditionen sehr schwierig ist, ein einheitliches Arbeitsmarktmodell zu schaffen;
vertritt deshalb die Auffassung, dass in den Fällen, in denen bestimmte Mitgliedstaaten
in besonderem Maße betroffen sind, auf nationaler Ebene in Konsultation mit den Sozi-
alpartnern eine detaillierte Folgenabschätzung der oben genannten Urteile vorgenom-
men werden sollte; 28.stellt fest, dass es wichtig ist, dass die Regeln auf dem EU-Arbeitsmarkt transpa-
rent und für alle gleichwertig sind, dass es aber aufgrund der unterschiedlichen politi-
schen Traditionen sehr schwierig ist, ein einheitliches Arbeitsmarktmodell zu schaffen;
vertritt deshalb die Auffassung, dass in den Fällen, in denen bestimmte Mitgliedstaaten
in besonderem Maße betroffen sind, auf nationaler Ebene in Konsultation mit den Sozi-
alpartnern eine detaillierte Folgenabschätzung der oben genannten Urteile vorgenom-
men werden sollte; 29. begrüßt die Aussage der Kommission, dass sie nun bereit ist, die Auswirkungen
des Binnenmarkts auf die Arbeitnehmerrechte und die Tarifverhandlungen einer erneu-
ten Prüfung zu unterziehen; 30. Entschließung des Europäischen Parlaments spricht sich dafür aus, dass dabei eine teilweise Überarbeitung der Entsendericht-
linie nicht ausgeschlossen werden sollte. Eine etwaige Überarbeitung dieser Richtlinie
sollte erst nach einer sorgfältigen Analyse der tatsächlichen Herausforderungen für die
unterschiedlichen Systeme der Tarifverhandlungen auf nationaler Ebene erfolgen. Die
Überarbeitung sollte, sofern sie für sinnvoll erachtet wird, insbesondere Fragen betref-
fen wie die geltenden Arbeitsbedingungen, die Lohnniveaus, den Grundsatz der Gleich-
behandlung der Arbeitnehmer im Zusammenhang mit der Dienstleistungsfreiheit, die
Einhaltung der unterschiedlichen Arbeitsmodelle und die Dauer der Entsendung; 30. spricht sich dafür aus, dass dabei eine teilweise Überarbeitung der Entsendericht-
linie nicht ausgeschlossen werden sollte. Eine etwaige Überarbeitung dieser Richtlinie
sollte erst nach einer sorgfältigen Analyse der tatsächlichen Herausforderungen für die
unterschiedlichen Systeme der Tarifverhandlungen auf nationaler Ebene erfolgen. Die
Überarbeitung sollte, sofern sie für sinnvoll erachtet wird, insbesondere Fragen betref-
f
i di
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d
A b it b di
di L h
i
d
G
d
t d
Gl i h 31. ist der Ansicht, dass die Ausübung der in den Mitgliedstaaten, in den ILO-
Übereinkommen und in der Charta der Grundrechte der Europäischen Union anerkann-
ten Grundrechte einschließlich des Rechts, Tarifverträge auszuhandeln, abzuschließen
und durchzusetzen sowie Arbeitskampfmaßnahmen zu ergreifen, nicht gefährdet wer-
den sollte; 32. betont, dass unmissverständlich klargestellt werden muss, dass die Entsendericht-
linie und andere Richtlinien die Mitgliedstaaten und Sozialpartner nicht daran hindern,
günstigere Bedingungen mit Blick auf die Gleichbehandlung der Arbeitnehmer zu ver-
langen, und dass gewährleistet ist, dass die gemeinschaftlichen Rechtsvorschriften auf
der Grundlage aller bestehenden Arbeitsmarktmodelle angewandt werden können; 155 Entschließung des Europäischen Parlaments 1 cf. on the judgement in the Viking case e.g. Rebhahn, Grundfreiheit vor Arbeitskampf – der Fall Vi-
king, ZESAR 2008, 109-117 Entschließung des Europäischen Parlaments 33. fordert die Kommission auf, die Beschlüsse des Rates betreffend die Schaffung
eines elektronischen Systems für den Austausch von Informationen dringend umzuset-
zen, da es die Mitgliedstaaten in die Lage versetzen könnte, Missbrauch wirksamer zu
bekämpfen; 33. fordert die Kommission auf, die Beschlüsse des Rates betreffend die Schaffung
eines elektronischen Systems für den Austausch von Informationen dringend umzuset-
zen, da es die Mitgliedstaaten in die Lage versetzen könnte, Missbrauch wirksamer zu
bekämpfen; 34. fordert die Mitgliedstaaten und die Kommission auf, Maßnahmen zu ergreifen,
um den Missbrauch zu bekämpfen, insbesondere bezüglich der Tätigkeit von Briefkas-
tenfirmen, die im Niederlassungsland keine echte und effektive Geschäftstätigkeit ent-
falten, sondern - manchmal sogar direkt vom Hauptauftragnehmer im Gastland - aus-
schließlich zu dem Zweck gegründet wurden, eine Geschäftstätigkeit im Gastland aus-
zuüben, um die uneingeschränkte Anwendung der Vorschriften und Regelungen des
Gastlandes, insbesondere im Hinblick auf Löhne und Arbeitsbedingungen, zu umgehen;
fordert die Kommission auf, in ihrem Verhaltenskodex für Unternehmen im Rahmen
der Dienstleistungsrichtlinie eindeutige Vorschriften zur Bekämpfung von Briefkasten-
firmen festzulegen; g
35. bekräftigt, dass die grundlegenden sozialen Rechte nicht in einer Hierarchie der
Grundfreiheiten unterhalb der wirtschaftlichen Rechte anzusiedeln sind; fordert deshalb,
dass im Primärrecht das Gleichgewicht zwischen den Grundrechten und den wirtschaft-
lichen Freiheiten erneut festgestellt wird, um so ein Wettrennen um niedrigere Sozial-
standards zu verhindern; 36. begrüßt den Gemeinsamen Standpunkt des Rates im Hinblick auf eine neue
Richtlinie über Leiharbeitnehmer, die eine nichtdiskriminierende Behandlung ab dem
ersten Tag der Beschäftigung vorsehen würde, sofern die Sozialpartner nicht etwas an-
deres vereinbaren; 37. fordert die Kommission auf, die seit langem erwartete Mitteilung über län-
derübergreifende Tarifverhandlungen vorzulegen, in der die Schaffung eines Rechts-
rahmens für länderübergreifende Tarifvereinbarungen vorgeschlagen wird; 38. beauftragt seinen Präsidenten, diese Entschließung dem Rat und der Kommission
sowie den Regierungen und Parlamenten der Mitgliedstaaten zu übermitteln. 38. beauftragt seinen Präsidenten, diese Entschließung dem Rat und der Kommission
sowie den Regierungen und Parlamenten der Mitgliedstaaten zu übermitteln. 156 I. Introduction
II. Fundamental Rights and Freedoms in General
III. Recent Decisions and Fundamental Rights
IV. Assessment and Outlook I. Introduction
II. Fundamental Rights and Freedoms in General
III. Recent Decisions and Fundamental Rights
IV. Assessment and Outlook I. Introduction
II. Fundamental Rights and Freedoms in General
III. Recent Decisions and Fundamental Rights
IV. Assessment and Outlook The recent jurisprudence of the ECJ viewed against the
background of the relation between Fundamental
Freedoms and Community Fundamental Rights Robert Rebhahn 2 ECJ of 12/6/2003, Schmidberger, Case C-112/00, Rec. 2003, I-5659. I. Introduction Many Member States protect collective bargaining and industrial dispute by making
them fundamental rights. However, the ECJ has so far never allowed Community law to
be subordinated to national fundamental rights since this would threaten unity. In
Community law, national fundamental rights as such can only be relevant in so far as
they give Member States the possibility of restrictions. Hence, only Community fundamental rights qualify as a counterweight to fundamen-
tal freedoms. To date, the Community has not succeeded in producing a binding cata-
logue of fundamental rights. Such a catalogue is long overdue since without it, funda-
mental freedoms continue to occupy the front stage when it comes to fundamental val-
ues, both in written primary legislation and in court practice. However, with regard to
the enormous increase in competencies and provisions, this is no longer adequate. The
ECJ at least recognises unwritten fundamental rights as general principles. However, the
absence of any statement on the fundamental right to free collective bargaining in the
Albany decision of 1999 is telling in itself. Recently, the decisions in the cases Viking
and Laval expressly acknowledge “the right to take collective action including the right
to strike as a fundamental right”.1 This is a major step in its own right, although the 157 Robert Rebhahn Robert Rebhahn ECJ’s reasoning in these specific cases may not be found to be fully convincing from a
legal doctrine point of view. An affirmation of this fundamental right of Community
law makes it all the more compelling to also affirm free collective bargaining as a simi-
lar right. Admittedly, the fundamental right to collective measures does not seem to play
a noticeable role in the reasoning of the judgements and the results. For this fundamen-
tal right, no legal doctrine has been established by the ECJ to date, either. The – albeit
minor – significance that this right does have is fully attributable to court practice as
established thus far. II. Fundamental Rights and Freedoms in General II. Fundamental Rights and Freedoms in General The relationship between fundamental freedoms and fundamental rights of Commu-
nity law in case of collision is a rather obscure one. It is not so much theoretical propo-
sitions that help determine this relationship but rather the ECJ’s practice. A judgement
may examine interventions in fundamental freedoms or fundamental rights. The few
judgements on collisions available to date relate to restrictions of a fundamental free-
dom. Fundamental rights have featured only as a potential restriction of the fundamental
freedom in question. There have hardly been any cases where the ECJ had to examine
whether the interference in a Community fundamental right may be justified by the
Community or an individual state in the interest of a fundamental freedom. Even in Vi-
king and Laval the issue under review was not whether the right to strike could be re-
stricted on account of a fundamental freedom but rather the contrary. However, the way
a problem is presented will always have an impact on perception, which may in turn
influence the legal statements that are to be made. If collisions are perceived mostly in
the context of a restriction of fundamental freedoms through the exercising of funda-
mental rights, this may soon lead to an exercising of fundamental rights only “in accor-
dance with the requirements of” fundamental freedoms. Fundamental freedoms and fundamental rights would be treated on an equal footing
only if, in case of a collision, both were assumed to be interests protected under primary
legislation which would then have to be “balanced“, either on the level which defines
the extent of protection or on the level where barriers to intervention are identified. However, this would have to be stated expressly by the court. Decisions taken by the
ECJ to date have not visibly reviewed and considered both positions on the same level
but have placed fundamental freedoms first. The Schmidberger judgement alone seemed
to place both rights on the same level, at least linguistically.2 This approximation in
ranking is not discernible at all in either Viking or Laval, as the fundamental right to
collective action that has just been acknowledged is not brought into play to justify the
restriction of the fundamental freedom concerned. III. Recent Decisions and Fundamental Rights The conclusion from Viking and Laval is that at least collective measures targeted
against relocation of plants to a different Member State or against the posting of work-
ers are to be measured against freedom of establishment and freedom to provide ser-
vices. Exercising a Community fundamental right cannot protect trade unions against
being subordinated to fundamental freedoms. If that is the case, the exercising of a fun-
damental right would have to be considered subsequently, which is not done. Even leav-
ing aside the fundamental right, there is arguably no conclusive justification for submit-
ting workers and trade unions to the fundamental freedom concerned. At one point it is
being said that it were sufficient for action to be targeted at a normative provision – the
collective agreement – whereas later it is being stated that this were irrelevant. A con-
clusive proposition would be that any factually substantial impairment of a fundamental
freedom, also by private entities, leads to a restriction of that freedom which is only
admissible if it can be justified. It may well be that the judgements are indeed based on
this rather broad proposition – if so, this is not made explicit, however. Restriction of a fundamental freedom is legitimate only if it can be justified. It is also
subject to the imperative of proportionality. Applied to strikes, this criterion amounts to
a profound alteration of national fundamental rights for many northern and southern
Member States, and for some southern Member States it even means that national fun-
damental rights are being curtailed. Unfortunately, when it comes to reviewing for pro-
portionality there is, in most cases, no way of telling what the result will be before the
judgement is pronounced. This uncertainty places a burden on those wishing to take
action, in this case the bearers of fundamental rights. According to the ECJ a justifica-
tion is conceivable not only for strikes of those employees whose working conditions
are at stake, but also for supportive boycotts and thus for sympathetic measures. This,
again, is a novelty for some states. The grounds of justification named in the judgements are protection of employees,
and, in more concrete terms, the aims of EC social policy. The fundamental freedom is
to be balanced against these aims. II. Fundamental Rights and Freedoms in General At the beginning of the deliberations 158 10.5771
– Relation between Fundamental Freedoms and Fundamental Rights in the recent ECJ’s jurisprudence on the issue it is indeed stated that the “exercising of the right to collective action” may
well be of overriding general interest. However, the judgement does not designate this
as a fundamental right. Only protection of working conditions and the EC’s social pol-
icy are mentioned as concrete examples of general interest, whereas the specific signifi-
cance of free collective bargaining and industrial dispute – thus, collective representa-
tion of interests, by application of pressure if necessary – is being left aside. III. Recent Decisions and Fundamental Rights It seems, however, that, in accordance with the Vi-
king judgement, a strike can only be justified if jobs or working conditions are in danger
or under serious threat, which is a narrower definition than found in many national fun- 159 Robert Rebhahn damental rights, and this appears to be the case not only for strikes related to social pol-
icy legislation. It probably is a novelty in Community law that the ECJ accepts interests
that are primarily economic in nature for justification in this case. However, this does
not compensate for the fundamental right being eclipsed at least verbally. Even if there is a danger or a threat, the national court must review whether the
measure is suitable and necessary to ensure attainment of the objective. Presumably, the
imperative of necessity is to be neglected if national law offers other means to attain a
collective agreement, which restrict the freedom of establishment to a lesser extent. This
could lead to the conclusion that the parties are to submit themselves to voluntary arbi-
tration first, which would, again, mean stricter limits than those set out in national fun-
damental rights. Moreover, the Viking decision states that the enterprise whose funda-
mental freedom is being infringed may confer rights against trade unions from this. In
some countries, this will, for the first time ever, necessitate effective sanctions against
strikes. The fact that the persons acting are bearers of fundamental rights is hardly ex-
pressed in the judgement. The decisions in Laval and Rüffert relate to the posting of workers, thus putting the
Posting Directive in the foreground. The Laval judgement is the first to expressly state
in this context that the Directive exclusively regulates the possibilities of the state of
work to make its labour law compulsory for posted workers. I was and still am of the
opinion that this interpretation is easily justifiable on account of the competence for the
internal market claimed by the EC. However, it is not compelling and some more justi-
fication would have been necessary. It is at any rate doubtful how this relates to em-
ployees’ fundamental rights. In Laval, the matter under review was not a strike of posted workers but a blockade
of Swedish workers against posting, since the Swedes considered their working condi-
tions to be in danger. III. Recent Decisions and Fundamental Rights In essence, the judgement places this boycott, which is more than
an industrial struggle out of sympathy, into the sphere of protection of the fundamental
right as well. This is a novelty for many Member States. At this point, at the very latest,
it becomes apparent that the decision (also) neglects entrepreneurial interests other than
the fundamental freedoms, which may be arguments against a boycott. The conflict of
interest between boycotters and posted employees is not expressly mentioned either. The ECJ, in any case, restricts its review of whether the boycott is admissible to the
Posting Directive. However, Sweden did not make use of all possibilities offered by the
Directive as it has not prescribed for posting companies also those collective agreements
which are not generally binding but are indeed generally applicable. In accordance with
the judgement it is at any rate inadmissible for the national peace obligation to be trig-
gered by national collective agreements alone since this would amount to unequal
treatment of foreign collective agreements. It is also inadmissible to impose an obliga-
tion on posting companies to negotiate minimum wages with trade unions first. The judgement appears to be coherent. However, certain doubts arise upon closer
scrutiny. The Posting Directive itself relies on the principle that foreign collective 160 /10.5771/978
– /10.5771/
– Relation between Fundamental Freedoms and Fundamental Rights in the recent ECJ’s jurisprudence agreements and national collective agreements will not always be treated equally. Moreover, the judgement assumes that free collective bargaining and industrial dispute
are guaranteed only in accordance with the requirements set out in the Posting Direc-
tive. However, the question could have been asked and indeed should have been asked
whether Community fundamental rights to collective measures and free collective bar-
gaining are sufficiently considered in the Posting Directive, in particular taking into
account that, according to the prevailing opinion, the Community is not allowed to regu-
late the collective bargaining system. The judgement does not specifically mention pro-
tection of national collective bargaining systems as a justification for the restriction but
essentially leaves the issue aside. y
The Rüffert judgement relates to a law on compliance with collective agreements. The requirements of the law in question go beyond those of the Posting Directive. III. Recent Decisions and Fundamental Rights The
ECJ finds this to be in contradiction with the freedom to provide services, in particular
because it were not clear why a posted worker would require stronger protection when
working for a public employer. Apparently, this is to say that this distinction does not
constitute a factual justification. This kind of “consideration” may well be used to ques-
tion all labour legislation provisions which assign more duties exclusively to certain
entities - such as the state - when they act as an employer or customer, as is the case for
example in anti-discrimination law. Moreover, the Rüffert decision also fails to deliber-
ate whether the law requiring compliance with collective agreements serves the purpose
of collective bargaining and thus a Community fundamental right. The German Federal
Constitutional Court, in contrast, explained in justifying the law on compliance with
collective agreements against freedom of occupation that this kind of legislation sup-
ported the regulatory function of collective agreements and the right to free collective
bargaining as a protected fundamental right. The ECJ has not reacted to this, despite the
much-praised dialogue with national supreme courts. The need to deal with this is not
rendered obsolete, either, by claiming that the Federal Constitutional Court’s concern
was to protect the national right to collective bargaining. After all, a Community fun-
damental right to collective bargaining could also encompass protection of the different
options existing nationally. IV. Assessment and Outlook From a labour law point of view, it is conspicuous that the judgements referred to do
not treat collective action and collective agreements as a regulatory problem and as in-
stitutions of working life but as marginal phenomena of the internal market. There is no
discernible effort to deliberate in how far collective arrangements and actions may be
reasonable or necessary in either abstract or concrete terms on account of an asserted
imbalance in the labour market. These phenomena are seen as mere disturbances in the 161 Robert Rebhahn exchange of services. This seems to be a simplistic view of things which may explain
why blockades are simply treated in the same way as strikes for peoples’ own working
conditions, meaning that employers’ interests other than the fundamental freedoms are
ignored. Neither is the following contradiction taken into account: Community law restricts
only actions by employees in situations created by the internal market. These actions
often function as a surrogate for the lack of a secure legal framework for collective
agreements and strikes across internal borders, not least because the Community is not
authorised to regulate strikes and may not be authorised to regulate collective agree-
ments either. However, for as long as the Community fails to regulate these areas it
should exercise constraint in restricting national possibilities. Measured against traditional standards of not only legal doctrine but also general re-
quirements with regard to statement of reasons, the deliberations on the relationship
between fundamental freedoms and fundamental rights in Viking and Laval are little
convincing. The ECJ’s attention and concern is focused exclusively on the fundamental
freedom in question whereas other aspects are relegated to the fringe and all possible
arguments are used in favour of the fundamental freedom. This broad understanding of
fundamental freedoms does indeed correspond to the phrase of effet utile. However,
with regard to Community fundamental rights a similar eagerness for effet utile seems
to be missing, unless even the acknowledgement of unwritten Community fundamental
rights were to be seen as an expression of this principle of interpretation. Fundamental
freedoms rank higher than fundamental rights up until this day, at least in terms of their
effect. The ECJ can thus hardly be considered a guardian of fundamental rights. This is
barely desirable for as much as it may be inherent to existing positive law. IV. Assessment and Outlook A subordination of fundamental rights to fundamental freedoms also becomes appar-
ent when the problem is transposed to national constitutional law. Freedom of estab-
lishment and freedom to provide services are material forms of entrepreneurial freedom. No normal state would consistently favour certain entrepreneurial rights above funda-
mental rights simply because they affect action that goes beyond internal borders. Why,
then, should an organisation that does not even aspire to be a state do it? This paper will leave aside the question whether the ECJ would come to the same
conclusions of its judgements if all missing aspects that have been indicated were to be
included. However, a comprehensive reasoning would have to make obvious the merit
of the ECJ taking seriously the problems that affect many people in the Member States. Moreover, this would also make it easier to accept the results even for those who do not
wish them to turn out exactly as they do for reasons of legal policy in the first place. Justice should not only be done, but should manifestly be seen to be done. European law experts admittedly claim that the ECJ’s decisions should not be meas-
ured by traditional standards. It is said that no mere legal doctrine or even orientation
towards the intentions of the legislature should be expected, but that, apart from the le-
gal surface, the deep structure should be considered and law should be perceived within 162 Relation between Fundamental Freedoms and Fundamental Rights in the recent ECJ’s jurisprudence its political context. These recent decisions clearly favour competition with low labour
costs, also across borders, thereby benefiting certain Member States. This effect is in-
herent in the Posting Directive in particular, if understood in the same way as done by
the ECJ. However, this is not a compelling interpretation. The fact that after enlarge-
ment twelve new Member States are now represented, also in the ECJ, may thus be
relevant for a better understanding of these decisions. The majority of these states will
expect Community law to support their competitive position, at least where postings are
concerned, now that they are after all requested to open up to an inflow of capital, goods
and managers. IV. Assessment and Outlook Observers will therefore claim that these decisions tie in with the expec-
tations of these countries and thus promote integration and that the ECJ would thus be
seen to promote integration and the internal market, even at the expense of traditional
workers’ rights. Acknowledgement of a fundamental right void of concrete conse-
quences then seems to be a means of increasing acceptance. The primacy of context before legal doctrine raises questions about the role of the
ECJ. At a time when political integration and legislative action stalled the ECJ was
hailed as a motor of integration. However, for a number of years now there can be no
talk of constraint in Community legislation. The question is whether the ECJ should
continue to focus its efforts on promoting integration through its decisions even if that
promotion does not result from a compelling inference. If it continues to drive integra-
tion regardless, there is a danger that it will no longer be perceived as a guarantor of law
but as a “courageous“ actor in politics. As a court, the ECJ would be in a position to
reduce the reservations against European policy maintained by many citizens of the Un-
ion. When acting primarily as a political player, however, it will only increase these
concerns. In doing so, it will reduce trust and confidence among a substantial share of
the EU population rather than building it. Übersetzung: Nele Faßnacht 163 https://doi.org/10.5771/9783845219165, am 24.10.2024, 06:46:57
Open Access –
- https://www.nomos-elibrary.de/agb 783845219165, am 24.10.2024, 06:46:57
- https://www.nomos-elibrary.de/agb https://doi.org/10.5771/97838
Open Access –
- ∗ The author would like to thank her Référendaire Ms Ioanna Dervisopoulos for her support in prepar-
ing the manuscript. The ECJ's Interpretation of the Posting Directive in
the Laval and Rüffert Judgements∗ Juliane Kokott I. Introduction
165
II. Protection of workers in the rulings of the European Court of Justice
166
III. Interpretation of the Posting Directive by the European Court of Justice
167
IV. Outlook
171 1 See, inter alia, Case C-303/98 Simap [2008] ECR I-7963); Case C-151/02 Jaeger [2003] ECR I-
8389); Joined cases C-397/01 to C-403/01 Pfeiffer and Others [2004] ECR I-8835; Case C-484/04
Commission v United Kingdom [2006] ECR I-7471; and my Opinion of 9 March 1006 in the latter
case. 3 See, inter alia, Case C-212/04 Adeneler and Others [2006] ECR I-6057; Case C-268/06 Impact
[2008], not yet published in the ECR; as well as my Opinions of 27 October 2005 and 9 January
2008 respectively in these cases. 2 See, inter alia, Case C-173/99 BECTU [2008] ECR I-4881; Case C-124/05 Federatie Nederlandse
Vakbeweging [2006] ECR I-3423; and my Opinion of 12 March 2006 in the latter case. Juliane Kokott However, smooth operation of this Common Market is a difficult undertaking. The
strengthening of competition within the Community, despite being profitable for the
national economies of the Union as a whole, may entail – at least temporary – disadvan-
tages for individual players. The increased competitive pressure involves a risk for
workers to have to accept changed terms and conditions of work or even to lose their
jobs. It is therefore important to make specific efforts to protect their interests. Especially when viewed against the background of the considerable gap in prosperity
that currently still exists within the Union, the process of further developing European
law involves particular challenges such as striking a fair balance between the realisation
of fundamental freedoms on the one hand and the protection and further development of
social standards on the other hand. According to Article 2 of the EU Treaty, the promo-
tion of economic and social progress is one of the Union’s basic objectives. Further-
more, it is an essential concern of the Lisbon Treaty to place more emphasis on the so-
cial dimension of the European Union. I. Introduction The relevance of the social dimension in European Union law and in the jurispru-
dence of the European Court of Justice is a topical and important issue, which is cur-
rently being keenly discussed, especially in the aftermath of some judgments delivered
by the ECJ. This is not surprising. What matters in this respect is to find a fair balance
between the diverse competing interests. It is with great pleasure that I have accepted
the invitation to today’s symposium – not only because of the relevance of the questions
raised – but also because I am of the opinion that the representatives of the European
Institutions should face a dialogue with science and the public. However, the office I hold demands a certain reserve. Although I was not involved as
an Advocate General in the Rüffert and Laval proceedings, which I am going to elabo-
rate on, I may in future handle similar cases. Therefore, I will focus my deliberations on
the basic ideas of the rulings of the European Court of Justice and refrain from com-
menting too much on the judgments. The fundamental idea of European integration is to promote economic progress by
diminishing national hurdles for the movement of persons, goods, services and capital,
thus creating a Common Market. Ultimately, this progress is for the benefit of all citi-
zens in all Member States. 165 II. Protection of workers in the rulings of the European Court of Justice Before I embark on the subject of my lecture today, I would like to call to mind the
diverse contributions that the European Court of Justice has made in the past decades to
the protection of workers. The European Court of Justice realised early on that social rights need strengthening
on the way to a common market. In underlining social rights as an indispensable ele-
ment of the European Union’s legal order, it has strengthened a counterbalance to the
market forces and thus played a vital part in shaping the social dimension of the Euro-
pean Union. Worth mentioning in this respect are firstly the comprehensive rulings on the protec-
tion of workers in the different areas. I would like to quote as examples the Court’s de-
cisions on working hours1, on the granting of holidays2, on fixed-term employment3 166 – The ECJ’s Interpretation of the Posting Directive in the Laval and Rüffert Judgements and on part-time employment4. Or think of the rulings of the Court of Justice, by which
it imposed equal treatment of male and female workers.5 Also the basic principles of the treatment of posted workers were originally devel-
oped in rulings of the European Court of Justice. According to these, Community law
does not in principle preclude the Member States from applying their legal provisions or
the collective agreements on minimum wages concluded between trade unions and em-
ployers to any person who is employed, even temporarily, within their territory.6 How-
ever, the application of such provisions must serve the protection of workers and be
proportionate.7 This jurisprudence has been considered in the 12th recital of the Posting Directive8,
which I would now like to turn to. As was reconfirmed through the lengthy legislative
procedure connected with this directive, we are faced with difficult issues: the realisa-
tion of the free movement of services9 must be harmonised with the protection of work-
ers10 and fair competition11, and thus with the prevention of social dumping. III. Interpretation of the Posting Directive by the
European Court of Justice 4 See, inter alia, Case 171/88 Rinner-Kühn [1989] ECR 2743, and Case C-300/06 Voß [2008], not yet
published in the ECR.
5 See, inter alia, Case 43/75 Defrenne [1976] ECR 455; Joined Cases C-231/06 to C-233/06 Jonk-
mann and Others [2007] ECR I-5149); and my opinion of 29 March 2007 in the latter case
6 See, in particular, Joined Cases 62/81 and 63/81 Seco and Desquenne & Giral [1982] ECR 223, pa-
ragraph 14, and Case C-164/99 Portugaia Construções [2002] ECR I-787, paragraph 21.
7 Cf. in this respect Joined Cases C-369/96 and C-376/96 Arblade and Others [1999] ECR I-8453, pa-
ragraph 36, and Case C-341/02 Commission v Germany [2005] ECR I-2733, paragraph 24.
8 Directive 96/71/EC of the European Parliament and of the Council of 16 December 1996 concern-
ing the posting of workers in the framework of the provision of services, OJ 1997, L 18, p. 1, here-
after: Posting Directive or Directive 96/71.
9 See first recital of Directive 96/71.
10 See first recital of Directive 96/71.
11 See first recital of Directive 96/71. published in the ECR.
5 See, inter alia, Case 43/75 Defrenne [1976] ECR 455; Joined Cases C-231/06 to C-233/06 Jonk-
mann and Others [2007] ECR I-5149); and my opinion of 29 March 2007 in the latter case
6 See, in particular, Joined Cases 62/81 and 63/81 Seco and Desquenne & Giral [1982] ECR 223, pa-
ragraph 14, and Case C-164/99 Portugaia Construções [2002] ECR I-787, paragraph 21.
7 Cf. in this respect Joined Cases C-369/96 and C-376/96 Arblade and Others [1999] ECR I-8453, pa-
ragraph 36, and Case C-341/02 Commission v Germany [2005] ECR I-2733, paragraph 24.
8 Directive 96/71/EC of the European Parliament and of the Council of 16 December 1996 concern-
ing the posting of workers in the framework of the provision of services, OJ 1997, L 18, p. 1, here-
after: Posting Directive or Directive 96/71. 11 See first recital of Directive 96/71. 10 See first recital of Directive 96/71. 9 See first recital of Directive 96/71. III. Interpretation of the Posting Directive by the
European Court of Justice In the Rüffert case, the referring court had asked the Court of Justice whether a
clause guaranteeing that collective wage rates are respected, as provided for in the Pub-
lic Procurement Act of Lower Saxony, constitutes an unjustified limitation of the free
movement of services. The Laval case, apart from touching upon the issue of workers’
strikes, treated the question of whether the Swedish system for determining wage rates
and other terms and conditions of work is in accordance with the guidelines of the Post-
ing Directive and with the free movement of services. According to Article 3 of the Posting Directive, Member States shall ensure that un-
dertakings guarantee workers posted to their territory specific terms and conditions of 8 Directive 96/71/EC of the European Parliament and of the Council of 16 December 1996 concern-
ing the posting of workers in the framework of the provision of services, OJ 1997, L 18, p. 1, here-
after: Posting Directive or Directive 96/71. 8 Directive 96/71/EC of the European Parliament and of the Council of 16 December 1996 concern-
ing the posting of workers in the framework of the provision of services, OJ 1997, L 18, p. 1, here-
after: Posting Directive or Directive 96/71. 9 See first recital of Directive 96/71. 11 See first recital of Directive 96/71. 167 Juliane Kokott work, which, in the recipient Member State, are laid down by law or collective agree-
ments which have been declared universally applicable. Several terms and conditions of
work are then listed in the directive, e.g. maximum work periods or minimum wages. Article 3, paragraph 7 of the Posting Directive states that it is possible to apply terms
and conditions of work and employment that are more favourable to workers. As I underlined at the beginning, I was not involved as an Advocate General in the
cases under discussion. My colleague Advocate General Bot delivered the opinion in the
Rüffert case. Advocate General Bot had pleaded that the Posting Directive be inter-
preted in such a way that it does not prevent a Member State from providing improved
protection of the posted workers at national level12. Advocate General Mengozzi argued
in a similar way in the Laval case.13 I also strongly sympathise with this interpretation
of the Posting Directive. 12 Opinion of Advocate General Bot in Case C-346/06 Rüffert [2008], not yet published in the ECR.
13 Opinion of Advocate General Mengozzi in Case C- 341/05 Laval [2007], not yet published in the
ECR, in particular at points 147 and 151.
14 Opinion of Advocate General Bot in Rüffert (cited in footnote 13, point 70).
15 Opinion of Advocate General Bot in Rüffert (cited in footnote 13, point 94).
16 Opinion of Advocate General Bot in Rüffert (cited in footnote 13, points 114 et seq.). The ECJ’s Interpretation of the Posting Directive in the Laval and Rüffert Judgements The Court of Justice did not follow the Advocate General’s suggestion. In the Court
of Justice’s opinion, the directive provides for a limited set of rules for the protection of
posted workers. In its view, the directive lists those modalities that allow national legis-
lators to extend their provisions on terms and conditions of work to include posted
workers. According to this interpretation, a minimum wage can be determined either by
law or by declaring a collective agreement universally applicable.17 In the Court of Justice’s opinion, Article 3, paragraph 7 of the Posting Directive does
not authorise Member States to impose terms and conditions of work and employment
that are not listed in paragraph 1. In its view, only such interpretation of the directive
prevents the directive from being to no avail. The listing of certain areas and certain
modalities by which terms and conditions of work could be extended to include posted
workers would be meaningless if, by means of paragraph 7, more favourable terms and
conditions of work and employment, such as collective wage rates exceeding the mini-
mum wage rates, could be imposed unilaterally using clauses guaranteeing that collec-
tive wage rates are respected. A different interpretation would result in depriving the
Posting Directive of its effectiveness.18 According to the Court of Justice, any protec-
tion reaching beyond the provisions of Article 3, paragraph 1 of the Posting Directive
seems therefore to be admissible only in exceptional cases pursuant to Article 3, para-
graph 10 if they are provisions relating to the area of public order.19 Therefore, in the Court of Justice’s opinion, the Posting Directive does not entitle a
Member State to decree a clause guaranteeing that collective wage rates are respected. The Court subsequently examined whether this interpretation of the directive was con-
firmed by its appreciation in the light of the free movement of services.20 The Court re-
examines the clause guaranteeing that collective wage rates are respected once again on
the basis of the free movement of services. Like Advocate General Bot, it affirms a
limitation of the free movement of services. However, unlike the Advocate General, the
Court denies the justification of the limitation for compelling reasons of the general
good. 17 In case the national legal system does not provide for a declaration of universal application, Mem-
ber States, pursuant to Article 3, paragraph 8 of Directive 96/71, can chose the following means:
universally applicable collective agreements or representative collective agreements. 17 In case the national legal system does not provide for a declaration of universal application, Mem-
ber States, pursuant to Article 3, paragraph 8 of Directive 96/71, can chose the following means:
universally applicable collective agreements or representative collective agreements.
18 Case C-341/05 Laval [2007], not yet published in the ECR, paragraph 80, and Case C-346/06 Rüf-
fert [2008], not yet published in the ECR, paragraph 33.
19 See Laval (cited in footnote 19, paragraph 82) and Case C-319/06 Commission v Luxembourg
[2008], not yet published in the ECR, paragraph 27.
20 Rüffert (cited in footnote 18, paragraph 36 et seq.).
21 Rüffert (cited in footnote 18, paragraph 40). 21 Rüffert (cited in footnote 18, paragraph 40). III. Interpretation of the Posting Directive by the
European Court of Justice Advocate General Bot sees the directive as a “hard core” of protection provisions that
must be regarded as mere minimum guarantees to the benefit of posted workers. In his
view, the directive transformed an option for regulation, as it was given to the Member
States until then, into an obligation for regulation. As I have set forth, the Court of Jus-
tice had found even before the Posting Directive was adopted that Member States may
on certain conditions apply its legal provisions to posted workers as well. In the Advo-
cate General’s opinion, the Posting Directive now turned this permission into an obliga-
tion with regard to certain minimum provisions for the protection of workers.14 In the
Advocate General’s view, it can be concluded from the directive’s Article 3, paragraph
7 that Member States may grant posted workers protection exceeding these require-
ments. A clause guaranteeing that collective wage rates are respected therefore constitutes,
according to Advocate General Bot, an admissible additional means of protection at
national level as defined by Article 3, paragraph 7 of the Posting Directive.15 Any pro-
tection measure taken by Member States that reaches beyond the directive’s provisions
must, however, be compatible with fundamental freedoms as well. It is, in Bot’s view,
beyond doubt that such a regulation is a limitation of the free movement of services. However, he regards this limitation as justified because it guarantees the protection of
workers and prevents social dumping.16 Ultimately, Advocate General Bot had there-
fore proposed to the Court of Justice to interpret the Posting Directive and the free
movement of services to allow a clause guaranteeing that collective wage rates are re-
spected, provided that this clause respect the prohibition of discrimination based on na-
tionality as well as the principle of transparency. 168 10.577
– The ECJ’s Interpretation of the Posting Directive in the Laval and Rüffert Judgements 18 Case C-341/05 Laval [2007], not yet published in the ECR, paragraph 80, and Case C-346/06 Rüf-
fert [2008], not yet published in the ECR, paragraph 33. [
],
y
p
, p
g p
19 See Laval (cited in footnote 19, paragraph 82) and Case C-319/06 Commission v Luxembourg
[2008], not yet published in the ECR, paragraph 27. 20 Rüffert (cited in footnote 18, paragraph 36 et seq.). ber States, pursuant to Article 3, paragraph 8 of Directive 96/71, can chose the following means:
universally applicable collective agreements or representative collective agreements.
18 Case C-341/05 Laval [2007], not yet published in the ECR, paragraph 80, and Case C-346/06 Rüf-
fert [2008], not yet published in the ECR, paragraph 33.
19 See Laval (cited in footnote 19, paragraph 82) and Case C-319/06 Commission v Luxembourg
[2008], not yet published in the ECR, paragraph 27.
20 Rüffert (cited in footnote 18, paragraph 36 et seq.).
21 Rüffert (cited in footnote 18, paragraph 40). The ECJ’s Interpretation of the Posting Directive in the Laval and Rüffert Judgements It bases its arguments in particular on its opinion that the clause guaranteeing that
collective wage rates are respected only applies to public contract awarders and not to
private principals at the same time.21 This line of reasoning is certainly not compulsive. It can be concluded from the Court of Justice’s deliberations in the Laval and Rüffert
judgments that it regards the options for regulation listed in the Posting Directive as
final and considers in principle inadmissible any measures reaching beyond them. It 17 In case the national legal system does not provide for a declaration of universal application, Mem-
ber States, pursuant to Article 3, paragraph 8 of Directive 96/71, can chose the following means:
universally applicable collective agreements or representative collective agreements. 18 Case C-341/05 Laval [2007], not yet published in the ECR, paragraph 80, and Case C-346/06 Rüf-
fert [2008], not yet published in the ECR, paragraph 33. 169 /10.5771/9
– Juliane Kokott therefore seems to me that, in the view of the Court of Justice, the directive constitutes a
final codified weighing up of the free movement of services on the one hand and the
protection of workers on the other hand. In its Commission/Luxembourg judgment of last week the Court of Justice seems to
substantiate its view of the final nature of the Posting Directive.22 This case was,
among other things, about a Luxembourg provision which extended the automatic ad-
justment of wages to the level of the cost of living to include posted workers. The
Commission did not find fault with the minimum wage rates being adjusted to the cost
of living. In its view, this is admissible pursuant to the Posting Directive. However, it
considered the fact that the indexation concerned all wages, including those that were
not minimum wages, to violate the directive. This was confirmed by the Court of Jus-
tice.23 In its view, such indexation measure is not provided for in the Posting Directive,
which gives an exhaustive list of those aspects with regard to which Member States can
extend their national provisions to include posted workers. 23 Commission v Luxembourg (cited in footnote 20, paragraph 47). 22 Commission v Luxembourg (cited in footnote 20, paragraph 26). 25 In case the national legal system does not provide for a declaration of universal application, Mem-
ber States, pursuant to Article 3, paragraph 8 of Directive 96/71, can chose the following means:
universally applicable collective agreements or representative collective agreements. 24 Commission v Luxembourg (cited in footnote 20, paragraph 54). IV. Outlook The Court of Justice’s judgments in the Viking, Laval and Rüffert cases are currently
strongly criticised. Previous speakers have also voiced various concerns today. Fur-
thermore, the submissions of Advocates General Bot and Mengozzi show that there is no
unanimous opinion even within the Court of Justice. In this respect, I would like to emphasise one particular aspect to which many critics,
who, for example, consider the Laval judgment an indicator of the demise of the right to
strike, do not pay sufficient attention in my view. One should be aware of the fact that
the Court of Justice, in the relevant judgments, ruled only on the particular question
referred for a preliminary ruling. The origin of a preliminary ruling procedure is always
a concrete legal dispute with its various particularities. When reading the judgments
carefully, one establishes that the Court of Justice puts particular emphasis on these par-
ticularities and bases its judgment on them. For example, in the Laval judgment it un-
derlined the national context in which the collective measures were embedded. It was
characteristic of this specific context that there were no provisions that would have been
sufficiently detailed and accessible to allow for an undertaking to establish which obli-
gation it has to respect with regard to the minimum wage.26 The Court of Justice has not made a general statement on the relation between fun-
damental freedoms and fundamental rights. In particular, it has not found in its recent
rulings in question that the right to strike or, worse, human dignity must in principle
take second place to fundamental freedoms. Rather, fundamental freedoms and funda-
mental Community rights are at the same level and must be harmonised. By placing particular emphasis on the specific characteristics of the cases, the Court
of Justice, in my view, clearly shows its intention to diversify its rulings on the basis of
the particularities of future proceedings referred for preliminary rulings. Let me stress the following in conclusion: in spite of all the criticism concerning the
discussed judgments it must not be overlooked that in them the Court of Justice makes
in principle also positive statements from a social point of view: it recognises the right
to strike as a fundamental right. The ECJ’s Interpretation of the Posting Directive in the Laval and Rüffert Judgements The ECJ’s Interpretation of the Posting Directive in the Laval and Rüffert Judgements 26 Laval (cited in footnote 19, paragraph 110).
27 Paragraph 103 of Laval (cited in footnote 19) states verbatim: “… that the right to take collective
action for the protection of the workers of the host State against possible social dumping may
constitute an overriding reason of public interest“. In this respect, see currently only Case C-244/04
Commission v Germany [2006] ECR I-885, paragraph 61. The ECJ’s Interpretation of the Posting Directive in the Laval and Rüffert Judgements Furthermore, the Court states
that Luxembourg has not provided sufficient proof of the fact that this provision is a
rule pertaining to the area of public order as defined by the Posting Directive.24 In its
judgment the Court of Justice now examines the measure only on the basis of the direc-
tive and, unlike in the Rüffert case, not additionally on the basis of the free movement
of services. What room for manoeuvre for the protection of workers does the Court of Justice’s
interpretation consequently leave to the Member States? What is undoubtedly admissi-
ble according to rulings of the Court of Justice are regulations regarding those aspects
of the terms and conditions of work that are explicitly listed in the directive. As regards
the manner in which these terms and conditions of work may be extended to include
posted workers, Member States have two options to choose from: statutory regulation or
a collective agreement declared universally applicable.25 Posting undertakings certainly benefit from the Court of Justice’s interpretation inso-
far as it provides increased legal certainty and predictability of the wages to be paid for
services rendered in another Member State. There is, however, the risk of wages in the
recipient Member State to drop to a minimum standard. 170 10.577
– 26 Laval (cited in footnote 19, paragraph 110). IV. Outlook And, remarkably, in the Laval judgment it takes up the
concept of social dumping and regards the right to strike for the protection of workers
against social dumping as an overriding reason of public interest.27 I am now coming to my concluding remarks. In my opinion, there is a risk, espe-
cially in public discussion, of overrating the Rüffert and Laval decisions. Both decisions
are not turning points at the end of a long line of decisions by the Court of Justice per- 171 /10.5771/97
– Juliane Kokott taining to the area of fundamental freedoms and social rights. As this symposium will
also certainly show, it must rather be established that these decisions are examples of
the European Court of Justice’s rulings, striving to strike the right balance in a fast-
evolving Union and to take new aspects into account on a case-by-case basis. If we know one thing for sure, it is that the Court of Justice will further develop and
differentiate its rulings. The public discussion among experts and discussion forums as
we are having one here today make an important contribution in this respect. It is a
valuable prospect that this event today opens up the opportunity to get to know not only
the German point of view but, thanks to numerous participants from other Member
States, also that of others. Übersetzung: Bundesministerium für Arbeit und Soziales 172
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Impact of the Marie Curie Cancer Care Delivering Choice Programme in Somerset and North Somerset on place of death and hospital usage: a retrospective cohort study
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BMJ supportive & palliative care
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Purdy, S., Lasseter, G., Griffin, T. P., & Wye, L. (2015). Impact of the
Marie Curie Cancer Care Delivering Choice Programme in Somerset
and North Somerset on place of death and hospital usage: a
retrospective cohort study. BMJ Supportive and Palliative Care, 5(1),
34-39. https://doi.org/10.1136/bmjspcare-2013-000645 Publisher's PDF, also known as Version of record
License (if available):
CC BY
Link to published version (if available):
10.1136/bmjspcare-2013-000645 Publisher's PDF, also known as Version of record
License (if available):
CC BY
Link to published version (if available):
10.1136/bmjspcare-2013-000645
Link to publication record on the Bristol Research Porta
PDF-document Link to publication record on the Bristol Research Portal
PDF-document This is the final published version of the article (version of record). It first appeared online via BMJ at
http://spcare.bmj.com/content/5/1/34. Please refer to any applicable terms of use of the publisher. Purdy, S., Lasseter, G., Griffin, T. P., & Wye, L. (2015). Impact of the
Marie Curie Cancer Care Delivering Choice Programme in Somerset
and North Somerset on place of death and hospital usage: a
retrospective cohort study. BMJ Supportive and Palliative Care, 5(1),
34-39. https://doi.org/10.1136/bmjspcare-2013-000645 Purdy, S., Lasseter, G., Griffin, T. P., & Wye, L. (2015). Impact of the
Marie Curie Cancer Care Delivering Choice Programme in Somerset
and North Somerset on place of death and hospital usage: a
retrospective cohort study. BMJ Supportive and Palliative Care, 5(1),
34-39. https://doi.org/10.1136/bmjspcare-2013-000645 ABSTRACT supporting people to die in their own
homes.1 From 2008 to 2011, Marie
Curie Cancer Care worked with local
professionals to develop end-of-life ser-
vices in several parts of the UK, including
North Somerset (North Somerset) and
Somerset,
two
counties
in
southwest
England.2 All palliative patients, regard-
less of condition, were eligible for the
Delivering
Choice
Programme
(DCP),
which included. Objectives The Marie Curie Cancer Care
Delivering Choice Programme (DCP) aims to help
palliative patients be cared for in their place of
choice. In this study, new palliative care services
delivered in two counties in England included
end-of-life care coordination centres, an out-of-
hours telephone line and discharge in-reach
nurses. The study aimed to investigate the
impact of DCP on place of death and hospital
usage (emergency department (ED) and
admissions). Correspondence to
Dr Sarah Purdy, School of Social
and Community Medicine,
University of Bristol, Canynge
Hall, 39 Whatley Road,
Bristol BS8 2PS, UK;
Sarah.Purdy@bristol.ac.uk Correspondence to
Dr Sarah Purdy, School of Social
and Community Medicine,
University of Bristol, Canynge
Hall, 39 Whatley Road,
Bristol BS8 2PS, UK;
Sarah.Purdy@bristol.ac.uk Correspondence to
Dr Sarah Purdy, School of Social
and Community Medicine,
University of Bristol, Canynge
Hall, 39 Whatley Road,
Bristol BS8 2PS, UK;
Sarah.Purdy@bristol.ac.uk ▸
An
out-of-hours
(OOH)
advice
and
response line manned by specialist pallia-
tive care nurses from 17:00 to 1:00, week-
ends and bank holidays, who responded to
calls from professionals, family carers and
patients (Somerset). An abstract outlining the results
of this quantitative evaluation
and the accompanying
qualitative evaluation of the
Delivering Choice Programme
was previously published as a
conference abstract of the 42nd
Society for Academic Primary
Care Annual Conference 3rd to
5th July 2013, Nottingham:
Purdy S, Wye L, Lasseter G,
et al. Improving care and
reducing hospital use at the end
of life: Results of a mixed
methods evaluation of Marie
Curie’s Delivering Choice
Programme. Prim Health Care
Res Dev 2013;14(Suppl 1):65–6. Methods Retrospective cohort of all eligible
palliative patients who died over a 6-month
period in two counties (n=3594). Participants
were those who died of conditions considered to
be eligible for end-of-life care, as defined by the
Public Health England National End of Life Care
Intelligence Network. The sample included
people who did and did not access DCP services. DCP service, hospital admission and ED use data,
demographic and death data were collected on
all eligible participants. ABSTRACT Data were analysed using
descriptive statistics and logistic regression. Results After adjusting for potential
confounders, those using Delivering Choice were
at least 30% less likely to die in hospital or have
an emergency hospital admission or ED visit in the
last 30 or 7 days of life than those who did not. Conclusions Recipients of DCP services were less
likely to die in or use hospital services. Those
considering new ways of providing end-of-life
care could explore the possibility of adopting
similar services and evaluating the outcomes from
patient, carer and system perspectives. ▸
Two
‘front
of
house’
hospital-based
end-of-life care discharge-in-reach nurses
who identified patients who wanted a non-
hospital death
and facilitated fast dis-
charges accordingly (Somerset). ▸
Two end-of-life care coordination centres
(one in each county) that took referrals
from community, hospital and hospice
staff to organise packages of care including
equipment,
night
nurses
and
personal
carers. In both counties, personal care was
supplied from local agencies, but North
Somerset
also
had
its
own
in-house
generic support team. Received 31 December 2013
Revised 8 April 2014
Accepted 30 April 2014
Published Online First
16 May 2014 ▸
These services were supported by an elec-
tronic end-of-life care register (EOL regis-
ter) to record advanced care wishes. In addition, in North Somerset, two
end-of-life
care
facilitators
were
employed. These staff provided specific
training, primarily to care homes and
community
teams,
on
issues
such
as
advance care planning, end-of-life care INTRODUCTION 1Centre for Academic Primary
Care, School of Social and
Community Medicine, University
of Bristol, Bristol, UK
2School for Policy Studies,
University of Bristol, Bristol, UK Sarah Purdy,1 Gemma Lasseter,1 Thomas Griffin,2 Lesley Wye1 Sarah Purdy,1 Gemma Lasseter,1 Thomas Griffin,2 Lesley Wye1 Sarah Purdy,1 Gemma Lasseter,1 Thomas Griffin,2 Lesley Wye1 1Centre for Academic Primary
Care, School of Social and
Community Medicine, University
of Bristol, Bristol, UK
2School for Policy Studies,
University of Bristol, Bristol, UK
Correspondence to
Dr Sarah Purdy, School of Social
and Community Medicine,
University of Bristol, Canynge
Hall, 39 Whatley Road,
Bristol BS8 2PS, UK;
Sarah.Purdy@bristol.ac.uk 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/ group.bmj.com
on March 27, 2017 - Published by
http://spcare.bmj.com/
Downloaded from group.bmj.com
on March 27, 2017 - Published by
http://spcare.bmj.com/
Downloaded from Research 1Centre for Academic Primary
Care, School of Social and
Community Medicine, University
of Bristol, Bristol, UK
2School for Policy Studies,
University of Bristol, Bristol, UK
Correspondence to
Dr Sarah Purdy, School of Social
and Community Medicine,
University of Bristol, Canynge
Hall, 39 Whatley Road,
Bristol BS8 2PS, UK;
Sarah.Purdy@bristol.ac.uk Participant characteristics From 1 September 2011 to 29 February 2012, 1022
people potentially eligible for end-of-life care died in
North Somerset and 2572 in Somerset (table 1). The
overall mean age at death was 81 (North Somerset) and
82 (Somerset) with comparable proportions of women
(54% and 55%) and men (46% and 45%). Commonest
causes of death were cancer (North Somerset 28%,
Somerset 29%), heart disease (18%, 18%), respiratory
disease (15%, 13%) and dementia (15%, 13%). Hospital admissions In the last 30 days of life, emergency hospital admis-
sions were proportionally lower in both counties
among those receiving a Delivering Choice interven-
tion compared with non-users (table 1). For both Duration of use of services
h
d In North Somerset, median time for accessing the care
coordination centre was 8 days before death (IQR 4–21). In Somerset, median time for accessing the care coordin-
ation centre was 9.5 days before death (IQR 4–20),
while median time to death after first contact for the
discharge in-reach service was 6 days (IQR 2–23) and for
the OOH line 10 days (IQR 2–31). INTRODUCTION Since the publication of the End of Life
Care Strategy in the UK in 2008, there
has
been
increasing
emphasis
on 34 Purdy S, et al. BMJ Supportive & Palliative Care 2015;5:34–39. doi:10.1136/bmjspcare-2013-000645 group.bmj.com
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ination centre (153, 15%), followed by entry on the
EOL register (93, 9%) (table 2). Less than 5% of
people had a key worker listed on the EOL register
(34, 4%) or accessed a generic support worker (GSW)
(25, 2%). Data on who used GSW services were only
available for 101 of the potential 181 days of the
study. pathways, use of the electronic end-of-life care register
and medication including syringe drivers. No patient-
level outcome data were linked to this intervention. The aim of this study was to investigate the impact
of Delivering Choice on place of death and hospital
admissions and emergency department (ED) use. A
parallel qualitative evaluation has been published
elsewhere.3 Of the 2572 people in Somerset who were eligible,
616 (24%) used one or more DCP intervention (table 2). The most frequently used were entry on the EOL register
(331, 13%), the care coordination centre (294, 11%) and
the OOH advice line (243, 9%). Fewer people had a key
worker listed on the EOL register (156, 6%) or were
seen by a discharge in-reach nurse (144, 6%). METHODS
h
l The sample was a retrospective cohort of people who
died between 1 September 2011 and 29 February
2012 in North Somerset and Somerset and whose
deaths were expected and potentially eligible for
end-of-life care according to criteria derived by the
UK National End of Life Care Intelligence Network.4
Quantitative data on use of Delivering Choice services
were collected and linked via NHS number, age and
postcode by National Health Service analysts to the
Primary Care Mortality Database, in-patient hospital
data and ED data obtained from NHS Connecting for
Health. All patient data were pseudonymised during
linkage. Differences between Delivering Choice and
non-Delivering Choice users for place of death, emer-
gency hospital admissions and ED visits in the last 30
and 7 days of life were explored. In addition, length
of time between date of first contact with a Delivering
Choice service and date of death was calculated, as
was a Charlson Comorbidity Index for those people
where
data
on
comorbidities
were
available.5
Univariable analyses and multivariable logistic regres-
sion were undertaken using STATA V.12. The regres-
sion models adjusted for a priori confounding factors,
including gender, age, deprivation, cause of death and
other DCP services received. A much higher proportion of DCP users died from
cancer than non-DCP users (table 1). The distribution
of deprivation scores relative to England as a whole was
similar for DCP users and non-users in both counties. Data on comorbidities were only available for those
who had a hospital admission during the 6 months
before death. Among these people, those who accessed
DCP services had higher levels of comorbidity than
those who did not access DCP (table 1). Place of death
Th
i i Those receiving Delivering Choice were less likely to
die in hospital (table 1). In North Somerset, the OR
for dying in hospital was 0.33 (95% CI 0.21 to 0.50,
p≤0.001) after adjusting for gender, age, deprivation
and cause of death (table 3). In Somerset, the OR was
0.20 (95% CI 0.17 to 0.27, p<0.001). Looking at
individual interventions, in North Somerset those
who used the care coordination centre were less likely
to die in hospital (OR 0.42, 95% CI 0.25 to 0.69,
p<0.001) as were those patients entered on the EOL
register (OR 0.30, 95% CI 0.13 to 0.69, p=0.005). In Somerset, patients accessing the care coordination
centre were less likely to die in hospital (OR 0.11,
95% CI 0.06 to 0.22, p<0.001) as were those who
used the OOH advice line (OR 0.34, 95% CI 0.20 to
0.57, p<0.001) or were entered on the EOL register
(OR 0.22, 95% CI 0.12 to 0.40, p<0.001). The study was approved by local NHS Research and
Development managers as a service evaluation, there-
fore NHS Research Ethics Committee and Research
Governance approvals were not required. However,
the study was reviewed by the ethics committee of the
Faculty of Medicine at the University of Bristol and
relevant Caldicott permissions were obtained. Using Delivering Choice Of the 1022 individuals in North Somerset who were
eligible, 213 (21%) used one or more Delivering 35 Purdy S, et al. BMJ Supportive & Palliative Care 2015;5:34–39. Using Delivering Choice doi:10.1136/bmjspcare-2013-000645 group.bmj.com
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Characteristics of end-of-life populations, place of death and hospital use
North Somerset
(n=1022) (%)*
Somerset
(n=2572) (%)*
Delivering choice
Yes
No
Yes
No
N
213
809
616
1956
Age at death (mean SD)
79.4 (10.7)
81.2 (12.8)
77.3 (12.5)
82.4 (11.4)
Gender male
109 (51)
361 (45)
301 (49)
864 (44)
Deprivation (index of multiple deprivation)
Least deprived
67 (31)
242 (30)
94 (15)
259 (13)
Below average deprivation
63 (30)
229 (28)
213 (35)
631 (32)
Average deprivation
33 (15)
119 (15)
178 (29)
629 (32)
Above average deprivation
10 (5)
65 (8)
100 (16)
345 (18)
Most deprived
37 (17)
139 (17)
29 (5)
80 (4)
Not available
3 (1)
15 (2)
2 (0)
12 (1)
Charlson score†
Number of individuals
168 (79)
506 (63)
513 (83)
1321 (68)
Weighted score
4.6
3.0
5.3
2.9
Cause of death
Cancer
145 (68)
142 (18)
440 (71)
314 (16)
Heart disease
15 (7)
170 (21)
44 (7)
430 (22)
Cerebrovascular
9 (4)
78 (10)
19 (3)
218 (11)
Respiratory
14 (7)
142 (18)
32 (5)
308 (16)
Dementia
12 (6)
145 (18)
30 (5)
316 (16)
Other
18 (8)
132 (16)
51 (8)
370 (19)
Place of death
Acute hospital
40 (19)
347 (43)
84 (14)
836 (43)
Home‡
88 (41)
225 (28)
337 (54)
779 (40)
Care home (not usual place of residence)
34 (16)
116 (14)
58 (9)
173 (9)
Hospice
34 (16)
30 (4)
98 (16)
55 (3)
Community hospital
N/A
N/A
31 (5)
31 (5)
Elsewhere
17 (8)
91 (11)
8 (1)
12 (1)
Hospital utilisation
Patients with one or more emergency admissions <30 days (SD)
61 (29)
335 (41)
233 (38)
875 (45)
Mean emergency admissions per patient <30 days (SD) [median, min, max]
0.31 (0.52)
[0,0,2]
0.47 (0.60)
[0,0,3]
0.53 (0.69)
[0,0,3]
0.45 (0.64)
[0,0,7]
Patients with one or more emergency admission <7 days (SD)
13 (6)
179 (22)
60 (10)
467 (24)
Mean emergency admissions per patient <7 days (SD) [median, min, max]
0.07 (0.27)
[0,0,2]
0.23 (0.43)
[0,0,2]
0.11 (0.33)
[0,0,2]
0.25 (0.46)
[0,0,2]
Patients with one or more ED attendance <30 days (SD)
54 (25)
363 (36)
159 (26)
712 (36)
Mean ED attendance per patient <30 days (SD) [median, min, max]
0.27 (0.50)
[0,0,4]
0.52 (0.64)
[0,0,2]
0.39 (0.51)
[0,0,3]
0.41 (0.60)
[0,0,5]
Patients with one or more ED attendance <7 days
13 (6)
213 (26)
43 (7)
432 (22)
Mean ED attendance per patient <7 days (SD) [median, min, max]
0.07 (0.29)
[0,0,2]
0.27 (0.47)
[0,0,2]
0.07 (0.27)
[0,0,2]
0.26 (0.43)
[0,0,3]
*Figure in parentheses refers to percentage unless otherwise indicated. Using Delivering Choice ED visits
In both counties, attendance at ED in the last 30 days
of life was proportionately lower among DCP users
Table 2
Delivering choice service use
Service
North Somerset
N=1022 (%)
Somerset
N=2572 (%)
Care coordination centre
153 (15)
294 (11)
EOL register
93 (9)
331 (13)
Key worker on EOL register
34 (4)
156 (6)
Generic support worker*
25 (2)
N/A
Out of hours advice line
N/A
243 (9)
Discharge in reach nurse
N/A
144 (6)
Total people using DCP
213 (21)
616 (24)
*Data were only available for 101 days. DCP, Delivering Choice Programme. Research
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in the last week of life only (OR 0.44, 95% CI 0.25
to 0.78, p=0.005). Entry on the EOL register in
Somerset was associated with lower risk of admission
(30 days: OR 0.41, 95% CI 0.28 to 0.60; 7 days: OR
0.57, 95% CI 0.33 to 0.98, p=0.043) but not in
North Somerset. Readmissions after accessing the dis-
charge in-reach service were low at 6%. Using Delivering Choice †Charlson score was calculated for those patients who had a hospital admission. ‡Home includes a care home where this was recorded as usual place of residence
Research coordination centres appeared to be the most effective
components of the interventions at both 30 days
(North Somerset: OR 0.55, 95% CI 0.34 to 0.90,
p=0.016; and Somerset: OR 0.58, 95% CI 0.42 to
0.80, p<0.001) and 7 days (North Somerset: OR
0.09, 95% CI 0.02 to 0.39, p<0.001; and Somerset:
OR, 0.26 95% CI 0.15 to 0.46, p<0.001). The OOH 30-day (North Somerset: OR 0.49, 95% CI 0.33 to
0.74, p<0.001; and Somerset: OR 0.61, 95% CI
0.48 to 0.76, p<0.001) and 7-day (North Somerset:
OR, 0.22 95% CI 0.12 to 0.44, p<0.001; Somerset:
OR 0.32, 95% CI 0.23 to 0.45, p<0.001) results,
these findings persist after adjusting for gender, age,
deprivation and cause of death (table 3). The two 30-day (North Somerset: OR 0.49, 95% CI 0.33 to
0.74, p<0.001; and Somerset: OR 0.61, 95% CI
0.48 to 0.76, p<0.001) and 7-day (North Somerset:
OR, 0.22 95% CI 0.12 to 0.44, p<0.001; Somerset:
OR 0.32, 95% CI 0.23 to 0.45, p<0.001) results,
these findings persist after adjusting for gender, age,
deprivation and cause of death (table 3). The two Purdy S, et al. BMJ Supportive & Palliative Care 2015;5:34–39. doi:10.1136/bmjspcare-2013-000645 36 group.bmj.com
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Downloaded from advice line was associated with lower risk of admission
in the last week of life only (OR 0.44, 95% CI 0.25
to 0.78, p=0.005). Entry on the EOL register in
Somerset was associated with lower risk of admission
(30 days: OR 0.41, 95% CI 0.28 to 0.60; 7 days: OR
0.57, 95% CI 0.33 to 0.98, p=0.043) but not in
North Somerset. Readmissions after accessing the dis-
charge in-reach service were low at 6%. Using Delivering Choice ED visits
In both counties, attendance at ED in the last 30 days
of life was proportionately lower among DCP users In both counties, attendance at ED in the last 30 days
of life was proportionately lower among DCP users Table 3
Impact of Delivering Choice services versus non-Delivering Choice services on deaths in hospital, emergency admissions and
A&E use*
North Somerset
Somerset
OR
95% CI
p Value
OR
95% CI
p Value
Death in hospital overall
0.33
0.21 to 0.50
<0.001
0.20
0.17 to 0.27
<0.001
Key worker
0.74
0.18 to 3.05
0.679
0.73
0.27 to 1.95
0.524
Care coordination centre
0.42
0.25 to 0.69
0.001
0.11
0.06 to 0.22
<0.001
Generic support workers
0.34
0.04 to 2.64
0.30
N/A
–
–
EOL register
0.30
0.13 to 0.69
0.005
0.22
0.12 to 0.40
<0.001
Out-of-hours advice line
N/A
–
–
0.34
0.20 to 0.57
<0.001
Discharge in reach nurses
N/A
–
–
1.6
0.98 to 2.60
0.06
Emergency admission <30 days
0.49
0.33 to 0.74
0.001
0.61
0.48 to 0.76
<0.001
Key worker
0.60
0.21 to 1.69
0.33
1.13
0.68 to 1.87
0.642
Care coordination centre
0.55
0.34 to 0.90
0.016
0.58
0.42 to 0.80
0.001
Generic support workers
0.35
0.08 to 1.59
0.175
N/A
–
–
EOL register
0.65
0.33 to 1.30
0.225
0.41
0.28 to 0.60
<0.001
Out-of-hours advice line
N/A
–
–
0.78
0.56 to 1.10
0.159
Discharge in reach nurses
N/A
–
–
4.15
2.68 to 6.43
<0.001
Emergency admission <7 days
0.22
0.12 to 0.44
<0.001
0.32
0.23 to 0.45
<0.001
Key worker
1.13
0.18 to 7.03
0.898
1.04
0.45 to 2.40
0.934
Care coordination centre
0.09
0.02 to 0.39
0.001
0.26
0.15 to 0.46
<0.001
Generic support workers
6.26
0.81 to 48.65
0.079
N/A
–
–
EOL register
0.39
0.11 to 1.34
0.136
0.57
0.33 to 0.98
0.043
Out-of-hours advice line
N/A
–
–
0.44
0.25 to 0.78
0.005
Discharge in reach nurses
N/A
–
–
1.54
0.95 to 2.50
0.081
A&E attendance <30 days
0.41
0.28 to 0.62
<0.001
0.66
0.51 to 0.85
0.001
Key worker
0.71
0.25 to 2.02
0.515
1.23
0.71 to 2.13
0.454
Care coordination centre
0.46
0.29 to 0.76
0.002
0.58
0.40 to 0.82
0.002
Generic support workers
0.45
0.10 to 2.01
0.295
N/A
–
–
EOL register
0.57
0.29 to 1.11
0.097
0.61
0.40 to 0.92
0.018
Out-of-hours advice line
N/A
–
–
0.60
0.41 to 0.87
0.007
Discharge in reach nurses
N/A
–
–
3.29
2.23 to 4.87
<0.001
A&E attendance <7 days
0.22
0.11 to 0.42
<0.001
0.32
0.22 to 0.47
<0.001
Key worker
1.16
0.17 to 7.84
0.878
0.85
0.32 to 2.20
0.730
Care coordination centre
0.15
0.05 to 0.43
<0.001
0.24
0.12 to 0.48
<0.001
Generic support workers
2.39
0.27 to 21.61
0.437
N/A
–
–
EOL register
0.38
0.12 to 1.27
0.116
0.79
0.44 to 1.41
0.427
Out-of-hours advice line
N/A
–
–
0.34
0.17 to 0.70
0.003
Discharge in reach nurses
N/A
–
–
1.25
0.74 to 2.11
0.427
*Adjusted for gender, age, deprivation and condition. Purdy S, et al. BMJ Supportive & Palliative Care 2015;5:34–39. doi:10.1136/bmjspcare-2013-000645 DISCUSSION Patients who used Delivering Choice services were
less likely to die in hospital, have emergency admis-
sions or visit ED within 30 or 7 days before death
than those who did not use Delivering Choice. The
two coordination centres appeared to have the biggest
impact. There are a number of limitations to this study. Randomisation of patients was not practical in this
service development. We addressed potential confoun-
ders such as age, gender, deprivation and cause of
death. However, there are other ways in which the
patient groups may have differed. For example, we were
unable to include data on comorbidities in the logistic
regression analyses. It is also possible that patients
referred to the Delivering Choice services were people
that a district nurse or GP considered were suitable to
die at home, or who possibly wished to die at home,
and the service was used to help facilitate this. This
potential confounding by indication would result in
increased uptake of the service by those who might have
died at home anyway. In view of the limitations of the
study design, we have reported associations but can
make no claims for causal relationships. As only a third of patients accessing Delivering
Choice had non-cancer diagnoses, this would suggest
that primary healthcare teams should be encouraged
to identify more patients for a palliative care approach
and service providers should also redesign services
that are manifestly appropriate for non-malignant
patients. The study also highlights the difficulties of
indentifying palliative patients in a timely way as half
of Delivering Choice patients came into contact with
the services just 6–20 days before death. Previous
research in Scotland has identified that encouraging
GPs to identify palliative care patients can be benefi-
cial as 60% of patients on the GP palliative care regis-
ter died at home, whereas 60% of people who were
not on the palliative care register died in hospital.9 10
However, GPs find early identification of non-cancer
palliative care patients to be particularly challenging.11
Therefore, other interventions to improve the identifi-
cation of patients for palliative care need to be put in
place, in addition to providing services such as
Delivering Choice for those already identified to be at
the end of life. If services can identify and care for
patients earlier, there is a possibility that hospital use Less than a quarter of all potential patients accessed
Delivering Choice services. Using Delivering Choice e services versus non-Delivering Choice services on deaths in hospital, emergency admissions and 37 Purdy S, et al. BMJ Supportive & Palliative Care 2015;5:34–39. doi:10.1136/bmjspcare-2013-000645 group.bmj.com
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Downloaded from Research The most effective components of the DCPs in both
counties appear to be the care coordination centres. A
qualitative
evaluation
of
the
components
of
Delivering Choice identified a number of factors that
contribute to further understanding of the effective-
ness of the individual interventions.2 The care coord-
ination centres in the two counties operated in
different ways. The North Somerset centre had an
in-house model that included a fast-track coordinator,
nurse assessors and its own team of personal care
workers (GSWs). This maximised their flexibility to
respond to patient and family needs, and this was par-
ticularly valued by families.3 Co-location with social
service staff meant that the centre was well placed to
identify potential patients early in the referral process
for end-of-life care. Although the Somerset Care
Coordination Centre had the same key function of
organising care packages as the North Somerset Care
Coordination Centre, the model was different. The
Somerset Care Coordination Centre was led by a
nurse
and
staffed
exclusively
by
administrators,
without any in-house care staff, additional nurses or
fast-track coordinator. Thus to make this model work,
the Somerset centre was heavily reliant on high-
quality management and good external relationships,
particularly with community and palliative care nurses
and private care agencies. However, both services
were valued for providing fast and efficient access to
care packages and equipment. Previous systematic
reviews have provided conflicting evidence with sug-
gestions that specialist palliative care input at home
can reduce general healthcare use and increase family
and patient satisfaction with care but not all the evi-
dence is supportive of this.7 8 (table 1). For both 30-day (North Somerset: OR 0.41,
95% CI 0.28 to 0.62, p<0.001; and Somerset: OR
0.66, 95% CI 0.51 to 0.85, p<0.001) and 7-day
(North Somerset: OR 0.22, 95% CI 0.11 to 0.42,
p<0.001; and Somerset: OR 0.32, 95% CI 0.22 to
0.47, p<0.001) results, these findings persist after
adjusting for confounders (table 3). Using Delivering Choice Again, the Care
Coordination Centres appeared to be the most effect-
ive component at 30 days (North Somerset: OR 0.46,
95% CI 0.29 to 0.76, p=0.002; and Somerset: OR
0.58, 95% CI 0.40 to 0.82, p=0.002) and 7 days
(North Somerset: OR 0.15, 95% CI 0.05 to 0.43,
p<0.001; and Somerset: OR 0.24, 95% CI 0.12 to
0.48, p<0.001). The OOH advice line was associated
with lower risk of ED attendance in the last week only
(OR 0.34, 95% CI 0.17 to 0.70, p=0.003). CONCLUSION
Th
l
f The results from this evaluation suggest that patients
using or supported by Delivering Choice were less
likely to die in hospital or call on hospital services. Those considering new ways of providing end-of-life
care could explore the possibility of adopting similar
services and evaluating the outcomes from patient,
carer and system perspectives. 4
Blackmore S, Pring A, Verne J. Predicting Death: estimating the
proportion of deaths that are unexpected. National End of Life
Care Intelligence Network, 2011. http://www.endoflifecare-
intelligence.org.uk (accessed Dec 2013). 5
Charlson ME, Pompei P, Ales KL, et al. A new method of
classifying prognostic comorbidity in longitudinal studies:
development and validation. J Chron Dis 1987;40:
373–83. 5
Charlson ME, Pompei P, Ales KL, et al. A new method of
classifying prognostic comorbidity in longitudinal studies:
development and validation. J Chron Dis 1987;40:
373–83. Acknowledgements We are grateful to the staff from the DCP
services, the local Marie Curie Cancer Care team and the PCT
staff who linked the data. 6
National end of life care Intelligence Network. National end of
life care profiles for primary care trusts. NHS National End of
Life Care Programme. http://www.endoflifecare-intelligence. org.uk/end_of_life_care_profiles/pct_pdf_profiles 6
National end of life care Intelligence Network. National end of
life care profiles for primary care trusts. NHS National End of
Life Care Programme. http://www.endoflifecare-intelligence. org.uk/end_of_life_care_profiles/pct_pdf_profiles Contributors All authors made substantial contributions to the
conception or design of the work; the acquisition, analysis or
interpretation of data for the work; drafting the work and
revising it critically for important intellectual content and
approved the version to be published. SP is the guarantor. 7
Candy B, Holman A, Leurent B, et al. Hospice care delivered
at home, in nursing homes and in dedicated hospice facilities:
a systematic review of quantitative and qualitative evidence. Int J Nurs Stud 2011;48:121–33. 7
Candy B, Holman A, Leurent B, et al. Hospice care delivered
at home, in nursing homes and in dedicated hospice facilities:
a systematic review of quantitative and qualitative evidence. Int J Nurs Stud 2011;48:121–33. Funding This evaluation was commissioned and funded by
Marie Curie Cancer Care. SP was funded by a Medical
Research Council Clinician Scientist Fellowship, number
G0501936. Funding This evaluation was commissioned and funded by
Marie Curie Cancer Care. SP was funded by a Medical
Research Council Clinician Scientist Fellowship, number
G0501936. 8
Luckett T, Davidson PM, Lam L, et al. DISCUSSION Moreover, about two-
thirds of Delivering Choice service users had cancer
while only about 30% of the total eligible population
died from this condition. It is possible that by includ-
ing a high proportion of patients with cancer, the
DCP achieved more home deaths. Indeed, in the years
2008–2010,
which
preceded
the
introduction
of
Delivering Choice, 26% of patients with cancer in
North Somerset and 28% of patients with cancer in
Somerset died at home compared with 18% and 20%
of all deaths being at home.6 However, among all
patients with DCP, the proportions of deaths at home
were 41% and 54%. Even among patients with cancer
this represented a substantial increase in proportions
of people dying at home. 38 Purdy S, et al. BMJ Supportive & Palliative Care 2015;5:34–39. doi:10.1136/bmjspcare-2013-000645 group.bmj.com
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Downloaded from Research Research REFERENCES at the end of life could be reduced further in line with
national policy.1 1
Department of Health. End of life care strategy: promoting
high quality of care for all adults at the end of life. London:
Department of Health, 2008. 1
Department of Health. End of life care strategy: promoting
high quality of care for all adults at the end of life. London:
Department of Health, 2008. In future studies, further evaluation of the compo-
nents of the DCP that seemed to be most beneficial
would be useful.2 Although randomised controlled
trials of complex interventions are costly and difficult
to undertake, the addition of trial evidence to the
current literature would provide more robust evidence
for those who commission and deliver services. 2
Wye L, Lasseter G, Percival J, et al. Independent Evaluation of
the Marie Curie Cancer Care Delivering Choice Programme in
Somerset and North Somerset, Final report. October 2012. http://www.mariecurie.org.uk/Documents/HEALTHCARE-
PROFESSIONALS/commissioning-services/Delivering-Choice-
Proramme-in-Somerset-and-North-Somerset-Final-Report.pdf
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Wye L, Lasseter G, Percival J, et al. Independent Evaluation of
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qualitative study of the domiciliary care worker role in meeting
the needs of families caring for those dying at home. Palliat
Support Care Published Online First: 21 Oct 2013
doi:10.1017/S147895151300076X 3
Percival J, Lasseter G, Purdy S, et al. “Earthly Angels”? A
qualitative study of the domiciliary care worker role in meeting
the needs of families caring for those dying at home. Palliat
Support Care Published Online First: 21 Oct 2013
doi:10.1017/S147895151300076X Purdy S, et al. BMJ Supportive & Palliative Care 2015;5:34–39. doi:10.1136/bmjspcare-2013-000645 hospital usage: a retrospective cohort study
and North Somerset on place of death and
Delivering Choice Programme in Somerset
Impact of the Marie Curie Cancer Care
Sarah Purdy, Gemma Lasseter, Thomas Griffin and Lesley Wye doi: 10.1136/bmjspcare-2013-000645
16, 2014
2015 5: 34-39 originally published online May
BMJ Support Palliat Care http://spcare.bmj.com/content/5/1/34
Updated information and services can be found at: CONCLUSION
Th
l
f Do community
specialist palliative care services that provide home nursing
increase rates of home death for people with life-limiting
illnesses? A systematic review and meta-analysis of
comparative studies. J Pain Symptom Manage 2013;45:
279–97. 8
Luckett T, Davidson PM, Lam L, et al. Do community
specialist palliative care services that provide home nursing
increase rates of home death for people with life-limiting
illnesses? A systematic review and meta-analysis of
comparative studies. J Pain Symptom Manage 2013;45:
279–97. Competing interests SP, LW, GL and TG received grant funding
support from Marie Curie Cancer Care to conduct this study. SP was funded by the Medical Research Council. Ethics approval University of Bristol Faculty of Medicine and
Dentistry Ethics Committee. Provenance and peer review Not commissioned; externally
peer reviewed. 9
Harrison N, Cavers D, Campbell C, et al. Are UK primary care
teams formally identifying patients for palliative care before
they die? Br J Gen Pract 2012;62:e344–52. Data sharing statement All data relating to this study and a
parallel qualitative evaluation are available at: http://www. bristol.ac.uk/primaryhealthcare/docs/reports/deliveringchoice. pdf Data sharing statement All data relating to this study and a
parallel qualitative evaluation are available at: http://www. bristol.ac.uk/primaryhealthcare/docs/reports/deliveringchoice. pdf 10
Zheng L, Finucane A, Oxenham D, et al. How good is UK
primary care at identifying patients for generalist and specialist
palliative care: a mixed methods study. Eu J Palliat Care
2013;20:216–20. Open Access This is an Open Access article distributed in
accordance withunder the terms of the Creative Commons
Attribution (CC BY 3.0) license, which permits others to
distribute, remix, adapt and build upon this work, for
commercial use, provided the original work is properly cited. See: http://creativecommons.org/licenses/by/3.0/ 11
Pocock L, Wye L, Purdy S. A qualitative study to evaluate the
process of indentifying patients at the end-of-life in primary
care. BMJ Supportive Palliat Care 2014;4:105. 39 Purdy S, et al. BMJ Supportive & Palliative Care 2015;5:34–39. doi:10.1136/bmjspcare-2013-000645 Purdy S, et al. BMJ Supportive & Palliative Care 2015;5:34–39. doi:10.1136/bmjspcare-2013-000645 group.bmj.com
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<span style="font-size: 10.5pt; font-family: Calibri, sans-serif;">A new NANOSATs propulsion system: swirling-combustion chamber and water electrolysis</span>
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AIMS energy
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* Correspondence: Email: angelo.minotti@uniroma1.it, info@angelominotti.com. Abstract: Defined for academic purposes (low budget projects), NanoSats become of interest in the
government and industry communities, since the deployment of swarms would able to act as a single
orbiting constellation, providing greater coverage and faster update rates than can be achieved using
conventional single satellite operations. On the other hand, that accomplishment is hampered by the
absence, due to the satellite’s small dimensions, of an independent efficient propulsion system. This
paper investigates the potentialities of a swirling-micropropulsion system that would permit NANOSATs
to be “propulsive-independent”. This would allow orbital operations, even at very low altitude,
and/or increasing also life-times. The propulsion system, patent pending, adopts H2/O2 obtained by
water electrolysis, a swirling combustion chamber and a sub-supersonic nozzle to optimize the thrust. The numerical combustion simulations, of the micro-thrust chamber, show that performances are
impressive, in particular if compared to the dimensions, and this would be of benefit in fields such as
intelligence, disaster monitoring, CBRNe observation, telecommunications and identification. Keywords: swirling microcombusion chambers; water electrolysis; cubesats; nanosats; orbit changing;
micropropulsion system; combustion simulations; H2/O2 detailed chemistry AIMS Energy, 6(3): 402–413.
DOI: 10.3934/energy.2018.3.402
Received: 12 January 2018
Accepted: 25 April 2018
Published: 04 May 2018 1 School of Aerospace Engineering, Sapienza University of Rome, Rome, Italy
2 Angelo Minotti ent., Rome, Italy 1 School of Aerospace Engineering, Sapienza University of Rome, Rome, Italy
2 Angelo Minotti ent., Rome, Italy 1 School of Aerospace Engineering, Sapienza University of Rome, Rome, Italy
2 Angelo Minotti ent., Rome, Italy * Correspondence: Email: angelo.minotti@uniroma1.it, info@angelominotti.com. 1.
Introduction In 1999 [1] the California Polytechnic State University and the Stanford University developed a
concept of satellite designed to utilize a standardised platform. Those satellites, known as 404 CubeSat (belonging to the nanosatellites family), are cubic modular satellites of “10 cm sides–1 kg
weight” for each module/unit. Defined for academic purposes and necessities (low budget projects),
a fundamental boost has been impressed by the MEMS fabrication technology (Micro Eectrical
Mechanical Systems), which has permitted components miniaturization [2]. Moreover, CubeSats
become of interest in the government and industry communities, since the benefits of swarm satellite
technologies have become more apparent [3]. A swarm of CubeSats would mean a group of dozens,
up to hundreds, satellites able to act as a single orbiting constellation, providing greater coverage and
faster update rates than can be achieved using conventional single satellite operations. That
accomplishment is hampered by the absence of a CubeSat independent propulsion system. In fact,
nowadays, most of the CubeSats operate in low Earth orbits determined by the deployment from the
launch vehicle [4] and none have incorporated propulsion capable of substantial ΔV in order to extend
their operability beyond Low Earth Orbit (LEO), or to orbits control, formation flying, proximity
operations, fine attitude control, or lifetime extension and de-orbiting. To realize these manoeuvres, a
propulsion system beyond the current state of the art is required. Such a propulsion system must be
consistent with the CubeSats cost and mass-volume limitations. Attitude control is generally, roughly
60% of small satellites, addressed adopting magnetic control and only 9% of operational small satellites
are equipped with an orbital control system [5] (the most popular being a cold gas propulsion system,
therefore limited by low specific impulse, low impulse density propellants, low ΔV and thrust). Only
one small satellite currently in orbit, as of 2010, has a chemical propulsion system [5]. Simultaneously, a
high-pressure bi-propellant micro-rocket engine is already being developed [6] using MEMS technology. High-pressure turbopumps and valves are incorporated onto the rocket chip and a spark ignites the
CH4-O2 mixture (the spark imposes chamber’s size limitation, because it cannot be shrunk proportionately
with the thruster). This combustor is of the order of centimetres with plans to operate at 12.7 MPa. Unfortunately,
high operating pressures are not practical for nanosatellite systems. 1.
Introduction Past [7] and actual [8] investigations try to solve those issues and/or to suggest solutions, even
in the manufacturing technologies, but all of them present technical drawbacks and/or low performance
and/or are not still ready to be mounted on satellites. Notwithstanding those limits, a business analysis [9] foresees 2400 nano/microsatellites
launches, within 2023, with a market of about 2 billion$ only in the 2019. In the light of the above, the present paper presents an innovative micro-propulsion system,
patent pending [10], which adopts H2/O2 as fuel/oxidizer, obtained by water electrolysis and intro-
duced into a micro-swirling combustion chamber (characteristic dimensions order of millimetres). The
relative hot gases are expanded in a sub-supersonic nozzle to optimize the provided thrust. Nowadays, there are examples, scientific and industrial (i.e., [11–13]), that show interest in
miniaturized “electrolysis propulsion”, but none of them adopts swirling combustion chambers. This
means that those thrust chambers, adopted in [11–13], are bigger than the swirling one reported in
the present manuscript, with the final consequence of shorter operability or lower power per unit
volume. Therefore, the miniaturized propulsion system, here reported, tries to overcome the current
nanosats’ limits, achieving orbit manoeuvres and lifetime extension, together with high combustion
efficiency in extremely small dimensions. Volume 6, Issue 3, 402–413. AIMS Energy AIMS Energy 405 The paper is structured as follows: section 2 reports the propulsion system’s main technologies,
section 3 reports the thrust chamber, section 4 reports the numerical/operating conditions and simulation
results, while section 5 reports the conclusions. 2.
The micro-propulsion system: main technologies The innovative nanosatellite propulsion system merges 3 main technologies: electrolysis [8], a
micro swirling combustion chamber and eventually catalysis [14]. In particular, liquid water is used
as propellant and separated, by electrolysis, in H2-O2 flows that are introduced into a swirling combustion
chamber, eventually characterized by catalytic walls in order to ignite the mixture without sparks (or
supporting the common spark ignition system). The aim is to overcome the actual nanosatellites limits
and to extend significantly their fields of application and operability to CBRNe, intelligence analysis,
disaster observation/prevention, climate analysis, planets observations-exploration and telecommunications. Figure 1. Micro propulsion system: overall structure, swirling chamber (left), electrolysis (right). Figure 1. Micro propulsion system: overall structure, swirling chamber (left), electrolysi There are many benefits: (1) water is unpressurized and safe during launch, poses no risk to the rocket or primary payload,
can operate in very low power modes, and leads to a relatively inexpensive system; (2) electricity for the electrolysis is generated by the solar cells (nominally 1.23 V are enough). Moreover, the propellant may serve as a battery, storing the electrical power with far greater
mass efficiency and none of the electrical losses associated with batteries; (3) electrolysis provides gaseous hydrogen, fuel, and oxygen, oxidant, at stoichiometric conditions;
this means obtaining the highest flame temperature and almost exclusively steam, as products of
combustion; (4) swirling combustion chamber and catalytic walls would permit to overcome the limitations
intrinsic of miniaturized chambers [15] (flame quenching, unstable combustion, low residence
time and low combustion efficiency) and then to realize a combustion chamber of the order of AIMS Energy AIMS Energy Volume 6, Issue 3, 402–413. 406 few millimetres with high combustion efficiency and, potentially, no-spark for the ignition. Hot
products are then expanded in a sub-supersonic micro-nozzle to generate the required
thrust (bipropellant thrusters, as opposed to monopropellant thrusters, offer higher specific impulse,
Isp, performances approaching 320–350 sec). 3.
The thrust chamber The miniaturized thrust chamber is composed of a cylindrical swirling combustion chamber and
a micronozzle. The combustion chamber’s dimensions, reported in the present study, are 6 × 6 mm
(D × H), the ducts have a diameter of 0.5 mm, while the micronozzle presents throat and exit radiuses
respectively equal to 0.15 mm and 1 mm (area expansion ratio equal to about 49); see Figure 2. The swirling motion is imposed by the inlet fluid dynamics, the oxidant duct, O2, is tangential to
the cylindrical wall while the fuel duct, H2, is perpendicular. The higher oxygen kinetic energy, see
section 4, pushes the fuel against the chamber wall forcing a helical path. The outlet duct, tangential
to the chamber’s wall, and positioned at its bottom, obliges the internal flow to create recirculation
before passing through the nozzle [16]. All that increases, significantly, the residence time, mixing
and providing very high combustion efficiency in very small devices [17]. In order to simplify the
ignition system, up to completely remove it, and to stress the miniaturization process, catalytic
material (i.e., platinum) can be deposited on the chamber’s internal walls. Catalysis lowers the activation
energy [18], required to ignite the hydrogen/oxygen mixing, permitting even smaller chamber’s
dimensions and maintaining complete combustion. The thrust chamber presents no particular difficulties to be manufactured and can be scaled
depending on the mission’s design necessities and on the required performance. Such a small device
(6 × 6 mm) may deliver thrust of the order up to 1 ÷ 10 N (the upper limit is defined by the inlet velocities,
which can be tuned modifying the operating pressure and the inlet section area). The following section 4 reports the case of a thrust chamber that provides about 3 × 10–2 N. Figure 2. Rendering of the thrust chamber. Figure 2. Rendering of the thrust chamber. Volume 6, Issue 3, 402–413. AIMS Energy AIMS Energy AIMS Energy 407 4.
Numerical-operating conditions and results The chamber, with a characteristic volume of 1.7 × 10–7 m3, has been reproduced by a mesh with
1.4 M cells (with a characteristic cell volume of 1.2 × 10–13 m3). It has been numerically improved
by “reordering, smoothing, and swapping” techniques [19]. Fluid dynamics has been solved adopting
the RANS k-eps turbulence approach, with the enhanced wall treatment [20]. The specific heat at
constant pressure, Cpi, is fitted by polynomials of temperature [21], while viscosity and thermal
conductivity, μ and k, are predicted by the gas kinetic theory [22]; mixtures are composition-
dependent according to Wilke’s formula [23]. The turbulence-combustion coupling is modelled
adopting the eddy dissipation concept (EDC) [24,25], while the hydrogen/oxygen kinetics by means
of a detailed mechanism, 9 species and 21 reactions [26]. The reactive Navier–Stokes equations are solved adopting a finite-volume solver (Ansys 17
CFD code), the PISO scheme [27] (for the pressure-velocity coupling) and the third-order MUSCL
scheme [28] (for the spatial discretization of all the variables). The computing resources are part of
the CRESCO/ENEAGRID High Performance Computing infrastructure and its staff [29]. Simulation
has been carried out assuming 8 × 10–6 kg/s and 1 × 10–6 kg/s of oxygen-hydrogen inlet mass flow
rates and inlet pressure of 3 atm, see Figure 3. These conditions impose a kinetic energy ratio (oxygen
kinetic energy/hydrogen kinetic energy) greater than 4. Figure 3. Pressure map, Pa. Figure 3. Pressure map, Pa. All this, leads to laminar inlet Reynolds numbers (respectively 1000 and 400 for O2 and H2). Finally, walls have been assumed adiabatic, to mimic protective material (as it would be in the real
case). Figures 4–5 report the internal temperature map and the zoom of the temperature at meeting
points. The maximum temperature, in the core of the chamber, is about 3200 K, while the meeting
point is characterized by a temperature close to 2400 K. Figures 6–7 report the velocity vectors at
meeting points, coloured by temperature, and the map of the H2 (fuel) mass fraction; it is evident
how the impinging zone, between fuel and oxidizer, creates a “tongue”, characterized by a high
temperature, close to 2400 K, that works as a premixing and ignition zone. It has been previously reported that the main characteristic of the swirling chamber is the possibility AIMS Energy AIMS Energy Volume 6, Issue 3, 402–413. 4.
Numerical-operating conditions and results 408 to miniaturize the system, maintaining high efficiency, thanks to the high recirculation and mixing
levels. to miniaturize the system, maintaining high efficiency, thanks to the high recirculation and mixing
levels. Figure 4. Temperature map, K. Figure 5. Zoom of the temperature map at the inlet plane, K. Figure 4. Temperature map, K. Figure 4. Temperature map, K. Figure 5. Zoom of the temperature map at the inlet plane, K. Figure 6. Zoom the velocity vectors, coloured by temperature, at inlet plane, K. Figure 4. Temperature map, K. Figure 4. Temperature map, K. Figure 5. Zoom of the temperature map at the inlet plane, K. Figure 5. Zoom of the temperature map at the inlet plane, K. Figure 6. Zoom the velocity vectors, coloured by temperature, at inlet plane, K. Figure 6. Zoom the velocity vectors, coloured by temperature, at inlet plane, K. Volume 6, Issue 3, 402–413. AIMS Energy 409 Figure 7. Zoom of the H2 map at the inlet plane. Figure 7. Zoom of the H2 map at the inlet plane. The following Figures 8–13 can prove all that. They report velocity vectors, velocity maps and
velocity path lines. In particular, the velocity vectors, Figure 8, show how the oxidizer pushes the
fuel against the wall, imposing swirling motion. This phenomenon is further highlighted by Figures
9–11, which show the velocity path-lines from, respectively, the oxidizer, the fuel inlets and their
combination. The helical path, vortexes and recirculation bubbles, just imposed by the inlet ducts relative
position, increase the residence time allowing complete combustion in a very small device. The final result is translated into the exhaust velocity values and, consequently, into the specific
impulse, shown in Figures 12–13. The exhaust velocity reaches values greater than 3300 m/s, with a specific impulse approaching
350 s, see Figures 12–13, and a thrust of about 3 × 10–2 N. Optimized bell shaped nozzle and greater inlet flows, to increase the performance, are under
investigations. Assuming 500 g of propellant and a lower specific impulse of 300 s, the thrust chamber
provides a V > 400 m/s. This V permits to form constellations at 200–300 km, from a 700 km
deployment, and to extend the relative satellites’ lifetime, according to mission requirements. Volume 6, Issue 3, 402–413. Figure 8. Velocity vectors at the inlet plane, m/s. Figure 8. Velocity vectors at the inlet plane, m/s. Figure 8. 4.
Numerical-operating conditions and results Velocity vectors at the inlet plane, m/s. Figure 8. Velocity vectors at the inlet plane, m/s. Volume 6, Issue 3, 402–413. Volume 6, Issue 3, 402–413. AIMS Energy AIMS Energy 410 Figure 9. Velocity path lines from the O2 inlet, m/s. Figure 9. Velocity path lines from the O2 inlet, m/s. Figure 10. Velocity path lines from the H2 inlet, m/s. Figure 11. Velocity path lines from the O2 and H2 inlet, m/s. Figure 10. Velocity path lines from the H2 inlet, m/s. Figure 10. Velocity path lines from the H2 inlet, m/s. Figure 10. Velocity path lines from the H2 inlet, m/s. Figure 11. Velocity path lines from the O2 and H2 inlet, m/s. Figure 11. Velocity path lines from the O2 and H2 inlet, m/s. Volume 6, Issue 3, 402–413. AIMS Energy AIMS Energy 411 Figure 12. Velocity map, m/s. Figure 13. Isp, sec., along of the exhaust nozzle section’s radius. Figure 12. Velocity map, m/s. Figure 12. Velocity map, m/s. Figure 12. Velocity map, m/s. Figure 12. Velocity map, m/s. Figure 13. Isp, sec., along of the exhaust nozzle section’s radius. Figure 13. Isp, sec., along of the exhaust nozzle section’s radius. 5.
Conclusions New satellite infrastructures (nanosats) are of interest for their low cost, easy of manufacturing
and their potentialities to be deployed as swarms in order to act as a single orbiting constellation. That accomplishment, which would provide greater coverage and faster update rates than can be
achieved using conventional single satellite operations, is hampered by the absence of an independent
propulsion system that must be simultaneously enough small, efficient and performing. This manuscript
presents an innovative miniaturized thrust chamber, fed by hydrogen and oxygen, obtained by electrolysis,
patent pending, that would overcome the limits of the current solutions, permitting, therefore, both
orbit constellations and fast manoeuvres. Even though the system is based on apparently simple
technologies, it fulfils the following design requirements: miniaturized dimensions, high combustion
efficiencies, scalability and high performance. Outcomes are extremely promising; simulation and technical improvements may be done,
respectively, adopting LES and an optimized bell shaped nozzle. Volume 6, Issue 3, 402–413. AIMS Energy AIMS Energy 412 Conflict of interests The author declares no conflict of interest in this paper. References 1. Puig-Suari J (2014) CubeSat Design Specification. California Polytechnic State University, San
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Attribution License (http://creativecommons.org/licenses/by/4.0) Volume 6, Issue 3, 402–413. AIMS Energy AIMS Energy
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PROPOSTA DE SISTEMA URBANO DE HORTAS VERTICAIS E TRANSIÇÃO ORGÂNICA
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Revista Ibero-Americana de Humanidades, Ciências e Educação
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Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE doi.org/10.51891/rease.v9i5.9949 1Estudante do curso superior de Tecnologia em Gestão da Produção Industrial da Faculdade de Tecnologia de Ferraz de
Vasconcelos, SP.
2Estudante do curso superior de Tecnologia em Gestão da Produção Industrial da Faculdade de Tecnologia de Ferraz de
Vasconcelos, SP.
3Estudante do curso superior de Tecnologia em Gestão da Produção Industrial da Faculdade de Tecnologia de Ferraz de
Vasconcelos, SP.
4Dra. em Comunicação e Semiótica pela PUC-SP e docente na Faculdade de Tecnologia de Ferraz de Vasconcelos, SP.
5 Mestre em Têxtil e Moda pela Universidade de São Paulo e docente na Faculdade de Tecnologia de Ferraz de Vasconcelos. 1.INTRODUÇÃO Esse trabalho trata-se da abordagem do método de implantação, gestão e
operacionalização eficientes do produto: Fazendas Verticais Modulares. Os autores, neste
artigo, abordam como utilizar os conceitos de Economia Circular, Logística Reversa e Supply
Chain Management para gerir estrategicamente a atividade econômica deste produto quando
devidamente implantado. Atualmente, à medida que o crescimento da população mundial
acelera, o aumento da demanda global por alimentos torna-se a principal consequência direta
desse cenário gerando necessidade de políticas públicas, cooperação internacional (conforme
a Agenda 2030 da ONU – Organização das Nações Unidas) e inovação. O conceito de desenvolvimento sustentável foi proposto em 1987 pela Comissão
Mundial do Desenvolvimento e Meio Ambiente em seu relatório final com o título de Nosso
Futuro Comum como sendo: atender às necessidades da geração presente sem comprometer
a habilidade das gerações futuras de atender suas próprias necessidades. (Lara et al, 2017). O uso de vastas terras para atividades de cultivo e pecuária, bem como a série de
mudanças na estrutura natural dos alimentos, tornando-os geneticamente modificados, e a
insistência no uso de pesticidas para melhorar a eficiência e rentabilidade da produção de
alimentos têm diminuído a qualidade nutricional dos alimentos em questão. 2060 Desse modo, este artigo se baseia no seguinte problema: É possível atender a
demanda global de alimentos em menor área de plantio, com menos produtos químicos e
maior eficiência? Para averiguar este problema, utilizou-se de pesquisa acadêmica em livros
e artigos científicos e projetos arquitetônicos voltados a estruturas de estufas verticais. O
foco desde trabalho está na abordagem de projetos de produção alimentícia em estufas-
edifícios e ferramentas de gestão de produção. PROPOSTA DE SISTEMA URBANO DE HORTAS VERTICAIS E TRANSIÇÃO
ORGÂNICA
PROPOSAL FOR AN URBAN SYSTEM FOR VERTICAL VEGETABLE GARDENS AND
ORGANIC TRANSITION Gabriel Moraes1
Erick Ferreira2
Kainan Alonso3
Luci Mendes de Melo Bonini4
Sandra Helena da Silva de Santis5 RESUMO: Trata-se da abordagem de uma proposta de implantação, gestão e operacionalização
eficientes do produto: Fazendas Verticais Modulares. Estuda-se como utilizar os conceitos de
Economia Circular, Logística Reversa e Supply Chain Management para gerir estrategicamente a
atividade econômica da produção de alimentos em centros urbanos. Atualmente, à medida que o
crescimento da população mundial acelera, o aumento da demanda global por alimentos torna-se a
principal consequência direta desse cenário gerando necessidade de políticas públicas, cooperação
internacional (conforme a Agenda 2030 da ONU – Organização das Nações Unidas) e inovação. Analisa-se uso de vastas terras para atividades de cultivo e pecuária, como a necessidade no uso de
pesticidas para melhorar a eficiência e rentabilidade da produção de alimentos têm diminuído a
qualidade nutricional dos alimentos para consumo direto humano. O artigo menciona projetos de
produção alimentícia em estufas-edifícios e ferramentas de gestão de produção e automação. 2059 Palavras-chave: Sustentabilidade. Produção de Alimentos. Economia Circular. ABSTRACT: This is the approach of a proposal for the efficient implementation, management and
operation of the product: Modular Vertical Farms. It studies how to use the concepts of Circular
Economy, Reverse Logistics and Supply Chain Management to strategically manage the economic
activity of food production in urban centers. Currently, as the growth of the world population
accelerates, the increase in the global demand for food becomes the main direct consequence of this
scenario, generating the need for public policies, international cooperation (according to the 2030
Agenda of the UN - United Nations Organization) and innovation. The use of vast lands for
cultivation and livestock activities is analyzed, as the need to use pesticides to improve the efficiency
and profitability of food production has reduced the nutritional quality of food for direct human
consumption. The article mentions food production projects in building greenhouses and production
management and automation tools. Keywords: Sustainability. Food production. Circular Economy. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. 2.REFERENCIAL TEÓRICO No cenário brasileiro, o avanço da atividade agrícola tradicional vai na contramão da
crescente adesão popular à temas de sustentabilidade e preservação de reservas
naturais/indígenas protegidas pelos órgãos ambientais. Esta situação se dá, por causa da
necessidade de grandes áreas para o cultivo de alimentos (Área Plantada), assim, para
garantir o abastecimento da demanda global, principalmente de soja e demais monoculturas,
existe um movimento de defesa da diminuição das áreas de preservação e aumento das áreas
disponíveis para atividade agrícola comercial, assim como outro movimento para Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE evista Ibero- Americana de Humanidades, Ciências e Educação- REASE manutenção e até mesmo aumento dessas áreas ambientais protegidas, causando
instabilidade política, social e econômica principalmente no Centro-oeste brasileiro, por ser
MT o estado líder na produção de soja (IMEA, 2019); e por ser divisa com a Floresta
Amazônica (Maior floresta tropical do mundo). Para aumentar a capacidade produtiva nesse sistema, logicamente a solução seria
expandir os territórios produtivos, gerando assim, captação de novos terrenos, muitas vezes,
pressionando a liberação de áreas que atualmente servem como reservas naturais e de
proteção ambiental. A vasta área aberta com a vegetação exposta gera a demanda por
agrotóxicos poderosos, conforme (VALADARES et al, 2020); e maiores incentivos ao
desenvolvimento de produtos transgênicos para uma “natural” resistência a pragas e
doenças. Se o alimento for produzido na cidade, em hortas verticais urbanas, as etapas
intermediárias seriam reduzidas e cerca de oitenta porcento da comida inutilizada seria
aproveitada. A produção nas áreas periurbanas deixaria de ser um sonho e os lucros
deixariam de concentrar-se nos centros atacadistas como o CEASA em Uberlândia e o
CEAGESP em São Paulo, (DE SÁ FILHO, Athayde Leite et al. 2021). 2061 Conforme (DE LUCENA et al, 2014) “O fato é que a evolução e o crescimento do
meio urbano e de seus arranha-céus foram condicionados a milhares de anos ao evento da
multiplicação da própria espécie humana, bem como, a necessidade desses em se aglomerar
num dado zoneamento a busca de segurança, habitat, alimentação e outros condicionantes”. Dessa forma a massa que demanda os alimentos estão nos centros urbanos, não na Zona
Rural que, por sua vez, é uma área de produção agrícola e não um centro de densidade
populacional. 2.1 MODELO EXISTENTE DE FAZENDAS VERTICAIS Fazendas Verticais são estruturas/edificações com a finalidade de proporcionar o
plantio de vegetais substituindo o modelo tradicional. Neste método de cultivo, a terra pode
ser excluída utilizando compostos/soluções nutritivas que proporcionem a germinação e
desenvolvimento das plantas diretamente aplicados às sementes, mudas e raízes sem necessidade de
enterrá-las, através de sistemas hidropónicos e/ou aeroponia. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE 2.2 PROCESSO SIMPLIFICADO DE PRODUÇÃO E MONTAGEM Para produzir os módulos são utilizados conectores em PVC devido o baixo custo,
disponibilidade no Mercado, durabilidade, maleabilidade do material e seu fácil transporte. Os módulos são conectados de acordo com a necessidade de capacidade de produção de
alimentos de cada projeto. O ambiente ideal é uma es tufa vertical de material
transparente/translúcido como vidro e com alta exposição à luz solar, sendo os mais
indicados em áreas urbanas: Parques abertos, em cima de edifícios, áreas verdes escolares e
similares. 2.3 ECONOMIA CIRCULAR DE RESÍDUOS ORGÂNICOS
Figura 01 – Conectores dos módulos
Figura 02 – Protótipo exemplar vazio de
suporte para mudas de hortaliças. Fonte – Autora: Gabriela Pastro. https://viveirosabordefazenda.wordp
ress.com/2014/03/11/passo-a-passo-
jardineira-de-pvc/
Fonte
–E-commerce
Shopee. https://shopee.com.br/CONEC
TOR-DE-COTOVELO-
CONJUNTO-DE-TUBOS-DE-
PVC-20mm-3-4-5-COR-branca-
i.198973203.6406842581. Figura 02 – Protótipo exemplar vazio de
suporte para mudas de hortaliças. Fonte – Autora: Gabriela Pastro. https://viveirosabordefazenda.wordp
ress.com/2014/03/11/passo-a-passo-
jardineira-de-pvc/ Figura 02 – Protótipo exemplar vazio de
suporte para mudas de hortaliças. Figura 02 – Protótipo exemplar vazio de
suporte para mudas de hortaliças. Figura 01 – Conectores dos módulos Figura 02 – Protótipo exemplar vazio de
suporte para mudas de hortaliças. Fonte
–E-commerce
Shopee. https://shopee.com.br/CONEC
TOR-DE-COTOVELO-
CONJUNTO-DE-TUBOS-DE-
PVC-20mm-3-4-5-COR-branca-
i.198973203.6406842581. 2062 Fonte
–E-commerce
Shopee. https://shopee.com.br/CONEC
TOR-DE-COTOVELO-
CONJUNTO-DE-TUBOS-DE-
PVC-20mm-3-4-5-COR-branca-
i 198973203 6406842581 Fonte – Autora: Gabriela Pastro. https://viveirosabordefazenda.wordp
ress.com/2014/03/11/passo-a-passo-
jardineira-de-pvc/ Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. 2.3 ECONOMIA CIRCULAR DE RESÍDUOS ORGÂNICOS A economia circular (EC) é uma noção que sustenta a circularidade responsável dos
recursos disponíveis para atingir os objetivos de desenvolvimento sustentável (Moraga et
al., 2019). Este conceito envolve um modelo regenerativo onde são feitos esforços para
minimizar os insumos (recursos) e desperdícios em qualquer forma de vazamento ou
descarte para fechar ou desacelerar o ciclo de materiais. A maioria das empresas e negócios
industriais segue os conceitos de economia linear em todo o mundo. Os produtos fabricados
na indústria são levados para uso público e descartados sem uso, seguindo o modelo de
consumo onde se produz, esse produto é levado ao consumidor e logo após descartado. Tal
prática não está alinhada com os objetivos de desenvolvimento sustentável, por esta razão a
economia circular está a redefinir os atuais métodos de gestão, onde o papel da estratégia de
gerenciamento dos resíduos alimentares é significativamente crucial na transição do modelo ,
ISSN - 2675 – 3375 ISSN - 2675 – 3375 ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE linear para esse modelo circular, criando novos ramos de negócios e movimentos ecológicos. Por exemplo, resíduos de alimentos podem ser utilizados para produção de composto
(fertilizante orgânico) (Waqas et al., 2018). Pensando nisso, o presente trabalho tem como idealização a utilização do método de
compostagem, fertilizantes orgânicos, dentro dos “módulos de fazendas verticais urbanas”. Esses insumos orgânicos viriam de parceiros (como restaurantes, feiras etc.) localizados nas
regiões próximas. Cada vez mais a preocupação referende à disposição final de resíduos
sólidos nos preocupa, pela falta de controle e a falta de locais para dispor esses resíduos no
final de sua cadeia (Jacobi & Besen, 2011). Assim com a ideia de parceria, ficaríamos ao fim
dessa cadeia, proporcionando um destino adequado a esses resíduos e, não somente isso,
colaborando para a criação de alimentos livres de agrotóxicos que possam prejudicar a saúde
do consumidor, promovendo o bem-estar da sociedade. Dessa forma proporcionalizamos a continuação do ciclo de vida dos resíduos
orgânicos, reciclando e transformando em insumos para recomeçar a cadeia produtiva,
empregando de maneira exímia e eficiente a logística reversa, que pode ser considerada como
parte da logística empresarial responsável pelo planejamento, operação e controle dos fluxos
reversos de diversas naturezas (LEITE, 2003). 2063 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023.
ISSN - 2675 – 3375 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. 2.4 LOGÍSTICA REVERSA E COMPOSTAGEM A compostagem reduz a quantidade de resíduos orgânicos que acaba em aterros,
juntamente com o chorume que produz (United States Environmental Protection Agency,
2008). Além disso, a compostagem tem o potencial de reduzir as emissões de gases com efeito
de estufa, como o metano (Favoino & Hogg, 2008), ao mesmo tempo que cria um
suplemento de solo comercializável que tem várias vantagens sobre os fertilizantes químicos
normalmente utilizados (Bulluck, 2002). Assim, a compostagem pode ajudar a atenuar as
alterações climáticas, reduzir a pressão sobre os aterros sanitários existentes (e a necessidade
de criar novos), e melhorar a qualidade dos solos agrícolas. Um método de encorajar a compostagem pode ser através do desenvolvimento de
uma norma social de compostagem. Uma vez estabelecida uma norma social, é provável que
o comportamento seja adotado por aqueles que ainda não o praticam, solidificando-o assim
ainda mais como prática comum. Novos indivíduos que se juntem ao grupo terão mais
probabilidades de adotar este comportamento normativo. Mas como pode uma norma social
ser estabelecida quando não existe para começar? Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Figura 03 – Tratamento Descentralizado de Resíduos com BSF Figura 03 – Tratamento Descentralizado de Resíduos com BSF Figura 03 – Tratamento Descentralizado de Resíduos com BSF Contudo, os projetos de sucesso precisam de ser flexíveis na concepção, adaptáveis e
operacionais de forma a melhor responderem às condições sociais, económicas e ambientais
atuais, que também são capazes de mudar ao longo do tempo e variam dependendo da área
geográfica do projeto (Zurbrügg et al., 2012). As famílias brasileiras, pequenas empresas comerciais e serviços de limpeza
produzem quase 94.335 toneladas dia-1 de resíduos orgânicos, o que representa 51% dos
resíduos sólidos urbanos produzidos (Brasil, 2012). Apenas 1% destes resíduos são destinados
a sistemas de compostagem, enquanto 59% são depositados em aterros e 39% são depositados
em aterros. A Política Nacional de Resíduos Sólidos (Brasil, 2010) tem orientações de zero
resíduos para resíduos, reciclagem, compostagem, incineração e eliminação em fim de vida
em aterros. O Plano Nacional de Resíduos Sólidos dá prioridade à compostagem como
alternativa para a gestão de resíduos orgânicos (Brasil, 2012). 2064 Os poucos sistemas de compostagem em funcionamento no Brasil são na sua maioria
a céu aberto, centralizados e à escala municipal. Existem poucos programas ou estudos
relativos à compostagem doméstica. A compostagem tem vantagens sobre a incineração e
eliminação em aterros devido a custos operacionais mais baixos, poluição ambiental
reduzida e, mais importante ainda, a utilização benéfica do produto final, que pode ser
utilizado como condicionador de solo ou fertilizante (Li et al., 2013). Aproximadamente 90% dos municípios brasileiros têm uma população de até 50.000
habitantes e existe a possibilidade de construir casas dentro de uma área que permite a
utilização de compostagem doméstica e a utilização de compostagem no local como ,
ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE fertilizante ou em áreas agrícolas próximas. Existem também regiões brasileiras em climas
semiáridos com pobres nutrientes do solo onde o composto poderia ser utilizado como
amaciador de solos. Além disso, existem áreas urbanas altamente povoadas onde a prática
da compostagem doméstica e a sua aplicação em locais próximos, tais como praças e parques,
poderia reduzir o custo do transporte de resíduos sólidos para aterros sanitários. Portanto, a
adopção de sistemas de compostagem doméstica no Brasil é muito atrativa. A compostagem é a decomposição e estabilização da fracção orgânica dos resíduos
sólidos, realizada por micróbios em condições aeróbias controladas. Figura 03 – Tratamento Descentralizado de Resíduos com BSF As variáveis mais
importantes no processo de compostagem são uma relação carbono/nitrogénio entre 20 e 40,
teor de humidade entre 50% e 70%, aeração adequada, tamanho de pequenas partículas e
transferência de massa adequada entre o ar e os sólidos (Chang et al., 2006). É evidente que
o sucesso da compostagem doméstica depende do desempenho do compositor doméstico e
da qualidade do produto, tendo sido realizadas experiências que têm em consideração o
desempenho do compositor doméstico em relação à aeração passiva e/ou forçada e à
alimentação por lotes e/ou semi-contínua (Chang et al., 2006; Karnchanawong e Suriyanon,
2011). 2065 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023.
ISSN - 2675 – 3375 3 MATERIAIS E MÉTODOS Este trabalho trata-se uma proposta de desenvolvimento de estufas verticais em
centros urbanos como as regiões metropolitanas de São Paulo e Campinas, por exemplo,
uma vez que os consumidores finais de hortaliças e legumes estão principalmente na Região
Urbana. Essa é uma pesquisa qualitativa de caráter descritivo, pois discorre sobre projetos
existentes de hortas verticais ao redor do mundo e propõe esse modelo para a realidade
brasileira incluindo a possibilidade de transição orgânica para este modelo. A pesquisa
qualitativa se preocupa com o nível de realidade que não pode ser quantificado, ou seja, ela
trabalha com o universo de significados, de motivações, aspirações, crenças, valores e
atitudes (MINAYO, 2014). Foi realiza algumas pesquisas (FORMS), com intuito de levantar informações sobre
tema em questão, a pesquisa realizada teve como público-alvo consumidores entre 19 a 40
anos, obtivemos a mais variadas respostas sobre o assunto, podendo criar uma base, para
sugestão de projeto, local apropriado, disposição de em criar e manter uma horta e dados
relacionados a saúde desses consumidores. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE 3.1 SUGESTÃO DE CULTURAS IDEAIS PARA PRODUÇÃO O sistema de estufa vertical é ideal para culturas como: Alface, rúcula, coentro,
cebolinha, salsa, tomate cereja, berinjela, abobrinha, espinafre, morango e similares. Essas
culturas são facilmente adaptáveis à hidroponia e já conhecidas por esse segmento, de forma
que também possuem histórico e estudos desenvolvidos para melhorar suas performances e
boas práticas de produção, além de serem populares na rotina dos brasileiros. No caso da
alface, por exemplo, pode-se atingir uma produtividade até seis vezes maior no sistema
hidropônico em relação ao cultivo tradicional. Recentemente, a técnica hidropônica está se tornando popular porque é limpa e
relativamente fácil, não há chance de doenças transmitidas pelo solo, infestação de insetos
ou pragas nas culturas, reduzindo assim ou eliminando o uso de pesticidas (SHARMA, Nisha
et al., 2018). Além disso, as plantas requerem menos tempo de crescimento em comparação
com a cultura cultivada no campo e o desenvolvimento da cultura é mais produtivo,
conforme dito anteriormente no caso da alface, pois não há impedimento mecânico às raízes
e todo o nutriente é prontamente absorvido pelas plantas. Esta técnica é muito útil para a
área onde há climas extremos como longas geadas e desertificação. Culturas em sistema
hidropônico não são influenciados pelas mudanças climáticas, portanto, podem ser
cultivadas o ano todo e é considerada como entressafra. A hidroponia economiza grande
quantidade de água. Maiores rendimentos podem ser obtidos desde que o número de plantas
por unidade é maior em comparação com a convencional agricultura. 2066 4 RESULTADO E DISCUSSÃO Após as pesquisas feitas, e estudo do artigo, podemos ver a viabilidade de fabricar
uma horta vertical nos centros urbanos, para trazer para mais perto a produção desses
alimentos, ao consumidor final. Fonte - Pesquisa Forms, Erick Ferreira, Gabriel Moraes e Kainan Alonso Fonte - Pesquisa Forms, Erick Ferreira, Gabriel Moraes e Kainan Alonso Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Para determinar a quantidade exata de material necessário para confeccionar a
estrutura, é preciso saber as dimensões e o tipo de configuração. Algumas variáveis que
precisam ser consideradas incluem: tamanho da estrutura, carga máxima suportada, clima
local, entre outras. Sem essas informações específicas, não é possível calcular a quantidade
necessária de material, visando obter maiores informações para obter uma ideia de protótipo
ideal, foram feitas pesquisas para fazer um levantamento dessas variáveis. Fonte - Pesquisa Forms, Erick Ferreira, Gabriel Moraes e Kainan Alonso. Fonte - Pesquisa Forms, Erick Ferreira, Gabriel Moraes e Kainan Alonso. Fonte - Pesquisa Forms, Erick Ferreira, Gabriel Moraes e Kainan Alonso. De acordo com a pesquisa realizada, foi selecionado por todos os entrevistados o
mesmo tamanho de horta vertical indicando um tamanho ideal para o protótipo proposto. Pensando nisso trabalharemos com um protótipo reduzido de uma horta vertical de
dois metros de altura por dois metros de comprimento (2m²), o que necessitará de: Pensando nisso trabalharemos com um protótipo reduzido de uma horta vertical de
dois metros de altura por dois metros de comprimento (2m²), o que necessitará de: 2067 •
Uma estrutura vertical resistente, como uma parede de madeira ou treliça,
ou suportes individuais para as barras de cano PVC;
•
Terra ou substrato para plantio em quantidade suficiente para preencher os
moldes;
•
Recipientes para plantio, como tubos de PVC;
•
Sementes ou mudas de plantas que se adaptem às condições de luminosidade
e clima local;
•
Adubo orgânico para fertilizar o solo;
•
Regador ou sistema de irrigação para molhar o plantio regularmente;
•
Ferramentas de jardinagem, como luvas, rastelos, pás e tesouras de poda;
•
Tela para proteção da estrutura contra sol forte, chuvas e evitar o ataque de
insetos; e
•
Acessórios opcionais, como placas para sinalizar o tipo de muda. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023.
ISSN - 2675 – 3375 4 RESULTADO E DISCUSSÃO •
Uma estrutura vertical resistente, como uma parede de madeira ou treliça,
ou suportes individuais para as barras de cano PVC; •
Terra ou substrato para plantio em quantidade suficiente para preencher os
moldes; ;
•
Recipientes para plantio, como tubos de PVC; Recipientes para plantio, como tubos de PVC; •
Sementes ou mudas de plantas que se adaptem às condições de luminosidade
e clima local; •
Adubo orgânico para fertilizar o solo; •
Adubo orgânico para fertilizar o solo; •
Regador ou sistema de irrigação para molhar o plantio regularmente; •
Ferramentas de jardinagem, como luvas, rastelos, pás e tesouras de poda; j
g
p
p
•
Tela para proteção da estrutura contra sol forte, chuvas e evitar o ataque de
insetos; e
•
Acessórios opcionais como placas para sinalizar o tipo de muda •
Tela para proteção da estrutura contra sol forte, chuvas e evitar o ataque de
insetos; e •
Acessórios opcionais, como placas para sinalizar o tipo de muda. Os canos de PVC são de 100 (cem) milímetros de diâmetro com 6 (seis) metros cada
barra. Serão necessárias 2 (duas) barras que serão divididas em pedaços de 2 (dois) metros
cada, totalizando 6 (seis) pedaços. Os canos de PVC são de 100 (cem) milímetros de diâmetro com 6 (seis) metros cada
barra. Serão necessárias 2 (duas) barras que serão divididas em pedaços de 2 (dois) metros
cada, totalizando 6 (seis) pedaços. Os canos serão perfurados em intervalos regulares de 50 (cinquenta) centímetros para
permitir o plantio, a drenagem da água e a ventilação das raízes das plantas. Os canos serão perfurados em intervalos regulares de 50 (cinquenta) centímetros para
permitir o plantio, a drenagem da água e a ventilação das raízes das plantas. Será adicionado uma camada de pedras no fundo dos canos para garantir uma
drenagem mais eficiente da água, sem a perca de material (terra). Será adicionado uma camada de pedras no fundo dos canos para garantir uma
drenagem mais eficiente da água, sem a perca de material (terra). Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Serão escolhidas plantas que se desenvolvem bem em vasos (recipientes) e que
tenham necessidades semelhantes de luz e água. Alguns exemplos de vegetais que se
desenvolvem bem nessas condições são: tomate cereja, pimenta, abobrinhas, berinjela,
rúcula, alface, cenoura e rabanete. 4 RESULTADO E DISCUSSÃO De acordo com as pesquisas feitas, temos uma grande aprovação nas compras de
produtos orgânicos e produzidos em hortas verticais, mesmo tendo um valor mais elevado
que as disponíveis no mercado, mas por ser de várias formas mais saudáveis, como está mais
próximo dos consumidores e tem rápido abastecimento, e sem conservantes, teve uma
aprovação muito maior nas pesquisas. Fonte - Pesquisa Forms, Erick Ferreira, Gabriel Moraes e Kainan Alonso. 2068 Fonte - Pesquisa Forms, Erick Ferreira, Gabriel Moraes e Kainan Alonso. Fonte - Pesquisa Forms, Erick Ferreira, Gabriel Moraes e Kainan Alonso. Fonte - Pesquisa Forms, Erick Ferreira, Gabriel Moraes e Kainan Alonso. O sistema de irrigação pode ser tanto automático, com um sistema conectado à um
temporizador de acordo com um cronograma pré-estabelecido, quanto manualmente por
meio de um regador e seguindo o mesmo cronograma. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. CONSIDERAÇÕES FINAIS A pesquisa destaca a importância de integrar conhecimentos de diversas áreas para a
implementação bem-sucedida de fazendas verticais e hortas urbanas. A arquitetura é
importante para a concepção dos edifícios e estruturas que abrigam as plantas e sistemas de
cultivo, enquanto o conhecimento biológico é necessário para selecionar as espécies de
plantas mais adequadas e entender suas necessidades de crescimento. Além disso, a pesquisa
enfatiza a importância de considerar as interações sociais e econômicas na implementação
desses projetos. A possibilidade de pensar e repensar novas metodologias de cultivo e que Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE proporcionem o aumento da eficiência e produtividade no fornecimento de alimentos. As
fazendas verticais e hortas urbanas podem ter um impacto significativo na comunidade
local, fornecendo alimentos frescos e sustentáveis e criando oportunidades de emprego. Portanto, é importante levar em consideração as necessidades e desafios específicos da
comunidade para garantir que esses projetos sejam viáveis e bem-sucedidos. Em resumo,
conectar conhecimentos arquitetônicos, biológicos, sociais e econômicos é crucial para
projetar e implementar fazendas verticais e hortas urbanas eficazes e sustentáveis, que
possam atender às necessidades das comunidades locais e contribuir para uma alimentação
mais saudável e sustentável. Com esse artigo é possível perceber que trazer a produção de
alimentos orgânicos para onde reside o principal público consumidor pode reduzir os custos
de produção de transporte democratizando o consumo de comida de qualidade
diversificando o prato do cidadão médio. Para o produtor isso significa aumento na
eficiência, diminuição no custo e maior oferta de produtos que atendam os clientes e as
expectativas do investimento. É verdade que o uso de estufas proporciona um ambiente
controlado que pode ser vantajoso para o controle de pragas e temperatura. Em um ambiente
fechado e controlado, é possível impedir a entrada de insetos e outras pragas que podem
prejudicar as plantas. Além disso, é possível controlar a temperatura e a umidade, criando
condições ideais para o crescimento das plantas. No entanto, é importante destacar que o
uso de estufas também apresenta desvantagens. Por exemplo, a ventilação pode ser um
desafio em um ambiente fechado, o que pode levar ao acúmulo de umidade e ao surgimento
de doenças fúngicas nas plantas. Além disso, é necessário monitorar cuidadosamente as
condições dentro da estufa e fazer ajustes regulares para garantir que as plantas estejam
recebendo as condições ideais 2069 REFERÊNCIAS LARA, Luiz Gustavo Alves de; OLIVEIRA, Samir Adamoglu de. A ideologia do crescimento econômico e o
discurso empresarial do desenvolvimento sustentável. Cadernos Ebape. br, v. 15, p. 326-348, 2017. DE LUCENA, Leandro Pessoa et al. Avaliação multicriterial das fazendas verticais canadenses como
modelos sustentáveis de agricultura urbana. RAI Revista de Administração e Inovação, v. 11, n. 1, p. 181-202,
2014. RODRIGUES, Andressa Caixeta; OLIVEIRA, Juliano Carlos Cecílio Batista. HORTAS URBANAS E
FAZENDAS VERTICAIS: A ARQUITETURA E A PRODUÇÃO DE ALIMENTOS. RODRIGUES, Andressa Caixeta; OLIVEIRA, Juliano Carlos Cecílio Batista. HORTAS URBANAS E
FAZENDAS VERTICAIS: A ARQUITETURA E A PRODUÇÃO DE ALIMENTOS. RODRIGUES, Andressa Caixeta; OLIVEIRA, Juliano Carlos Cecílio Batista. HORTAS URBANAS E
FAZENDAS VERTICAIS: A ARQUITETURA E A PRODUÇÃO DE ALIMENTOS. BENÍTEZ, Raúl Osvaldo. FAO, Perdas e desperdícios de alimentos na América Latina e no Caribe. Texto:
FAO, 2018 Raúl Osvaldo Benítez. Disponível em: www.fao.org/americas/noticias/ver/pt/c/239394/ Acesso
em: 20/11/2022 às 15:00. Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. ISSN - 2675 – 3375 Revista Ibero-Americana de Humanidades, Ciências e Educação. São Paulo, v.9.n.05. mai. 2023. ISSN - 2675 – 3375 Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE Revista Ibero- Americana de Humanidades, Ciências e Educação- REASE United States Environmental Protection Agency. (2008). Composting: Environmental benefits. Retrieved
from http://www.epa.gov/waste/conserve/rrr/composting/benefits.htm. Favoino, E., & Hogg, D. (2008). The potential role of compost in reducing greenhouse gases. Waste
Management & Research, 26, 61-69. Bulluck, L. R. (2002). Organic and synthetic fertility amendments influence soil microbial, physical and
chemical properties on organic and conventional farms. Applied Soil Ecology, 19, 147. Zurbrügg, C., Gfrerer, M., Ashadi, H., Brenner, W., Küper, D. 2012. Determinants of sustainability in solid
waste management - The Gianyar Waste Recovery Project in Indonesia. Waste Management. MORAGA, Gustavo et al. Circular economy indicators: What do they measure?. Resources, Conservation
and Recycling, v. 146, p. 452-461, 2019. WAQAS, M. et al. Optimization of food waste compost with the use of biochar. Journal of environmental
management, v. 216, p. 70-81, 2018. JACOBI, Pedro Roberto; BESEN, Gina Rizpah. Gestão de resíduos sólidos em São Paulo: desafios da
sustentabilidade. Estudos avançados, v. 25, p. 135-158, 2011. LEITE, L. F. C. et al. Estoques totais de carbono orgânico e seus compartimentos em Argissolo sob floresta
e sob milho cultivado com adubação mineral e orgânica. Revista Brasileira de Ciência do Solo, v. 27, p. 821-
832, 2003. BRASIL. Ministério do Meio Ambiente. Plano Nacional de Resíduos Sólidos. Brasília, 2012. BRASIL. Casa Civil. Lei nº 12.305, de 2 de agosto de 2010. Institui a Política Nacional de Resíduos Sólidos;
altera a Lei no 9.605, de 12 de fevereiro de 1998; e dá outras providências. Diário Oficial [da] União, Brasília,
03 ago. 2010. 2070 LI, Z.; LU, H.; REN, L.; HE, L. Experimental and modelling approaches for food waste composting. Chemosphere, v. 93, n. 7, p. 1247–1257, 2013. http://dx.doi.org/10.1016/j.resconrec.2011.01.006 CHANG, J. I.; TSAI, J. J.; WU, K. H. Composting of vegetable waste. Waste Management and Research,
v. 24, p. 354–362, 2006. http://dx.doi.org/10.1177/0734242X06065727 KARNCHANAWONG, S.; SURIYANON, N. RODRIGUES, Andressa Caixeta; OLIVEIRA, Juliano Carlos Cecílio Batista. HORTAS URBANAS E
FAZENDAS VERTICAIS: A ARQUITETURA E A PRODUÇÃO DE ALIMENTOS. Household organic waste composting using bins with
different types of passive aeration. Resources, Conservation and Recycling, v. 55, n. 5, p. 548–553, 2011. https://doi.org/10.1016/j.resconrec.2011.01.006 SHARMA, Nisha et al. Hydroponics as an advanced technique for vegetable production: An
overview. Journal of Soil and Water Conservation, v. 17, n. 4, p. 364-371, 2018. IMEA. Instituto Mato-Grossense de Economia Agropecuária. Estimativa de safra de soja. 4ª
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agrotóxicos: uma análise descritiva dos resultados de Censo Agropecuário 2017. 2020. DE SÁ FILHO, Athayde Leite et al. Hortas urbanas no Brasil: Evolução, desafios e perspectivas. Journal on
Innovation and Sustainability RISUS, v. 12, n. 1, p. 30-44, 2021. MINAYO, M. C. de S. (Org.). O desafio do conhecimento: pesquisa qualitativa em saúde. 14ª ed. Rio de
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https://openalex.org/W2280892688
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https://discovery.ucl.ac.uk/id/eprint/1486642/1/Lewis_Using%20Data%20Linkage%20to%20Investigate%20Inconsistent%20Reporting%20of%20Self-Harm%20and%20Questionnaire%20Non-Response.pdf
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English
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Using Data Linkage to Investigate Inconsistent Reporting of Self-Harm and Questionnaire Non-Response
|
Archives of Suicide Research
| 2,016
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cc-by
| 12,943
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# Becky Mars, Rosie Cornish, Jon Heron, Andy
Boyd, Catherine Crane, Keith Hawton, Glyn Lewis,
Kate Tilling, John Macleod, and David Gunnell
This is an Open Access article distributed under the
terms of the Creative Commons Attribution-Non-
Commercial License (http://creativecommons.org/
licenses/by-nc/3.0/), which
permits
unrestricted
non-commercial use, distribution, and reproduction
in any medium, provided the original work is prop-
erly cited. The moral rights of the named author(s)
have been asserted. Using Data Linkage
to Investigate Inconsistent
Reporting of Self-Harm
and Questionnaire
Non-Response Becky Mars, Rosie Cornish, Jon Heron, Andy Boyd, Catherine
Crane, Keith Hawton, Glyn Lewis, Kate Tilling, John Macleod, and
David Gunnell The objective of this study was to examine agreement between self-reported and
medically recorded self-harm, and investigate whether the prevalence of self-harm
differs in questionnaire responders vs. non-responders. A total of 4,810 participants
from the Avon Longitudinal Study of Parents and Children (ALSPAC) completed
a self-harm questionnaire at age 16 years. Data from consenting participants were
linked to medical records (number available for analyses ranges from 205–
3,027). The prevalence of self-harm leading to hospital admission was somewhat
higher in questionnaire non-responders than responders (2.0 vs. 1.2%). Hospital
attendance with self-harm was under-reported on the questionnaire. One third
reported self-harm inconsistently over time; inconsistent reporters were less likely to
have depression and fewer had self-harmed with suicidal intent. Self-harm prevalence
estimates derived from self-report may be underestimated; more accurate figures may
come from combining data from multiple sources. Keywords
agreement, ALSPAC, consistency, data linkage, self-harm, suicide attempt Keywords
agreement, ALSPAC, consistency, data linkage, self-harm, suicide attempt Archives of Suicide Research, 20:113–141, 2016
Published with license by Taylor & Francis
ISSN: 1381-1118 print=1543-6136 online
DOI: 10.1080/13811118.2015.1033121 Archives of Suicide Research, 20:113–141, 2016
Published with license by Taylor & Francis
ISSN: 1381-1118 print=1543-6136 online
DOI: 10.1080/13811118.2015.1033121 Consistency in Reports of Self-Harm in Adolescence et al., 2006; Hart, Musci, Ialongo, Ballard, &
Wilcox, 2013). However, the absence of a
gold standard means it is not possible to tell
which measure=assessment is more accurate
(see Table 1 for a summary of studies). There is some evidence to suggest that
individuals with more severe psychopath-
ology are more likely to report self-harm
consistently over time (Christl et al., 2006;
Eikelenboom, Smit, Beekman, Kerkhof, &
Penninx, 2014). and measures of association. The extent to
which this occurs in the case of self-harm is
not currently known. y
Non-response and loss to follow-up
occur more frequently among individuals
with particular characteristics (Kidger et al.,
2012; Wolke et al., 2009). For example, in
the Early Developmental Stages of Psycho-
pathology Study (Christl, Wittchen, Pfister,
Lieb, & Bronisch, 2006), participation rates
at follow-up were lower among those who
had attempted suicide compared to those
without suicidal thoughts or attempts. This
skewed pattern of participation would have
led to underestimates of the prevalence of
suicide attempts. )
Whereas previous studies have typically
compared self-report questionnaire and
interview responses, the present study
compares self-reported self-harm with data
from medical records. This data is external
and objective, although it cannot be con-
sidered to be free from error. We linked
data from medical records with data repor-
ted by participants in the Avon Longitudi-
nal Study of Parents and Children, a
longitudinal population-based birth cohort
(Boyd et al., 2013). Our aims were to: In addition, as information on self-
harm is typically collected retrospectively
via self-report, the accuracy of responses
may be affected by issues such as denial,
reinterpretation,
problems
with
recall,
current mood, or by misinterpretation of
the study questions (Velting, Rathus, &
Asnis, 1998). There is also evidence to
suggest that concerns over social desir-
ability may encourage under-reporting, as
adolescents have been found to report
suicide attempts two to three times more
frequently under conditions of anonymity
(Safer, 1997). 1. Investigate
whether
the
prevalence
of
self-harm
recorded
in
medical
records
differs
between
responders
and non-responders to the self-harm
questionnaire. 2. Investigate
the
level
of
agreement
between
self-report
and
medically
recorded self-harm events. The agreement between different sour-
ces of data on self-reported self-harm in
adolescents has previously been investigated
(Bja¨rehed, Pettersson, Wa˚ngby-Lundh, &
Lundh,
2013;
O’Sullivan
&
Fitzgerald,
1998; Ougrin & Boege, 2013; Ross & Heath,
2002; Velting et al., 1998). INTRODUCTION Community studies of self-harm are vital as
the majority of self-harm episodes do not
present
to
clinical
services
(Hawton,
Rodham,
Evans,
&
Weatherall,
2002;
Kidger, Heron, Lewis, Evans, & Gunnell,
2012; Ystgaard et al., 2009). However, such
studies are subject to a number of limita-
tions
such
as
misreporting
and
non-
response (Grimes & Schulz, 2002), which
can lead to bias in estimates of prevalence 113 Consistency in Reports of Self-Harm in Adolescence 20
(20%) reported at least one
episode of self-harm on the
questionnaire that was not
recorded in the clinical
record
88.5% inconsistently reported
a suicide attempt at some
point during the study;
65.3% were inconsistent the
year following the self-harm
event
Consistent and inconsistent
reporters did not differ on
clinical or demographic
variables, but consistent
reporters had higher lifetime
suicidal ideation
97 adolescents were selected
for interview. 32=66 (48%)
participants who reported
(Continued ) TABLE 1. Studies Investigating Consistency in Self-Harm Reports: Across Different Measures or Over Time
Publication
Country
Sample
Measure
Comparison of
different measures
Consistency in
reports over time
O
de
Adolescent samples
Ougrin and
Boege (2013)
United
Kingdom
Adolescent
inpatients and
outpatients
n ¼ 100, 12–17 years
Self-harm
Yes
Self-report ques-
tionnaire and
clinical record
(reported during
the clinical
assessment)
No
No
Hart et al. (2013)
United
States
Longitudinal
community
sample of
adolescents
n ¼ 678, Assessed
annually between
age 12 and 22
years
Suicide attempts
No
Yes
Also examined char-
acteristics associa-
ted with discrepant
reporting
No
Bja¨rehed et al. (2013)
Sweden
Adolescent
community
sample
Non-suicidal self-
injury
Yes
Self-report
questionnaire and
No
No
ARCHIVES OF SUICIDE RESEARCH
11 No Consistency in Reports of Self-Harm in Adolescence Most have focu-
sed on suicide attempts, which comprise
only a portion of episodes (Hawton et al.,
2002; Kidger et al., 2012; Muehlenkamp &
Gutierrez, 2004). Studies have reported
inconsistency across different self-report
methods, e.g., interviews vs. questionnaires
(Bja¨rehed
et
al.,
2013;
O’Sullivan
&
Fitzgerald, 1998; Ougrin & Boege, 2013;
Ross & Heath, 2002; Velting et al., 1998),
and also when using the same self-report
method across repeated assessments (Christl 3. Examine consistency in the reporting of
self-harm in ALSPAC over time, by
comparing questionnaire responses at
age 16 and 18 years. 4. Identify characteristics associated with
inconsistent reporting of self-harm over
time. We hypothesize that the prevalence of
self-harm recorded in medical records will
be
higher
among
questionnaire
non-
responders than responders, and that recall
of self-harm episodes over time will be
most consistent in individuals with more
severe
mental
health
problems=self-
harm. VOLUME 20 NUMBER 2 2016 114 Studies Investigating Consistency in Self-Harm Reports: Across Different Measures or Over Time
Country
Sample
Measure
Comparison of
different measures
Consistency in
reports over time
Other
design
Results
amples
3)
United
Kingdom
Adolescent
inpatients and
outpatients
n ¼ 100, 12–17 years
Self-harm
Yes
Self-report ques-
tionnaire and
clinical record
(reported during
the clinical
assessment)
No
No
3 (3%) indicated in the clinical
record they had self-harmed
but did not report self-harm
on the questionnaire. 20
(20%) reported at least one
episode of self-harm on the
questionnaire that was not
recorded in the clinical
record
United
States
Longitudinal
community
sample of
adolescents
n ¼ 678, Assessed
annually between
age 12 and 22
years
Suicide attempts
No
Yes
Also examined char-
acteristics associa-
ted with discrepant
reporting
No
88.5% inconsistently reported
a suicide attempt at some
point during the study;
65.3% were inconsistent the
year following the self-harm
event
Consistent and inconsistent
reporters did not differ on
clinical or demographic
variables, but consistent
reporters had higher lifetime
suicidal ideation
l. Sweden
Adolescent
community
sample
Non-suicidal self-
injury
Yes
Self-report
questionnaire and
No
No
97 adolescents were selected
for interview. 32=66 (48%)
participants who reported
(Continued ) Results
3 (3%) indicated in the clinical
record they had self-harmed
but did not report self-harm
on the questionnaire. ARCHIVES OF SUICIDE RESEARCH 115 115 Publication
Country
Sample
Measure
Comparison of
different measures
Consistency in
reports over time
Other
design
Results
n ¼ 1,052, from
grade 7 (mean age
13.7 years) and 8
(mean age 14.7
years)
follow-up inter-
view
self-harm on the
questionnaire did not
disclose self-harm during
the follow-up interview. Kidger et al. (2012)
United
Kingdom
Adolescent
community
sample
n ¼ 4810, 16 years
Suicide attempts
No
No
Yes
Examined suicidal
thoughts among
those with suicidal
self-harm
Approximately 10% of those
who reported wanting to die
during the most recent
episode of self-harm said
they had never had thoughts
of killing themselves
Christl et al. (2006)
Germany
Longitudinal
community
sample of
adolescents=
young adults
n ¼ 3021, 14–24
years at baseline
Suicide attempts
No
Yes
Also examined char-
acteristics associa-
ted with discrepant
reporting
Yes
Compared drop-out
rates among those
with and without
suicidal behaviour
One third of baseline suicide
attempters (n ¼ 15/45), did
not report a suicide attempt
at follow-up 4 years later
81% of discrepant reporters
were female and 59% were
aged 14–17 at baseline. LE 1. Continued
cation
Country
Sample
Measure
Comparison of
different measures
Consistency in
reports over time
Other
design
Results
n ¼ 1,052, from
grade 7 (mean age
13.7 years) and 8
(mean age 14.7
years)
follow-up inter-
view
self-harm on the
questionnaire did not
disclose self-harm during
the follow-up interview. r et al. 12)
United
Kingdom
Adolescent
community
sample
n ¼ 4810, 16 years
Suicide attempts
No
No
Yes
Examined suicidal
thoughts among
those with suicidal
self-harm
Approximately 10% of those
who reported wanting to die
during the most recent
episode of self-harm said
they had never had thoughts
of killing themselves
tl et al. 06)
Germany
Longitudinal
community
sample of
adolescents=
young adults
n ¼ 3021, 14–24
years at baseline
Suicide attempts
No
Yes
Also examined char-
acteristics associa-
ted with discrepant
reporting
Yes
Compared drop-out
rates among those
with and without
suicidal behaviour
One third of baseline suicide
attempters (n ¼ 15/45), did
not report a suicide attempt
at follow-up 4 years later
81% of discrepant reporters
were female and 59% were
aged 14–17 at baseline. ARCHIVES OF SUICIDE RESEARCH Greater consistency in report-
ing was associated with a
higher number of psychi-
atric disorders
Those with a suicide attempt
at baseline were at least 1.6
times more likely to drop
out of the study then those ication
Country
Sample
Measure
Comparison of
different measures
Consistency in
reports over time
Other
design
Results
n ¼ 1,052, from
grade 7 (mean age
13.7 years) and 8
(mean age 14.7
years)
follow-up inter-
view
self-harm on the
questionnaire did not
disclose self-harm during
the follow-up interview. er et al. 012)
United
Kingdom
Adolescent
community
sample
n ¼ 4810, 16 years
Suicide attempts
No
No
Yes
Examined suicidal
thoughts among
those with suicidal
self-harm
Approximately 10% of those
who reported wanting to die
during the most recent
episode of self-harm said
they had never had thoughts
of killing themselves
tl et al. 006)
Germany
Longitudinal
community
sample of
adolescents=
young adults
n ¼ 3021, 14–24
years at baseline
Suicide attempts
No
Yes
Also examined char-
acteristics associa-
ted with discrepant
reporting
Yes
Compared drop-out
rates among those
with and without
suicidal behaviour
One third of baseline suicide
attempters (n ¼ 15/45), did
not report a suicide attempt
at follow-up 4 years later
81% of discrepant reporters
were female and 59% were
aged 14–17 at baseline. Greater consistency in report-
ing was associated with a
higher number of psychi-
atric disorders
Those with a suicide attempt
at baseline were at least 1.6
times more likely to drop
out of the study then those Results without suicide attempts or
ideas
002)
Not
specified
Adolescent
community
sample
n ¼ 440 (from 2
schools), average
age 14–15 years
Self-mutilation
(defined as
deliberate
alteration or
destruction of
body tissue
without suicidal
intent)
Yes
Self-report screen-
ing questionnaire
and follow-up
interview
No
No
School sample 1: 38.8% who
reported self-harm on the
questionnaire were not
classified as having self-
harmed following the
interview (19=49)
School sample 2: 24.4% who
reported self-harm on the
questionnaire were not
classified as having self-
harmed following the
interview (10=41)
l. United
States
Adolescent
outpatients
n ¼ 48, 12–20 years,
mean 15.3 years
Suicide attempts
Yes
Self-report ques-
tionnaire and
interview
Also investigated
explanations and
characteristics
associated with
discrepant
reporting
No
No
Discrepancies in reporting
were found amongst 50% of
the sample (24=48). ARCHIVES OF SUICIDE RESEARCH Discrepancies primarily due to
confusion with the oper-
ational definition of suicidal
behaviour (i.e., confused
attempt and ideation or con-
fused attempt and gesture). The discrepant and non-dis-
crepant groups were com-
parable on measures of
suicidal intent, ideation, and
hopelessness and on their
diagnostic profiles
(Continued ) without suicide attempts or
ideas
School sample 1: 38.8% who
reported self-harm on the
questionnaire were not
classified as having self-
harmed following the
interview (19=49)
School sample 2: 24.4% who
reported self-harm on the
questionnaire were not
classified as having self-
harmed following the
interview (10=41)
Discrepancies in reporting
were found amongst 50% of
the sample (24=48). Discrepancies primarily due to
confusion with the oper-
ational definition of suicidal
behaviour (i.e., confused
attempt and ideation or con-
fused attempt and gesture). The discrepant and non-dis-
crepant groups were com-
parable on measures of
suicidal intent, ideation, and
hopelessness and on their
diagnostic profiles No No Ross and
Heath (2002)
Not
specified
Adolescent
community
sample
n ¼ 440 (from 2
schools), average
age 14–15 years
Self-mutilation
(defined as
deliberate
alteration or
destruction of
body tissue
without suicidal
intent)
Yes
Self-report screen-
ing questionnaire
and follow-up
interview
No
Velting et al. (1998)
United
States
Adolescent
outpatients
n ¼ 48, 12–20 years,
mean 15.3 years
Suicide attempts
Yes
Self-report ques-
tionnaire and
interview
Also investigated
explanations and
characteristics
associated with
discrepant
reporting
No
ARCHIVES OF SUICIDE RESEARCH No No ARCHIVES OF SUICIDE RESEARCH ARCHIVES OF SUICIDE RESEARCH 117 Country
Sample
Measure
Comparison of
different measures
Consistency in
reports over time
Other
design
Results
nd
Ireland
Adolescent
community
sample
n ¼ 88 age 13–14
years
Suicide attempt
Yes
Self-report
screening
questionnaire and
follow-up
interview
No
No
45 adolescents completed a
follow-up interview. 5=7
(71%) participants who
reported a suicide attempt
on the questionnaire did not
disclose self-harm during
the follow-up interview. es
m
4)
The
Nether-
lands
Longitudinal cohort
of adults with
depressive or
anxiety disorders
n ¼ 1973, aged 18–
65 years at base-
line, (mean age
42.4 years)
Suicide attempts
No
Yes
Also examined char-
acteristics associa-
ted with discrepant
reporting
No
23% of baseline suicide
attempters, did not report
their attempt at follow-up 2
years later (63=274)
Consistent reporting was
associated with a greater
number of suicide attempts,
and more severe current
psychopathology. No
differences were found for
recency of the event, age,
sex, or education
t al. Denmark
Patients admitted to
hospital following
a suicide attempt
n ¼ 243, age 12þ,
mean age 31 years
Suicide attempts,
assessed 1 year
after baseline
Yes
Self-report (tele-
phone interview)
and hospital
records
No
No
Seven suicide attempts listed in
the hospital records were
not reported by participants. Nine patients reported a
suicide attempt that was not
listed in the hospital records Country
Sample
Measure
Comparison of
different measures
Consistency in
reports over time
Other
design
Results
nd
Ireland
Adolescent
community
sample
n ¼ 88 age 13–14
years
Suicide attempt
Yes
Self-report
screening
questionnaire and
follow-up
interview
No
No
45 adolescents completed a
follow-up interview. 5=7
(71%) participants who
reported a suicide attempt
on the questionnaire did not
disclose self-harm during
the follow-up interview. es
m
4)
The
Nether-
lands
Longitudinal cohort
of adults with
depressive or
anxiety disorders
n ¼ 1973, aged 18–
65 years at base-
line, (mean age
42.4 years)
Suicide attempts
No
Yes
Also examined char-
acteristics associa-
ted with discrepant
reporting
No
23% of baseline suicide
attempters, did not report
their attempt at follow-up 2
years later (63=274)
Consistent reporting was
associated with a greater
number of suicide attempts,
and more severe current
psychopathology. No
differences were found for Results
45 adolescents completed a
follow-up interview. 5=7
(71%) participants who
reported a suicide attempt
on the questionnaire did not
disclose self-harm during
the follow-up interview. ARCHIVES OF SUICIDE RESEARCH Austria
Adult community
sample
n ¼ 1385, age 18–84
years, Mean 37.8
years
Suicide attempts
No
No
Yes
Examined intent to
die among those
reporting suicidal
self-harm
One quarter (15=60) of
individuals reporting a
suicide attempt were false
positives (lacked intent or
attempt aborted)
0.8% (n ¼ 11) were identified
as false negatives (reported
no suicide attempt on the
screen question but reported
a self-harm event with intent
to die in follow-up ques-
tions). 2=11 (18%) false
negatives resulted in injuries
requiring hospital treatment
There were no differences
between true positives and
false positives regarding age
or education or lethality of
method
al. United
States
Adult clinical
sample: Five
cohorts, three
with borderline
personality
disorder
Self-harm
Yes
Self-report interview
and
1) therapist notes
2) participant diary
cards
3) medical records
No
No
Agreement with therapist
notes (presence=absence of
self-harm) was 83%
Good agreement with diary
cards (mean 4.5 acts at
interview vs. mean 4.3 acts
on diary cards)
82% of episodes reported by
participants as being
(Continued ) l. Austria
Adult community
sample
n ¼ 1385, age 18–84
years, Mean 37.8
years
Suicide attempts
No
No
Yes
Examined intent to
die among those
reporting suicidal
self-harm
One quarter (15=60) of
individuals reporting a
suicide attempt were false
positives (lacked intent or
attempt aborted)
0.8% (n ¼ 11) were identified
as false negatives (reported
no suicide attempt on the
screen question but reported
a self-harm event with intent
to die in follow-up ques-
tions). 2=11 (18%) false
negatives resulted in injuries
requiring hospital treatment
There were no differences
between true positives and
false positives regarding age
or education or lethality of
method
al. United
States
Adult clinical
sample: Five
cohorts, three
with borderline
personality
disorder
Self-harm
Yes
Self-report interview
and
1) therapist notes
2) participant diary
cards
3) medical records
No
No
Agreement with therapist
notes (presence=absence of
self-harm) was 83%
Good agreement with diary
cards (mean 4.5 acts at
interview vs. mean 4.3 acts
on diary cards)
82% of episodes reported by
participants as being
(Continued ) Yes
Examined intent to
die among those
reporting suicidal
self-harm
One quarter (15=60) of
individuals reporting a
suicide attempt were false
positives (lacked intent or
attempt aborted)
0.8% (n ¼ 11) were identified
as false negatives (reported
no suicide attempt on the
screen question but reported
a self-harm event with intent
to die in follow-up ques-
tions). ARCHIVES OF SUICIDE RESEARCH 23% of baseline suicide
attempters, did not report
their attempt at follow-up 2
years later (63=274)
Consistent reporting was
associated with a greater
number of suicide attempts,
and more severe current
psychopathology. No
differences were found for
recency of the event, age,
sex, or education
Seven suicide attempts listed in
the hospital records were
not reported by participants. Nine patients reported a
suicide attempt that was not
listed in the hospital records TABLE 1. Continued
Publication
Country
Sample
Measure
Comparison of
different measures
Consistency in
reports over time
O
d
O’Sullivan and
Fitzgerald
(1998)
Ireland
Adolescent
community
sample
n ¼ 88 age 13–14
years
Suicide attempt
Yes
Self-report
screening
questionnaire and
follow-up
interview
No
No
Adult samples
Eikelenboom
et al. (2014)
The
Nether-
lands
Longitudinal cohort
of adults with
depressive or
anxiety disorders
n ¼ 1973, aged 18–
65 years at base-
line, (mean age
42.4 years)
Suicide attempts
No
Yes
Also examined char-
acteristics associa-
ted with discrepant
reporting
No
Morthorst et al. (2011)
Denmark
Patients admitted to
hospital following
a suicide attempt
n ¼ 243, age 12þ,
mean age 31 years
Suicide attempts,
assessed 1 year
after baseline
Yes
Self-report (tele-
phone interview)
and hospital
records
No
No
118
VOLUME 20 NUMBER 2 2016 TABLE 1. Continued
Publication
Country
Sample
Measure
Comparison of
different measures
Consistency in
reports over time
O’Sullivan and
Fitzgerald
(1998)
Ireland
Adolescent
community
sample
n ¼ 88 age 13–14
years
Suicide attempt
Yes
Self-report
screening
questionnaire and
follow-up
interview
No
No
Adult samples
Eikelenboom
et al. (2014)
The
Nether-
lands
Longitudinal cohort
of adults with
depressive or
anxiety disorders
n ¼ 1973, aged 18–
65 years at base-
line, (mean age
42.4 years)
Suicide attempts
No
Yes
Also examined char-
acteristics associa-
ted with discrepant
reporting
No
Morthorst et al. (2011)
Denmark
Patients admitted to
hospital following
a suicide attempt
n ¼ 243, age 12þ,
mean age 31 years
Suicide attempts,
assessed 1 year
after baseline
Yes
Self-report (tele-
phone interview)
and hospital
records
No
No
118
VOLUME 20 NUMBER 2 2016 No No VOLUME 20 NUMBER 2 2016 118 l. Sample The Avon Longitudinal Study of Parents and
Children
(ALSPAC). ALSPAC
is
a
population-based
birth
cohort
study
examining influences on health and develop-
ment across the lifecourse. The ALSPAC
core enrolled sample consists of 14,541
pregnant women resident in the former
county of Avon in South West England
(United Kingdom), with expected delivery
dates between April 1, 1991 and December
31, 1992 (Boyd et al., 2013). Of the 14,062
live
births,
13,798
were
singletons=
first-born of twins and were alive at 1 year
of age. Participants have been followed up
since recruitment through regular question-
naires and research clinics. Detailed infor-
mation about ALSPAC is available on the
study
website
(http://www.bristol.ac.uk/
alspac), which includes a fully searchable
data-dictionary of available data (http://
www.bris.ac.uk/alspac/researchers/data-
access/data-dictionary). Self-harm
was
assessed via self-report questionnaire at
age 16 years (mean age of respondents 16
years 8 months, standard deviation [SD]
approximately 3 months). The postal ques-
tionnaire was sent to 9,383 participants of
whom
4,855
(51.7%)
returned
it
and
4,810 completed the self-harm items (Kidger
et al., 2012). Ethical approval for the study
was obtained from the ALSPAC Law and
Ethics committee and local research ethics
committees
(NHS
Haydock
REC:
10=
H1010=70). The
Hospital
Episode
Statistics
Database
(HES). The HES database (Copyright
# 2012, re-used with the permission of
The Health and Social Care Information
Centre. All rights reserved) contains infor-
mation about hospital presentations and
admissions
for
all
NHS
hospitals
in
England; it contains admissions data from
1989 onwards, outpatient data from 2003
onwards
and
A&E
data
from
2007
onwards
(http://www.hscic.gov.uk/hes). Of the 3,027 individuals who consented
to data linkage (see above) 2,957 indivi-
duals (97.7%) had an existing linkage to
the NHS central register, which in turn
provided a means to identify the indivi-
duals’ secondary care records contained in
the HES database. The remaining 70 cases
were linked to HES using NHS ID num-
ber, name, and date of birth. In this scen-
ario ‘‘linkage’’ refers to the process of
testing if the ALSPAC participants had
any HES records rather than the actual
identification and extraction of a record. We make this distinction as some indivi-
duals will genuinely not have any HES
records, while others may have a HES Linkage. ARCHIVES OF SUICIDE RESEARCH 2=11 (18%) false
negatives resulted in injuries
requiring hospital treatment
There were no differences
between true positives and
false positives regarding age
or education or lethality of
method
No
Agreement with therapist
notes (presence=absence of
self-harm) was 83%
Good agreement with diary
cards (mean 4.5 acts at
interview vs. mean 4.3 acts
on diary cards)
82% of episodes reported by
participants as being
(Continued ) No Yes
Self-report interview
and
1) therapist notes
2) participant diary
cards
3) medical records
No No ARCHIVES OF SUICIDE RESEARCH 119 119 ication
Country
Sample
Measure
Comparison of
different measures
Consistency in
reports over time
Other
design
Results
medically treated had a cor-
responding medical record. There were no false nega-
tives —all medically treated
episodes were reported by
participants. k and
essler
006)
United
States
Predominately
Adult community
sample
n ¼ 5,877, aged
15–54 years
Suicide attempts
No
No
Yes
Examined intent to
die among those
reporting suicidal
self-harm
112=268 (42%) of those
reporting a lifetime history
of suicide attempt reported
no intent to die VOLUME 20 NUMBER 2 2016 120 B. Mars et al. B. Mars et al. METHODS When the ALSPAC children reached
adulthood (age 18), they were invited to
enroll in the study in their own right and
to consent to the extraction and use of
their health records. Through the Project
to Enhance ALSPAC through Record
Linkage
(PEARL)
http://www.bristol. ac.uk/alspac/participants/playingyourpart/
information
and
consent
forms
were
posted to 12,385 of the participants eligible
to
be
included
in
this
investigation
(singletons=first
born
twins
from
the
ALSPAC core enrolled sample who were
alive at 1 year. See Figure 1). Of those
invited
to
consent
(n ¼ 12,385),
3,027
(24.4%) consented to data linkage by the
study cut-off date, 8,905 (71.9%) did not
respond to the consent request, and 82
(0.7%) returned an incomplete consent
form. Only 371 (3.0%) declined to consent. Sample The
Health
and
Social
Care
Information
Centre
(HSCIC)
linked
ALSPAC
participants
with
the
NHS
Central Register, with a 99% match rate
(Boyd et al., 2013); this was done on the
basis of NHS ID number, name, date of
birth, and postcode using deterministic
linkage. ARCHIVES OF SUICIDE RESEARCH 121 Consistency in Reports of Self-Harm in Adolescence Consistency in Reports of Self-Harm in Adolescence FIGURE 1. Flow-chart of linkage between the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort and
the Hospital Episode Statistics database (HES). FIGURE 1. Flow-chart of linkage between the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort and
the Hospital Episode Statistics database (HES). 5 million (8%) patients in the UK. Link-
age between ALSPAC and the CPRD was
conducted by the NHS Information Centre
(NHS IC) as a trusted third party. With
approval from the NIGB Ethics and Confi-
dentiality Committee, the NHS IC ident-
ified ALSPAC eligible individuals who also
appeared in the CPRD, and sent an anon-
ymized linking dataset to be stored securely
at the CPRD where the data were merged
and analyzed. This particular linkage does record which we failed to identify during
the linkage process. In March 2013 the
NHS Health and Social Care Information
Centre (HSCIC) extracted the hospital
admissions records of 2,988 participants,
although we consider the denominator to
be the 3,027 cases tested for linkage. The
Clinical
Practice
Research
Datalink
(CPRD). The CPRD is an anonymized
database of primary care records of around VOLUME 20 NUMBER 2 2016 122 B. Mars et al. B. Mars et al. CPRD records before the age of 10 years,
as self-harm before this age is rare. CPRD records before the age of 10 years,
as self-harm before this age is rare. not require consent above and beyond the
consent
obtained
for
participation
in
ALSPAC. However, any participants who
did not agree to their health records being
extracted (via the PEARL consent request
described above) were excluded (n ¼ 3). Consistency in Reports of Self-Harm in Adolescence study (Madge et al., 2008). Participants who
responded positively to the item ‘‘have you
ever hurt yourself on purpose in any way
(e.g., by taking an overdose of pills or by
cutting yourself)?’’ were classified as having
a lifetime history of self-harm. measured during pregnancy (O-levels and
A-levels are school qualifications taken
around age 16 and 18 years respectively),
(5) child IQ assessed using the Wechsler
intelligence test for children (WISC-III)
(Wechsler,
1991)
at
age
8
years
(6)
depression symptoms, assessed at age 16
and 18 years using the short Moods and
Feelings Questionnaire (SMFQ), a score
of 11 or more on the SMFQ was taken as
indicative of depressive symptoms (Patton
et al., 2008) and (7) depressive disorder,
assessed at age 18 years using the CIS-R. Those who answered ‘‘yes’’ to having
self-harmed were then asked further closed
response questions, including how long ago
they last hurt themselves (in the last week,
more than a week ago but in the last year,
more than a year ago), the reasons for
self-harm the last time they hurt themselves
on purpose (six response categories), and
whether they had ever seriously wanted to
kill themselves when self-harming (Kidger
et al., 2012). Participants were classified as
having a lifetime history of suicidal self-
harm if they selected ‘‘I wanted to die’’ as
a reason for harming themselves on the
most recent occasion, or if they reported
they had ever seriously wanted to kill them-
selves when self-harming (Mars et al.,
2014). The
Hospital
Episode
Statistics
Database
(HES). We used an extract of the HES
data including hospital admissions for
self-harm
(ICD
10
codes
Y10–Y34,
X60–X84 and X40–X49), A&E atten-
dances for self-harm (A&E diagnostic
codes 141=142 ‘‘poisoning (inc overdose)
due to prescriptive=proprietary drugs,’’ or
reason for A&E attendance coded as
‘‘deliberate
self-harm’’)
and
hospital
admissions
for
a
mental
health
con-
dition(s)
(ICD-10
codes
F00–F99). Further details can be found in Appendix
1. While X40–X49 are coded as accidental
poisoning, previous studies indicate that
they are also used for self-harm. The date
of
hospital
attendance
was
cross-
referenced with the date of questionnaire
completion to identify whether events
occurred before or after completion of
the
self-harm
questionnaire. Although
A&E data is recorded in HES, it is only
available from 2007 onwards and is likely
to be under-reported. Measures History of self-harm was assessed in
the ALSPAC cohort, the Hospital Episode
Statistics Database (Secondary Care) and
the Clinical Practice Research Datalink
(Primary Care). The methods of assessment
for each data source are described below. Data on psychosocial characteristics were
also collected in ALSPAC. Of the live births linked by the NHS
IC that appeared in the CPRD, 520
were in the sub-sample eligible for this
investigation (singletons=first born twins
from the ALSPAC core enrolled sample
who were alive at 1 year). The sample
was further restricted to individuals who
were registered with a CPRD-contributing
practice for the entire period between age
10 and 17 years (n ¼ 205) (Figure 2), to
ensure that there were no breaks in the
patients’ records. We did not examine The Avon Longitudinal Study of Parents and
Children (ALSPAC). The self-harm ques-
tions used in the age 16 self-report question-
naire were based on those used in the CASE FIGURE 2. Flow-chart of linkage between the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort and
the Clinical Practice Research Datalink (CPRD). FIGURE 2. Flow-chart of linkage between the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort and
the Clinical Practice Research Datalink (CPRD). ARCHIVES OF SUICIDE RESEARCH 123 Consistency in Reports of Self-Harm in Adolescence For example, in
the extracted data, all but two self-harm
hospital admissions were recorded as hav-
ing come via A&E (the remaining two
admissions were emergency referrals by
GP);
however,
two-thirds
of
hospital
admissions had no corresponding A&E
record for self-harm. For this reason, we
have focused primarily on hospital admis-
sions data in this paper, as this is known to
be more complete. The findings for A&E The same question was used to assess
lifetime self-harm at age 18 years, using
the self-administered computerized version
of the Clinical Interview Schedule-Revised
(CIS-R) (Lewis, Pelosi, Araya, & Dunn,
1992). There is close agreement between
the self-administered computerized version
and the interviewer administrated versions
of the CIS-R (Bell, Watson, Sharp, Lyons,
& Lewis, 2005; Lewis, 1994; Patton et al.,
1999). Psychosocial
Characteristics. We
examined
key psychosocial characteristics assessed
previously in ALSPAC to identify factors
associated with inconsistencies in reporting
self-harm over time. The following vari-
ables were used: (1) participant’s gender,
(2)
ethnicity,
(3)
parent
social
class
(professional=managerial or other occupa-
tions; the highest of maternal or paternal
social class was used), (4) highest maternal
educational attainment (less than O-level,
O-level,
A-level,
or
university
degree) VOLUME 20 NUMBER 2 2016 124 B. Mars et al. only data are also presented, but need to
be interpreted with caution. 41 participants with at least one recorded
hospital admission for self-harm, and 18
(0.6%) with at least one recorded ‘‘A&E
only’’ attendance for self-harm (i.e., A&E
attendance
without
subsequent
hospital
admission). It is notable that 66% of indivi-
duals who were admitted to hospital follow-
ing self-harm had no corresponding A&E
record for self-harm. Eighty-two (2.7%)
had at least one hospital admission for a
mental health condition recorded in HES. Of the 3,027 individuals tested for linkage,
2,363 (78.1%) completed the self-harm
questionnaire at age 16 years. The
Clinical
Practice
Research
Datalink
(CPRD). Cases of self-harm occurring in
the CPRD until December 31, 2011 were
identified using appropriate Read codes
for attempted suicide and self-harm (see
Appendix 2) (Thomas et al., 2013). Analysis Plan 0.5%, difference¼0.6%,
95% CI 0.2–1.4%, P¼ 0.081). Consistency in Self-Report Over Time. We
investigated inconsistency in reporting of
lifetime self-harm over time between age
16 and 18 years in ALSPAC cohort. Parti-
cipants who reported no self-harm, or
reported self-harm for the first time at age
18 years were excluded from these analyses. Characteristics associated with incon-
sistent reporting of self-harm over time were
also examined using logistic regression. Consistency in Self-Report Over Time. We
investigated inconsistency in reporting of
lifetime self-harm over time between age
16 and 18 years in ALSPAC cohort. Parti-
cipants who reported no self-harm, or
reported self-harm for the first time at age
18 years were excluded from these analyses. Characteristics associated with incon-
sistent reporting of self-harm over time were
also examined using logistic regression. RESULTS
Self-Harm
HES. Of the 3,027 ALSPAC participants
tested for linkage with HES (hospital
records), 54 (1.8%) had one or more
self-harm events recorded in HES, including RESULTS CPRD. Two of the 6 individuals with a
self-harm Read code recorded in the CPRD
completed the age 16 self-harm question-
naire. There was no evidence of a differ-
ence in prevalence between questionnaire
responders and non-responders (2.8% in
non-responders vs. 3.1% in responders, Analysis Plan Non-Response. We examined whether there
was an association between questionnaire
response and medically recorded self-harm
by comparing the prevalence of self-harm
in HES and the CPRD among those who
completed and did not complete the self-
harm questionnaire at age 16 years. CPRD. Of the 205 ALSPAC participants
registered with a CPRD contributing prac-
tice between age 10 and 17 years, 64
(31.2%) completed the self-harm question-
naire at age 16 years. Only 6 participants
(2.9%) had a relevant self-harm Read code
recorded in the CPRD. Agreement Between Self-Report and Medically
Recorded Self-Harm Events. We compared
self-reported
self-harm
episodes
with
events recorded in HES and the CPRD,
in order to identify instances in which
self-harm was inconsistently reported. Non-Response HES. The
prevalence
of
hospital
admissions for self-harm and mental health
conditions recorded in HES was higher
among those who did not complete the
self-harm questionnaire at age 16 years than
among those who did (Table 2) (self-harm
hospital
admissions:
2.0%
in
non-
responders
vs. 1.2%
in
responders,
difference ¼ 0.8%, 95% CI 0.4–1.9%,
P ¼ 0.128; mental health hospital admis-
sions 4.8 vs. 2.1%, difference ¼ 2.7%, 95%
CI 1.0–4.4%, P < 0.001). The same pattern
of results was found for A&E only self-harm
attendances (1.1 vs. 0.5%, difference¼0.6%,
95% CI 0.2–1.4%, P¼ 0.081). HES. The
prevalence
of
hospital
admissions for self-harm and mental health
conditions recorded in HES was higher
among those who did not complete the
self-harm questionnaire at age 16 years than
among those who did (Table 2) (self-harm
hospital
admissions:
2.0%
in
non-
responders
vs. 1.2%
in
responders,
difference ¼ 0.8%, 95% CI 0.4–1.9%,
P ¼ 0.128; mental health hospital admis-
sions 4.8 vs. 2.1%, difference ¼ 2.7%, 95%
CI 1.0–4.4%, P < 0.001). The same pattern
of results was found for A&E only self-harm
attendances (1.1 vs. 0.5%, difference¼0.6%,
95% CI 0.2–1.4%, P¼ 0.081). HES. The
prevalence
of
hospital
admissions for self-harm and mental health
conditions recorded in HES was higher
among those who did not complete the
self-harm questionnaire at age 16 years than
among those who did (Table 2) (self-harm
hospital
admissions:
2.0%
in
non-
responders
vs. 1.2%
in
responders,
difference ¼ 0.8%, 95% CI 0.4–1.9%,
P ¼ 0.128; mental health hospital admis-
sions 4.8 vs. 2.1%, difference ¼ 2.7%, 95%
CI 1.0–4.4%, P < 0.001). The same pattern
of results was found for A&E only self-harm
attendances (1.1 vs. Self-Harm ARCHIVES OF SUICIDE RESEARCH 125 Consistency in Reports of Self-Harm in Adolescence difference ¼ 0.3%, 95% CI 4.8–5.4%,
P ¼ 0.910). These findings need to be
interpreted with caution, given the small
number of ALSPAC individuals with a
self-harm Read code recorded in the CPRD
(n ¼ 6). Agreement Between Self-Report and
Medical Records
HES. Of the 2,363 individuals tested for
linkage
who
completed
the
self-harm
TABLE 2. Differences In Prevalence of Hospital Admissions For Self-Harm and Mental Health
Conditions in the Hospital Episode Statistics Database Among Those Who Completed vs. Those Who Did Not Complete the Age 16 Year Self-Harm Questionnaire
Self-harm
questionnaire data
n ¼ 2,363 N (%)
No self-harm
questionnaire data
n ¼ 664 N (%)
Difference
(95% CI)
P value
Admitted to hospital for self-harm
28 (1.2%)
13 (2.0%)
0.8% (0.4%, 1.9%)
0.128
Admitted to hospital for mental
health problem
50 (2.1%)
32 (4.8%)
2.7% (1.0%, 4.4%)
<0.001
TABLE 3. Psychosocial Characteristics Associated With Inconsistent Reporting of Self-Harm
Episodes Over Time
Self-harm
reported
consistently
(n ¼ 385)
Self-harm
reported
inconsistently
(n ¼ 204)
OR
[95%CI]
P value
Female gender, n (%)
312 (81.0%)
162 (79.4%)
0.90 [0.59, 1.38]
0.636
Parental social class, (pregnancy), n (%)
Other
131 (36.1%)
62 (32.5%)
Professional=managerial
232 (63.9%)
129 (67.5%)
0.85 [0.59, 1.23]
0.395
Mother’s education (pregnancy), n (%)a
<O-level
60 (15.9%)
29 (14.6%)
O-level
141 (37.4%)
69 (35.2%)
Degree=A level
176 (46.7%)
100 (50.2%)
0.91 [0.72, 1.16]
0.441
Ethnicity
White
361 (96.8%)
190 (96.5%)
Non-white
12 (3.2%)
7 (3.5%)
0.90 [0.34, 2.33]
0.832
IQ, age 8 years, mean (SD)
108.7 (15.6)
110.6 (14.5)
1.01 [1.00, 1.02]
0.166
Depression symptoms: SMFQ score 11þ,
age 16 years, n (%)
167 (44.2%)
62 (30.7%)
0.56 [0.39, 0.80]
0.002
Depression symptoms: SMFQ score 11þ,
age 18 years, n (%)
159 (46.4%)
59 (31.1%)
0.52 [0.36, 0.76]
0.001
Depressive disorder: CIS-R, age 18 years
83 (21.6%)
15 (7.4%)
0.29 [0.16, 0.52] <0.001 difference ¼ 0.3%, 95% CI 4.8–5.4%,
P ¼ 0.910). These findings need to be
interpreted with caution, given the small
number of ALSPAC individuals with a
self-harm Read code recorded in the CPRD
(n ¼ 6). HES. Of the 2,363 individuals tested for
linkage
who
completed
the
self-harm TABLE 3. aOR for maternal education assumes a linear trend across the education categories. Note. SMFQ: Short Mood and Feelings Questionnaire; CIS-R: Clinical Interview Schedule revised. DISCUSSION There were 15 self-harm hospital atten-
dances recorded in HES prior to completion of
the self-harm questionnaire (12 admissions and
3 A&E only attendances). Three (20%;
95% CI 4–48%) of these episodes were
not reported by ALSPAC participants on
the questionnaire (1=12 admissions and
2=3 A&E only attendances). Main Findings This study is, as far as we are aware,
the first to examine whether the prevalence
of medically recorded self-harm differs
from prevalence determined by question-
naire
response
in
a
community-based
sample of adolescents. We also investigated
the level of agreement between self-reported
self-harm history and data obtained from
medical records. CPRD. Both of the self-harm events
recorded in the CPRD were reported by
participants on the self-harm questionnaire;
however, neither participant reported hav-
ing sought help for self-harm from their
GP (a consultation with the GP would be
necessary in order for a self-harm Read
code to be recorded in the CPRD). We found some evidence for both
selective non-participation of individuals
with
self-harm,
and
for
discrepancies
between
self-reported
and
medically
recorded self-harm episodes; approximately
one-fifth of self-harm events recorded in
HES (hospital admissions or A&E presen-
tations) were not reported by participants
on the questionnaire. Taken together, these
findings suggest that prevalence estimates
derived from self-report may underestimate
the true rate of adolescent self-harm in the
community. Consistency of Reporting of
Self-Harm Over Time Consistency of Reporting of
Self-Harm Over Time Five hundred and eighty nine indivi-
duals reported lifetime self-harm at age 16
years
and
provided
information
on
self-harm at age 18 years. Of these, 385
(65.4%) reported self-harm consistently at
both time points, and 204 individuals
(34.6%) reported self-harm inconsistently,
i.e., reported lifetime self-harm at age 16
years but not at age 18 years. We additionally examined the consist-
ency of self-reported self-harm over time
and found that over a third of respondents
who reported self-harm at age 16 years said
they had never self-harmed when asked at
age 18 years. Those who reported self-
harm inconsistently over time were less
likely to have to have depressive disorder,
less likely to have harmed in the year prior
to the age 16 year questionnaire and were
less likely to have self-harmed with suicidal
intent. Characteristics Associated With Consistency in
Reporting of Self-Harm Over Time. Compared
with those who reported self-harm consist-
ently
over
time,
those
who
reported
self-harm inconsistently were less likely to
have evidence of depression at age 16 and
18
years,
were
less
likely
to
have
self-harmed in the year prior to the age
16 year questionnaire, and were less likely
to have harmed with suicidal intent by
age 16 years (Table 3). There was little evi-
dence for differences according to gender, Self-Harm Psychosocial Characteristics Associated With Inconsistent Reporting of Self-Harm
Episodes Over Time
Self-harm
reported
consistently
(n ¼ 385)
Self-harm
reported
inconsistently
(n ¼ 204)
OR
[95%CI]
P value
Female gender, n (%)
312 (81.0%)
162 (79.4%)
0.90 [0.59, 1.38]
0.636
Parental social class, (pregnancy), n (%)
Other
131 (36.1%)
62 (32.5%)
Professional=managerial
232 (63.9%)
129 (67.5%)
0.85 [0.59, 1.23]
0.395
Mother’s education (pregnancy), n (%)a
<O-level
60 (15.9%)
29 (14.6%)
O-level
141 (37.4%)
69 (35.2%)
Degree=A level
176 (46.7%)
100 (50.2%)
0.91 [0.72, 1.16]
0.441
Ethnicity
White
361 (96.8%)
190 (96.5%)
Non-white
12 (3.2%)
7 (3.5%)
0.90 [0.34, 2.33]
0.832
IQ, age 8 years, mean (SD)
108.7 (15.6)
110.6 (14.5)
1.01 [1.00, 1.02]
0.166
Depression symptoms: SMFQ score 11þ,
age 16 years, n (%)
167 (44.2%)
62 (30.7%)
0.56 [0.39, 0.80]
0.002
Depression symptoms: SMFQ score 11þ,
age 18 years, n (%)
159 (46.4%)
59 (31.1%)
0.52 [0.36, 0.76]
0.001
Depressive disorder: CIS-R, age 18 years
83 (21.6%)
15 (7.4%)
0.29 [0.16, 0.52] <0.001
Past year self-harm, age 16 years
237 (61.9%)
99 (49.3%)
0.60 [0.42, 0.84]
0.003
Lifetime self-harm with suicidal intent, age 16 years
154 (40.2%)
42 (20.9%)
0.39 [0.26, 0.58] <0.001
Note. SMFQ: Short Mood and Feelings Questionnaire; CIS-R: Clinical Interview Schedule revised. aOR f
l d
i
li
d
h
d
i
i al Characteristics Associated With Inconsistent Reporting of Self-Harm
ver Time BLE 3. Psychosocial Characteristics Associated With Inconsistent Reporting of Self-Harm
Episodes Over Time VOLUME 20 NUMBER 2 2016 126 B. Mars et al. B. Mars et al. questionnaire at age 16 years, 419 (17.7%)
reported a history of self-harm. Only 12
(2.9%; 95% CI 1.5–5.9%) of these episodes
were recorded in HES. social class, IQ, maternal education or
ethnicity (Table 3). Strengths and Limitations Strengths and Limitations ALSPAC is a large, population-based
study, which is important, given that less
than 20% of adolescents who self-harm ARCHIVES OF SUICIDE RESEARCH 127 Consistency in Reports of Self-Harm in Adolescence of self-harm may not have been recorded
in the CPRD (Thomas et al., 2013), or
may have been missed (i.e., if documented
as a free text response rather than a Read
code). Fourth, when extracting data from
the HES database, we included codes
related to accidental poisoning (ICD 10
codes X40–X49) as these codes are often
used to indicate self-harm. While some
may be true instances of accidental self-
poisoning, this is unusual in adolescence. present to medical services (Hawton et al.,
2002; Kidger et al., 2012). We investigated
the level of agreement in reports of
self-harm both across different sources
(self-report and medical records) and over
time. The findings need to be interpreted in
light of several limitations. First, we were
only able to compare reports among those
who had been admitted to hospital or had
consulted with their GP. We were also only
able to examine self-harm hospital admis-
sions among those who had consented to
data linkage (24% of the
sub-sample
invited to consent) and GP events for
those in the CPRD between age 10 and
17 years (1.5% of the sub-sample of
13,798
included
in
this
investigation). These sub samples with available linked
records may not be representative of the
whole ALSPAC cohort. The issue of
required consent has the potential to
induce bias in our findings, however using
questionnaire data we found little evidence
of an association between self-harm and
consent to data linkage and so this is
unlikely to be a problem in this study. Second, it is likely that cultural differences
influence self-reporting of self-harm. The
degree of stigma associated with mental ill-
ness and self-harm varies around the world
(Abdullah & Brown, 2011; Evans-Lacko,
Brohan, Mojtabai, & Thornicroft, 2012;
Reynders, Kerkhof, Molenberghs, & Van
Audenhove, 2014), therefore findings from
our study may not be generalizable outside
a UK context. Finally, self-harm in ALSPAC was
assessed via self-report questionnaire at
age 16 years and via a self-administered
computerized assessment at age 18 years. Although the question used at both time
points was identical, the difference in set-
ting may have contributed to the discrepan-
cies in reporting found in this study. Comparison With Previous Research Previous studies investigating inconsist-
ency in reporting of self-harm have typically
relied on comparisons between interview
and
questionnaire
responses
(Bja¨rehed
et al., 2013; Ougrin & Boege, 2013; Ross
& Heath, 2002; Velting et al., 1998). Lower
rates of self-harm are usually found when
using interview as opposed to questionnaire
measures (Evans, Hawton, Rodham, Psy-
chol,
&
Deeks,
2005). However,
the
absence of a gold standard assessment for
self-harm means that it is not possible to
identify
which
of
these
measurement
approaches is more accurate—the ability
to ask additional clarification questions
could help to eliminate false positives that
arise from inaccurate self-reports (Hawton
et al., 2002; Ross & Heath, 2002; Velting
et al., 1998), but it is also possible that the
loss of anonymity found with interview
assessments may result in under-reporting
of self-harm (Safer, 1997). Third,
the
number
of
individuals
with self-harm recorded in their medical
records
was
small,
particularly
in
the
CPRD. This precluded our ability to exam-
ine characteristics associated with incon-
sistent reporting, and limited power to
detect differences between questionnaire
responders and non-responders. Findings
therefore need to be interpreted with
caution, and require replication in a larger
sample. It is possible that some episodes In the Early Developmental stages
of Psychopathology Study, Christl et al. (2006) found some evidence for selective VOLUME 20 NUMBER 2 2016 128 B. Mars et al. those that are more recent are more likely
to be recalled by participants and may be
less subject to reinterpretation. However,
in their investigation of suicide attempts
in adults, Eikelenboom et al. (2014) found
no
association
between
consistency in
reporting and the recency of self-harm at
baseline. It is also possible that individuals
with psychopathology and those who have
harmed with suicidal intent may be more
likely to continue to self-harm as adults. The
reasons
for
discrepant
reporting
require further investigation and could
include denial, errors in recall, or reinterpre-
tation of the self-harm event. Reports may
also be influenced by current mood state,
for example depressed mood could lead
to enhanced recall of negative events, such
as self-harm. Unfortunately, it is not poss-
ible to determine in this, or other studies,
which of the assessments is more accurate
(i.e.,
whether
the
first
reporting
of
self-harm is a false positive or whether the
second reporting is a false negative). Comparison With Previous Research non-response as those who reported sui-
cide attempts at baseline were at least 1.6
times more likely to drop out of the study
than those without suicidal thoughts or
behavior. The use of data linkage allows
us to extend this work by objectively
comparing the prevalence of self-harm
among questionnaire responders and non-
responders. There was also some evidence
for inconsistency between self-reported
and medically recorded self-harm. Possible
reasons for discrepancies include concerns
over stigma, denial, or problems with recall. Individuals
may
also
suppress
painful
memories such as self-harm or suicidal
ideation, which has been suggested as a
possible
adaptive
defensive
mechanism
(Goldney, Winefield, Winefield, & Saebel,
2009; Klimes-Dougan, Safer, Ronsaville,
Tinsley, & Harris, 2007). Our finding that a third of adolescents
were discrepant in their reporting of life-
time self-harm over time is lower than the
proportion found by Hart et al. (2013)
(approximately two thirds disrepant 1 year
after reporting a self-harm event) but
similar
to
findings
of
other
previous
longitudinal research (Eikelenboom et al.,
2014, Christl et al., 2006), all of which
investigated reporting of suicide attempts. Inconsistent reporting has also been shown
for other stigmatized behaviors such as
drug
use
(Percy,
McAlister,
Higgins,
McCrystal, & Thornton, 2005). We extend
this research by examining consistency in
reporting of self-harm regardless of suicidal
intent, and by examining various charac-
teristics associated with discrepant report-
ing. Similar to Christl et al. (2006) and
Eikelenboom et al. (2014), we found great-
er consistency in reporting among those
with psychopathology. We also found indi-
viduals were more likely to report self-harm
consistently if they had harmed with suici-
dal intent during their lifetime, and if they
had self-harmed in the year prior to ques-
tionnaire completion. This could suggest
that more severe self-harm episodes and It is also important to note that while
selective non-participation of those with
self-harm and inconsistent reporting could
result in distorted prevalence estimates, this
does not necessarily lead to biased esti-
mates of associations between self-harm
and exposure variables (Wolke et al., 2009). Further research is planned to investigate
this
issue
in
more
detail
within
the
ALSPAC cohort. Consistency in Reports of Self-Harm in Adolescence David
Gunnell,
School
of
Social
and Community Medicine, University of
Bristol, Bristol, UK. this approach would require complete
coverage of medical records for the sample
in question. In practice achieving this may
be restricted by governance requirements
based on concerns around the protection
of privacy with regard to sensitive infor-
mation in the situation where individuals
have not provided explicit consent. Such
concerns may be offset by evidence that
data-linkage
as
we
describe
here
can
improve the validity of medical research
and thus enhance the potential of research
to improve the public good. Correspondence concerning this article
should be addressed to Dr. Becky Mars,
School of Social and Community Medicine,
University of Bristol, Oakfield House,
Bristol BS8 2BN, UK. E-mail: becky. mars@bristol.ac.uk We are extremely grateful to all the
families who took part in this study, the
midwives for their help in recruiting them,
and the whole ALSPAC team, which
includes interviewers, computer and lab-
oratory
technicians,
clerical
workers,
research scientists, volunteers, managers,
receptionists, and nurses. AUTHOR NOTE Becky
Mars,
School
of
Social
and
Community Medicine, University of Bristol,
Bristol, UK. Rosie Cornish, School of Social and
Community Medicine, University of Bristol, Becky
Mars,
School
of
Social
and
Community Medicine, University of Bristol, Bristol, UK. Rosie Cornish, School of Social and Rosie Cornish, School of Social and
Community Medicine, University of Bristol, The authors report no conflict of interests This publication is the work of the
authors who serve as guarantors for the
contents of this paper. The study sponsor
had no further role in the study design
and collection, analysis, and interpretation of
data or in the writing of the article and the
decision to submit it for publication. CONCLUSION In our analyses of the ALSPAC cohort, we
have shown that self-harm prevalence esti-
mates derived from self-report are affected
by non-response and inconsistent report-
ing, and likely underestimate the true level
of adolescent self-harm in the community. Our findings require replication, but sug-
gest
benefits
of
combining
self-report
self-harm data with data from medical
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Enhance ALSPAC through Record Link-
age (PEARL) linked to routine health and
social records. PEARL was funded by the
Welcome Trust with a £1.7million grant
which commenced in April 2009 (Principal
investigator, Prof John Macleod; grant
reference WT086118=Z=08=Z. KH, GL
and DG are National Institute for Health
Research (NIHR) Senior Investigators. Catherine
Crane,
Department
of
Psychiatry,
University
of
Oxford,
Oxford, UK. Catherine
Crane,
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of
Psychiatry,
University
of
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young adolescents’ drug use self-reports: A recanting 132 VOLUME 20 NUMBER 2 2016 B. Mars et al. B. Mars et al. Consistency in Reports of Self-Harm in Adolescence APPENDIX 1 List of ICD-10 Codes Used to Identify Hospital Admissions for Non-fatal Self-harm in the Hospital
Episodes Statistics database (HES)
ICD-10 code
Description
X40
Accidental poisoning by and exposure to nonopioid analgesics, antipyretics and
antirheumatics
X41
Accidental poisoning by and exposure to antiepileptic, sedative-hypnotic, antiparkinsonism
and psychotropic drugs, not elsewhere classified
X42
Accidental poisoning by and exposure to narcotics and psychodysleptics [hallucinogens], not
elsewhere classified
X43
Accidental poisoning by and exposure to other drugs acting on the autonomic nervous
system
X44
Accidental poisoning by and exposure to other and unspecified drugs, medicaments and
biological substances
X45
Accidental poisoning by and exposure to alcohol
X46
Accidental poisoning by and exposure to organic solvents and halogenated hydrocarbons and
their vapors
X47
Accidental poisoning by and exposure to other gases and vapors
X48
Accidental poisoning by and exposure to pesticides
X49
Accidental poisoning by and exposure to other and unspecified chemicals and noxious
substances
X60
Intentional self-poisoning by and exposure to nonopioid analgesics, antipyretics and
antirheumatics
X61
Intentional self-poisoning by and exposure to antiepileptic, sedative-hypnotic,
antiparkinsonism and psychotropic drugs, not elsewhere classified
X62
Intentional self-poisoning by and exposure to narcotics and psychodysleptics [hallucinogens],
not elsewhere classified
X63
Intentional self-poisoning by and exposure to other drugs acting on the autonomic nervous
system
X64
Intentional self-poisoning by and exposure to other and unspecified drugs, medicaments and
biological substances
X65
Intentional self-poisoning by and exposure to alcohol
X66
Intentional self-poisoning by and exposure to organic solvents and halogenated
hydrocarbons and their vapors
X67
Intentional self-poisoning by and exposure to other gases and vapors
X68
Intentional self-poisoning by and exposure to pesticides
X69
Intentional self-poisoning by and exposure to other and unspecified chemicals and noxious
substances
X70
Intentional self-harm by hanging, strangulation and suffocation
X71
Intentional self-harm by drowning and submersion
X72
Intentional self-harm by handgun discharge
d CD-10 Codes Used to Identify Hospital Admissions for Non-fatal Self-harm in the Hospital
Episodes Statistics database (HES) (Continued ) ARCHIVES OF SUICIDE RESEARCH 133 Consistency in Reports of Self-Harm in Adolescence Consistency in Reports of Self-Harm in Adolescence APPENDIX 1. Consistency in Reports of Self-Harm in Adolescence Continued
ICD-10 code
Description
X73
Intentional self-harm by rifle, shotgun and larger firearm discharge
X74
Intentional self-harm by other and unspecified firearm discharge
X75
Intentional self-harm by explosive material
X76
Intentional self-harm by smoke, fire and flames
X77
Intentional self-harm by steam, hot vapors and hot objects
X78
Intentional self-harm by sharp object
X79
Intentional self-harm by blunt object
X80
Intentional self-harm by jumping from a high place
ICD-10 code
Description
X81
Intentional self-harm by jumping or lying before moving object
X82
Intentional self-harm by crashing of motor vehicle
X83
Intentional self-harm by other specified means
X84
Intentional self-harm by unspecified means
Y10
Poisoning by and exposure to nonopioid analgesics, antipyretics and antirheumatics,
undetermined intent
Y11
Poisoning by and exposure to antiepileptic, sedative-hypnotic, antiparkinsonism and
psychotropic drugs, not elsewhere classified, undetermined intent
Y12
Poisoning by and exposure to narcotics and psychodysleptics [hallucinogens], not elsewhere
classified, undetermined intent
Y13
Poisoning by and exposure to other drugs acting on the autonomic nervous system,
undetermined intent
Y14
Poisoning by and exposure to other and unspecified drugs, medicaments and biological
substances, undetermined intent
Y15
Poisoning by and exposure to alcohol, undetermined intent
Y16
Poisoning by and exposure to organic solvents and halogenated hydrocarbons and their
vapors, undetermined intent
Y17
Poisoning by and exposure to other gases and vapors, undetermined intent
Y18
Poisoning by and exposure to pesticides, undetermined intent
Y19
Poisoning by and exposure to other and unspecified chemicals and noxious substances,
undetermined intent
Y20
Hanging, strangulation and suffocation, undetermined intent
Y21
Drowning and submersion, undetermined intent
Y22
Handgun discharge, undetermined intent
Y23
Rifle, shotgun and larger firearm discharge, undetermined intent
Y24
Other and unspecified firearm discharge, undetermined intent
Y25
Contact with explosive material, undetermined intent
Y26
Exposure to smoke, fire and flames, undetermined intent
Y27
Contact with steam, hot vapors and hot objects, undetermined intent
Y28
Contact with sharp object, undetermined intent
Y29
Contact with blunt object, undetermined intent
(Continued ) APPENDIX 1. Continued (Continued ) VOLUME 20 NUMBER 2 2016 134 B. Mars et al. APPENDIX 1. Continued
ICD-10 code
Description
Y30
Falling, jumping, or pushed from a high place, undetermined intent
Y31
Falling, lying, or running before or into moving object, undetermined intent
Y32
Crashing of motor vehicle, undetermined intent
Y33
Other specified events, undetermined intent
Y34
Unspecified event, undetermined intent APPENDIX 1. Continued APPENDIX 2 List of Read Codes Used to Identify Suicides and Non-fatal Self-harm in the Clinical Practice
Research Datalink (CPRD)
Read code
Description
SL..14
Overdose of biological substance
SL..15
Overdose of drug
SLHz.00
Drug and medicament poisoning not otherwise specified
TK..00
Suicide and self-inflicted injury
TK..11
Cause of overdose—deliberate
TK..12
Injury—self-inflicted
TK..13
Poisoning—self-inflicted
TK..14
Suicide and self-harm
TK..15
Attempted suicide
TK..17
Para-suicide
TK0.00
Suicide þ self-inflicted poisoning by solid=liquid substances
TK00.00
Suicide þ self-inflicted poisoning by analgesic=antipyretic
TK01.00
Suicide þ self-inflicted poisoning by barbiturates
TK01000
Suicide and self-inflicted injury by amylobarbitone
TK01100
Suicide and self-inflicted injury by barbitone
TK01400
Suicide and self-inflicted injury by phenobarbitone
TK02.00
Suicide þ self-inflicted poisoning by other sedatives=hypnotics
TK03.00
Suicide þ self-inflicted poisoning tranquillizer=psychotropic
TK04.00
Suicide þ self-inflicted poisoning by other drugs=medicines
TK05.00
Suicide þ self-inflicted poisoning by drug or medicine not otherwise specified
TK06.00
Suicide þ self-inflicted poisoning by agricultural chemical
TK07.00
Suicide þ self-inflicted poisoning by corrosive=caustic substance
TK0z.00
Suicide þ self-inflicted poisoning by solid=liquid substance not otherwise specified
TK1.00
Suicide þ self-inflicted poisoning by gases in domestic use
TK10.00
Suicide þ self-inflicted poisoning by gas via pipeline
TK11.00
Suicide þ self-inflicted poisoning by liquefied petrol gas Read Codes Used to Identify Suicides and Non-fatal Self-harm in the Clinical Practice
Research Datalink (CPRD) (Continued ) ARCHIVES OF SUICIDE RESEARCH 135 Consistency in Reports of Self-Harm in Adolescence Consistency in Reports of Self-Harm in Adolescence Consistency in Reports of Self-Harm in Adolescence APPENDIX 2. Continued
Read code
Description
TK1y.00
Suicide and self-inflicted poisoning by other utility gas
TK1z.00
Suicide þ self-inflicted poisoning by domestic gases not otherwise specified
TK2.00
Suicide þ self-inflicted poisoning by other gases and vapors
TK20.00
Suicide þ self-inflicted poisoning by motor vehicle exhaust gas
TK21.00
Suicide and self-inflicted poisoning by other carbon monoxide
TK2z.00
Suicide þ self-inflicted poisoning by gases and vapors not otherwise specified
TK3.00
Suicide þ self-inflicted injury by hang=strangulate=suffocate
TK30.00
Suicide and self-inflicted injury by hanging
TK31.00
Suicide þ self-inflicted injury by suffocation by plastic bag
TK3y.00
Suicide þ self-inflicted injury by other means than hang=strangle=suffocate
TK3z.00
Suicide þ self-inflicted injury by hang=strangle=suffocate not otherwise specified
TK4.00
Suicide and self-inflicted injury by drowning
TK5.00
Suicide and self-inflicted injury by firearms and explosives
TK51.00
Suicide and self-inflicted injury by shotgun
TK52.00
Suicide and self-inflicted injury by hunting rifle
TK54.00
Suicide and self-inflicted injury by other firearm
TK5z.00
Suicide and self-inflicted injury by firearms=explosives not otherwise specified
TK6.00
Suicide and self-inflicted injury by cutting and stabbing
TK60.00
Suicide and self-inflicted injury by cutting
TK60100
Self-inflicted lacerations to wrist
TK60111
Slashed wrists self-inflicted
TK61.00
Suicide and self-inflicted injury by stabbing
TK6z.00
Suicide and self-inflicted injury by cutting and stabbing not otherwise specified
TK7.00
Suicide and self-inflicted injury by jumping from high place
TK70.00
Suicide þ self-inflicted injury—jump from residential premises
TK71.00
Suicide þ self-inflicted injury—jump from other manmade structure
TK72.00
Suicide þ self-inflicted injury—jump from natural sites
TK7z.00
Suicide þ self-inflicted injury—jump from high place not otherwise specified
TKx.00
Suicide and self-inflicted injury by other means
TKx0.00
Suicide þ self-inflicted injury—jump=lie before moving object
TKx0000
Suicide þ self-inflicted injury—jumping before moving object
TKx1.00
Suicide and self-inflicted injury by burns or fire
TKx2.00
Suicide and self-inflicted injury by scald
TKx3.00
Suicide and self-inflicted injury by extremes of cold
TKx4.00
Suicide and self-inflicted injury by electrocution
TKx5.00
Suicide and self-inflicted injury by crashing motor vehicle
TKx6.00
Suicide and self-inflicted injury by crashing of aircraft
TKx7.00
Suicide and self-inflicted injury caustic substance, excluding poison
TKxy.00
Suicide and self-inflicted injury by other specified means
TKxz.00
Suicide and self-inflicted injury by other means not otherwise specified APPENDIX 2. Continued (Continued ) VOLUME 20 NUMBER 2 2016 136 B. Mars et al. B. Mars et al. APPENDIX 2. Continued APPENDIX 2. Consistency in Reports of Self-Harm in Adolescence Continued Read code
Description
TKy.00
Late effects of self-inflicted injury
TKz.00
Suicide and self-inflicted injury not otherwise specified
U2..00
[X]Intentional self-harm
U2..11
[X]Self-inflicted injury
U2..12
[X]Injury—self-inflicted
U2..13
[X]Suicide
U2..14
[X]Attempted suicide
U2..15
[X]Para-suicide
U20.00
[X]Intentional self-poisoning=exposure to noxious substances
U20.11
[X]Deliberate drug overdose=other poisoning
U200.00
[X]Intentional self-poisoning=exposure to non-opioid analgesic
U200.11
[X]Overdose—paracetamol
U200.12
[X]Overdose—ibuprofen
U200.13
[X]Overdose—aspirin
U200000
[X]Intentional self-poisoning=exposure to non-opioid analgesic at home
U200100
[X]Intentional self-poisoning non-opioid analgesic at residential institution
U200400
[X]Intentional self-poisoning non-opioid analgesic in street=highway
U200500
[X]Intentional self-poisoning non-opioid analgesic trade=service area
U200y00
[X]Intentional self-poisoning non-opioid analgesic other specified place
U200z00
[X]Intentional self-poisoning non-opioid analgesic unspecified place
U201.00
[X]Intentional self-poisoning=exposure to antiepileptic
U201000
[X]Intentional self-poisoning=exposure to antiepileptic at home
U201z00
[X]Intentional self-poisoning antiepileptic unspecified place
U202.00
[X]Intentional self-poisoning=exposure to sedative hypnotic
U202.11
[X]Overdose—sleeping tablets
U202.12
[X]Overdose—diazepam
U202.13
[X]Overdose—temazepam
U202.15
[X]Overdose—nitrazepam
U202.16
[X]Overdose—benzodiazepine
U202.17
[X]Overdose—barbiturate
U202.18
[X]Overdose—amobarbital
U202000
[X]Intentional self-poisoning =exposure to sedative hypnotic at home
U202400
[X]Intentional self-poisoning sedative hypnotic in street=highway
U202y00
[X]Intentional self-poisoning sedative hypnotic other specified place
U202z00
[X]Intentional self-poisoning sedative hypnotic unspecified place
U204.00
[X]Intentional self-poisoning=exposure to psychotropic drug
U204.11
[X]Overdose—antidepressant
U204.12
[X]Overdose—amitriptyline
U204.13
[X]Overdose—SSRI
U204000
[X]Intentional self-poisoning =exposure to psychotropic drug at home Description (Continued ) ARCHIVES OF SUICIDE RESEARCH 137 Consistency in Reports of Self-Harm in Adolescence Consistency in Reports of Self-Harm in Adolescence APPENDIX 2. Consistency in Reports of Self-Harm in Adolescence Continued
Read code
Description
U204100
[X]Intentional self-poisoning psychotropic drug at residential institution
U204y00
[X]Intentional self-poisoning psychotropic drug other specified place
U204z00
[X]Intentional self-poisoning psychotropic drug unspecified place
U205000
[X]Intentional self-poisoning=exposure to narcotic drug at home
U205y00
[X]Intentional self-poisoning narcotic drug other specified place
U205z00
[X]Intentional self-poisoning narcotic drug unspecified place
U206.00
[X]Intentional self-poisoning=exposure to hallucinogen
U206400
[X]Intentional self-poisoning hallucinogen in street=highway
U207.00
[X]Intentional self-poisoning=exposure to other autonomic drug
U207000
[X]Intentional self-poisoning=exposure to other autonomic drug at home
U207z00
[X]Intentional self-poisoning other autonomic drug unspecified place
U208.00
[X]Intentional self-poisoning=exposure to other=unspecified drug=medicament
U208400
[X]Intentional self-poisoning other=unspecified drug=medication in street=highway
U208y00
[X]Intentional self-poisoning other=unspecified drug=medication other specified place
U208z00
[X]Intentional self-poisoning other=unspecified drug=medication unspecified place
U20A.00
[X]Intentional self-poisoning organic solvent, halogen hydrocarbon
U20A.11
[X]Self-poisoning from glue solvent
U20A000
[X]Intentional self-poisoning organic solvent, halogen hydrocarbon, home
U20A400
[X]Intentional self-poisoning organic solvent, halogen hydrocarbon, in highway
U20Az00
[X]Intentional self-poisoning organic solvent, halogen hydrocarbon, unspecified place
U20B.00
[X]Intentional self-poisoning=exposure to other gas=vapor
U20B.11
[X]Self carbon monoxide poisoning
U20B000
[X]Intentional self-poisoning=exposure to other gas=vapor at home
U20B200
[X]Intentional self-poisoning other gas=vapor school=public admin area
U20By00
[X]Intentional self-poisoning other gas=vapor other specified place
U20Bz00
[X]Intentional self-poisoning other gas=vapor unspecified place
U20C.00
[X]Intentional self-poisoning=exposure to pesticide
U20C.11
[X]Self-poisoning with weedkiller
U20C.12
[X]Self-poisoning with paraquat
U20C000
[X]Intentional self-poisoning=exposure to pesticide at home
U20Cy00
[X]Intentional self-poisoning pesticide other specified place
U20y.00
[X]Intentional self-poisoning=exposure to unspecified chemical
U20y000
[X]Intentional self-poisoning=exposure to unspecified chemical at home
U20y200
[X]Intentional self-poisoning unspecified chemical school=public admin area
U20yz00
[X]Intentional self-poisoning unspecified chemical unspecified place
U21.00
[X]Intentional self-harm by hanging=strangulation=suffocation
U210.00
[X]Intentional self-harm by hanging=strangulation=suffocation at home
U211.00
[X]Intentional self-harm by hanging=strangulation=suffocation occurrence at residential
institution
U21y.00
[X]Intentional self-harm by hanging=strangulation=suffocation other specified place APPENDIX 2. Continued (Continued ) VOLUME 20 NUMBER 2 2016 138 B. Mars et al. APPENDIX 2. Consistency in Reports of Self-Harm in Adolescence Continued Read code
Description
U21z.00
[X]Intentional self-harm by hanging=strangulation=suffocation unspecified place
U22.00
[X]Intentional self-harm by drowning and submersion
U221.00
[X]Intentional self-harm by drowning=submersion occurrence at residential institution
U22y.00
[X]Intentional self-harm by drowning=submersion occurrence at other specified place
U22z.00
[X]Intentional self-harm by drowning=submersion occurrence at unspecified place
U24.00
[X]Intentional self-harm by rifle shotgun=larger firearm discharge
U241.00
[X]Intentional self-harm by rifle shotgun=larger firearm discharge occurrence at residential
institution
U242.00
[X]Intentional self-harm by rifle shotgun=larger firearm discharge in school=public admin
area
U25.00
[X]Intentional self-harm by other=unspecified firearm discharge
U250.00
[X]Intentional self-harm other=unspecified firearm discharge occurrence at home
U26.00
[X]Intentional self-harm by explosive material
U27.00
[X]Intentional self-harm by smoke, fire and flames
U270.00
[X]Intentional self-harm by smoke fire=flames occurrence at home
U274.00
[X]Intentional self-harm by smoke fire=flame occurrence in street=highway
U27z.00
[X]Intentional self-harm by smoke fire=flames occurrence in unspecified place
U28.00
[X]Intentional self-harm by steam hot vapors=hot objects
U280.00
[X]Intentional self-harm by steam hot vapors=hot objects occurrence at home
U28z.00
[X]Intentional self-harm by steam hot vapors=hot objects occurrence in unspecified place
U29.00
[X]Intentional self-harm by sharp object
U290.00
[X]Intentional self-harm by sharp object occurrence at home
U291.00
[X]Intentional self-harm by sharp object occurrence at residential institution
U294.00
[X]Intentional self-harm by sharp object occurrence in street=highway
U29y.00
[X]Intentional self-harm by sharp object occurrence at other specified place
U29z.00
[X]Intentional self-harm by sharp object occurrence at unspecified place
U2A.00
[X]Intentional self-harm by blunt object
U2A0.00
[X]Intentional self -arm by blunt object occurrence at home
U2A1.00
[X]Intentional self -arm by blunt object occurrence at residential institution
U2A3.00
[X]Intentional self -arm by blunt object occurrence at sports=athletic area
U2B.00
[X]Intentional self-harm by jumping from a high place
U2B0.00
[X]Intentional self-harm by jumping from high place occurrence at home
U2B4.00
[X]Intentional self-harm by jumping from high place occurring in street=highway
U2B6.00
[X]Intentional self-harm by jumping from high place industrial=construction area
U2By.00
[X]Intentional self-harm by jumping from high place occurrence other specified place
U2Bz.00
[X]Intentional self-harm by jumping from high place occurrence unspecified place
U2C.00
[X]Intentional self-harm by jumping=lying before moving object
U2C1.00
[X]Intentional self-harm by jumping=lying before moving object occurrence at residential
institution
U2C4.00
[X]Intentional self-harm by jumping=lying before moving object occurrence in street=
highway Description (Continued ) ARCHIVES OF SUICIDE RESEARCH 139 Consistency in Reports of Self-Harm in Adolescence Consistency in Reports of Self-Harm in Adolescence APPENDIX 2. Consistency in Reports of Self-Harm in Adolescence Continued
Read code
Description
U2Cy.00
[X]Intentional self-harm by jumping=lying before moving object occurrence other specified
place
U2D.00
[X]Intentional self-harm by crashing of motor vehicle
U2D0.00
[X]Intentional self-harm by crashing of motor vehicle occurrence at home
U2D4.00
[X]Intentional self-harm by crashing of motor vehicle occurrence in street=highway
U2D6.00
[X]Intentional self-harm by crashing of motor vehicle occurrence industrial=construction
area
U2E.00
[X]Self-mutilation
U2y.00
[X]Intentional self-harm by other specified means
U2y0.00
[X]Intentional self-harm by other specified means occurrence at home
U2y1.00
[X]Intentional self-harm by other specified means occurrence at residential institution
U2yz.00
[X]Intentional self-harm by other specified means occurrence at unspecified place
U2z.00
[X]Intentional self-harm by unspecified means
U2z0.00
[X]Intentional self-harm by unspecified means occurrence at home
U2z2.00
[X]Intentional self-harm by unspecified means occurrence school=institution=public
administrative area
U2zy.00
[X]Intentional self-harm by unspecified means occurrence other specified place
U2zz.00
[X]Intentional self-harm by unspecified means occurrence at unspecified place
U30.11
[X]Deliberate drug poisoning
U41.00
[X]Hanging strangulation þ suffocation undetermined intent
U44.00
[X]Rifle shotgun þ larger firearm discharge undetermined intent
U45.00
[X]Other þ unspecified firearm discharge undetermined intent
U4B.00
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Burning self
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(Continued ) APPENDIX 2. Continued (Continued ) VOLUME 20 NUMBER 2 2016 140 B. Mars et al. B. Mars et al. APPENDIX 2. Continued Read code
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Jumping from height
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Jumping from building
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Nipping self
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Pinching self
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Scratches self
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Self-asphyxiation
ZX1H100
Self-strangulation
ZX1H200
Self-suffocation
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Self-scalding
ZX1J.00
Self-electrocution
ZX1K.00
Self-incineration
ZX1K.11
Setting fire to self
ZX1K.12
Setting self alight
ZX1L.00
Self-mutilation
ZX1L100
Self-mutilation of hands
ZX1L200
Self-mutilation of genitalia
ZX1L300
Self-mutilation of penis
ZX1L600
Self-mutilation of ears
ZX1LD00
[X]Self-mutilation
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Shooting self
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Stabbing self
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Throwing self in front of train
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Jumping under train
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Throwing self in front of vehicle
ZX1S.00
Throwing self onto floor Description 141 ARCHIVES OF SUICIDE RESEARCH 141
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Journal of Autism and Developmental Disorders (2022) 52:490–507
https://doi.org/10.1007/s10803-021-04963-4
ORIGINAL PAPER
Autistic Adults May Be Erroneously Perceived as Deceptive
and Lacking Credibility
Alliyza Lim1
· Robyn L. Young1 · Neil Brewer1
Accepted: 3 March 2021 / Published online: 17 March 2021
© The Author(s) 2021
Abstract
We hypothesized that autistic adults may be erroneously judged as deceptive or lacking credibility due to demonstrating
unexpected and atypical behaviors. Thirty autistic and 29 neurotypical individuals participated in video-recorded interviews,
and we measured their demonstration of gaze aversion, repetitive body movements, literal interpretation of figurative language, poor reciprocity, and flat affect. Participants (N = 1410) viewed one of these videos and rated their perception of the
individual’s truthfulness or credibility. The hypothesis was partially supported, with autistic individuals perceived as more
deceptive and less credible than neurotypical individuals when telling the truth. However, this relationship was not influenced
by the presence of any of the target behaviors, but instead, by the individual’s overall presentation.
Keywords Autism · Perceptions · Deception · Credibility
Autistic Adults May Be Erroneously
Perceived as Deceptive and Lacking
Credibility
Interpersonal judgments frequently involve an attempt to
determine if another individual is trustworthy and believable or perhaps lying or attempting to deceive in some way.
Such judgments are important components of the task that
confronts judges and jurors, but they are also required in a
variety of other contexts. For example, is the salesperson
trying to sell you a car telling the truth about the history of
the car? Is the politician making promises during an election
campaign someone you can trust? When your adolescent son
or daughter tells you where they have been until 3 a.m., can
you rely on what they say?
How then do you spot a liar? If your answer was that liars
fidget and avoid eye contact, you would not be alone. An
international study conducted by the The Global Deception
The data and materials from the study are available at https://doi.
org/10.17605/OSF.IO/TEWSX
* Alliyza Lim
alliyza.lim@flinders.edu.au
1
College of Education, Psychology, and Social Work, Flinders
University, GPO Box 2100, Adelaide, South Australia 5001,
Australia
13
Vol:.(1234567890)
Research Team (2006) found that gaze aversion and fidgeting are the two behaviors most commonly perceived as
indicating deception. It is both coincidental and unfortunate
that, to varying degrees, these behaviors form part of the
diagnostic criteria for, and may characterize, individuals
with autism spectrum disorder (ASD). Research findings
appear to be unanimous that people on the autism spectrum
demonstrate more gaze aversion than their neurotypical
peers (Doherty-Sneddon et al., 2013; Klin et al., 2002; Riby
& Hancock, 2008). Furthermore, the repetitive patterns of
behavior often demonstrated by people with autism, which
include repetitive movements such as rocking, pacing, finger
flicking, and hand or foot tapping (Cunningham & Schreibman, 2008), have been associated with perceptions of dishonest behavior. We hypothesized that the marked similarity between perceived indicators of deception and common
autistic behaviors may cause people on the autism spectrum
to be judged as more deceptive than their neurotypical peers
when telling the truth.
Behavioral Markers of Deception
Research on behavioral markers of deception has demonstrated that, contrary to popular belief, there is no evidence
that gaze aversion and body movements are indicative of
deception (DePaulo et al., 2003; Mann et al., 2012, 2013;
Sporer & Schwandt, 2007; Vrij, 2019). In fact, a recent study
Journal of Autism and Developmental Disorders (2022) 52:490–507
by Luke (2019) suggests that there is currently insufficient
information in the literature to conclude the existence of any
reliable behavioral cue to deception. Yet, gaze aversion and
body movements remain pervasive stereotypes of cues to
deception, held even by presumed expert lie detectors such
as police officers, customs officers, prosecutors, and judges
(Akehurst et al., 1996; Bogaard & Meijer, 2018; Bogaard
et al., 2016; Delmas et al., 2019; Dickens & Curtis, 2019;
Strömwall & Granhag, 2003; Vrij & Semin, 1996; Vrij et al.,
2006).
Why Do We Rely on Unreliable Cues?
Attribution theories provide a widely accepted theoretical
framework to explain the way in which behavioral cues are
used in the formation of deception and credibility judgments.
Attribution theories suggest that individuals are naturally
driven to understand the causes of events and will use their
knowledge of an individual’s personal and situational characteristics to attribute an explanation for observed behaviors (Kelley, 1967). For example, the expectancy violations
theory (Burgoon, 1983; Burgoon & Hale, 1988; Burgoon &
Jones, 1976; Burgoon et al., 1989) suggests that people hold
expectations about how others behave in social interactions.
When an individual violates these expectancies, the attention of their communication partner is drawn away from the
interaction and toward the unexpected behavior. Observers
then interpret this behavior in light of personal and situational characteristics to form an appraisal of the violation;
if the behavior is interpreted as a negative violation, it results
in unfavorable communication outcomes (Burgoon & Hale,
1988).
Based on this premise, Levine et al. (2000) and Bond
et al. (1992) proposed the norm-violation model and expectancy-violation model of deception judgments, respectively.
These models state that, while nonverbal behaviors that are
expected or normative are accepted at face value, nonverbal
behaviors that are unexpected (Bond et al., 1992) or atypical (Levine et al., 2000) demand an explanation and raise
suspicion about the sender’s intentions. They argue that it is
not the specific behaviors of gaze aversion or fidgeting per
se that leads to deception judgments but the fact that these
behaviors violate social norms and expectations, therefore
prompting the observer to infer deception as a means of
explaining the behavior (Bond et al., 1992; Levine et al.,
2000).
In instances where multiple possible causes for a behavior exist, Kelley (1971) proposes that the observer employs
a discounting principle—as more plausible reasons for the
behavior come to light, the importance of each individual
cause is diminished. The relative importance of a particular cause is evaluated based on the number and perceived
significance of all possible alternative explanations. For
491
example, if an observer witnesses an individual fidgeting and avoiding eye contact while testifying in court, the
observer may assume that the individual is being deceptive. However, if the observer is aware that the individual
has been diagnosed with autism, the observer may be less
likely to attribute deception as the cause of the behavior.
Given autism is largely unidentifiable by physical appearance, observers are unlikely to be aware of an individual’s
diagnosis and unlikely to expect behaviors such as gaze
aversion and fidgeting. Therefore, when these, or other
autistic behaviors, are displayed, there is an incongruence
between the observer’s expectations and the individual’s
behavior, leading the observer to search for possible explanations for the behavior such as deception and low credibility. Thus, characteristics of autism that are both atypical and incongruent with social norms may be viewed as
indicative of deception.
Deception and Credibility
A concept that is closely related to deception is source
credibility. In a study of the relationship between perceived
deception and source credibility, O’Sullivan (2003) proposed that the performance of human lie detectors is subject
to the fundamental attribution error: that is, the tendency
to overestimate the importance of dispositional traits of
an individual and to underestimate the importance of situational factors (Ross & Nisbett, 1991). O’Sullivan (2003)
explained that, because individuals are often unable to distinguish between trait truthfulness (whether the individual
is trustworthy) and state truthfulness (whether the individual
is telling the truth in that specific instance), they tend to
assume that trustworthy individuals always tell the truth and
untrustworthy individuals are always deceptive. Therefore,
to understand perceptions of deception, it would also be
beneficial to consider factors that influence perceptions of
credibility, as these constructs are closely related.
One cue used to infer credibility is emotional expression (Heath, 2009; Kaufmann et al., 2003; Melinder et al.,
2016; Wessel et al., 2013). A meta-analysis of 20 studies by
Nitschke et al. (2019) found that the level of distress displayed by rape victims significantly influenced the degree to
which they were perceived as credible complainants, with
those who displayed congruent emotions (e.g., crying) being
perceived as more credible than those who displayed incongruent (e.g., smiling) or neutral emotions (e.g., flat affect).
This tendency to rely on emotional displays in forming credibility judgments once again raises the question of whether
autistic individuals, who are known to display reduced emotional expressivity (Stagg et al., 2014; Zantinge et al., 2019),
would be perceived as less credible than their neurotypical
peers.
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492
Journal of Autism and Developmental Disorders (2022) 52:490–507
Autistic Behaviors and Perceptions of Deception
and Credibility
to deception or low credibility with the individual seen as
trying to avoid or change the subject of conversation.
Given that many forms of social interaction involve some
degree of impression formation, misinterpretation of autistic behaviors has the potential to cause detrimental consequences for people on the autism spectrum (see Denault
& Jupe, 2018; Porter & ten Brinke, 2009; Vrij & Turgeon,
2018), particularly in situations such as interactions with the
criminal justice system. It is, therefore, important to understand the influence of autistic behaviors on the formation of
deception and credibility judgments. In addition to examining gaze aversion, repetitive body movements, and flat affect,
this study examines two characteristic behaviors of people
on the autism spectrum that could potentially be viewed as
indicative of deception or low credibility: poor reciprocity
and literal interpretation of figurative language. Although
little attention has been paid to these behaviors in the deception and credibility literature, their atypical and unexpected
nature may also cause them to be interpreted negatively.
As part of their difficulty with socioemotional reciprocity,
autistic individuals are often known to have trouble maintaining two-way conversation (American Psychiatric Association, 2013). Due to impairments in their ability to understand the perspective of their conversation partner, autistic
individuals may talk exclusively about their own interests,
failing to recognize that this may not be of interest to the
listener (Chin & Bernard-Opitz, 2000). Autistic individuals may also be less responsive to cues for turn-exchange
and have difficulty conforming to appropriate norms for the
timing and latency of turn-taking (Paul et al., 2009). Furthermore, research has demonstrated that autistic individuals
perform more poorly than their neurotypical counterparts on
tasks that require the understanding of figurative language
(Cheung et al., 2019; Saban-Bezalel et al., 2019; SabanBezalel & Mashal, 2019).
Turn-taking is a central component of any social conversation and there is evidence that children are able to make
predictions about the turn structure of a conversation from
as early as two years of age (Casillas & Frank, 2017). Likewise, studies on the use of figurative language have shown
that children aged eight to 10 years are able to understand
and use figurative language (Pollio & Pollio, 1974) and that
by age 11, children are able to identify the communicative
intent behind a range of figurative statements (Demorest
et al., 1983). This suggests the expectation that, by adulthood, one would have the ability to both predict and finish
their turn in a social conversation, as well as be proficient
in the use of figurative language. Thus, when individuals
exhibit poor reciprocity or respond inappropriately to figurative language, this expectation is violated. In the absence of
any apparent intellectual disability, language impairment,
or cultural differences, observers may attribute the behavior
The Study
13
This study examined the relationship between autism, autistic behaviors, and perceptions of deception and credibility.
We hypothesized that autistic individuals would be more
likely than neurotypical individuals to be judged as deceptive and lacking credibility, and that this difference would be
attributable to higher levels of gaze aversion, repetitive body
movements, literal interpretation of figurative language, poor
reciprocity, and flat affect displayed by autistic individuals.
We also hypothesized that the relationship between ASD
diagnosis and perceived deception and credibility would be
moderated by the knowledge that the target individual is
on the autism spectrum. This hypothesis is not only consistent with Kelley’s (1971) discounting principle but also
with emerging evidence that providing information on an
individual’s ASD diagnosis results in more positive interpersonal judgments (Maras, Crane, et al., 2019; Maras, Marshall, et al., 2019; Matthews et al., 2015; Sasson & Morrison, 2019). For example, Maras, Marshall, et al. (2019)
found that mock jurors who were informed that a defendant
was autistic perceived the defendant as more honest and less
guilty than those who were not informed of his diagnosis.
Follow-up qualitative analyses revealed that, consistent
with attribution theories, mock jurors who were told that
the defendant was autistic were more likely to attribute his
inappropriate behaviors to his autism, whereas mock jurors
who were not given any information on ASD diagnosis
reported that the defendant’s aggressive behaviors, body
language, and gaze aversion led them to believe that he was
being deceptive to protect his own interests (Maras, Marshall, et al., 2019).
Although gaze aversion, repetitive body movements, literal interpretation of figurative language, poor reciprocity,
and flat affect are generally considered to be characteristic
behaviors of autistic individuals and are reflected in diagnostic criteria, there are deficiencies in our knowledge base
regarding the prevalence of these behaviors among autistic adults. Much research in this area has been conducted
among children and adolescents rather than with adults. As
the specific behavioral manifestations of ASD diagnostic
criteria may vary over the lifespan (Fecteau et al., 2003;
Georgiades et al., 2017), it is possible that the current literature does not reflect the degree to which these behaviors are
displayed in an adult population. Furthermore, few studies
conducted with autistic adults have measured the behaviors
through direct observation, with research relying primarily
on self-report or informant-report measures. Therefore, to
test our hypotheses, we recruited autistic and neurotypical
individuals to participate as stimuli in short-video recorded
Journal of Autism and Developmental Disorders (2022) 52:490–507
interviews and measured the degree to which they displayed
each of the target behaviors. Participants were then randomly
allocated to view one of these videos and to rate the extent
to which they believed the target individual was deceptive
or credible.
Method
Participants and Design
Participants were 1726 adults recruited via the online crowdsourcing platform TurkPrime (Litman et al., 2017). Three
hundred and sixteen participants failed to pass one or more
attention checks (e.g., “Please spell the word ‘WORLD’
backwards”), and their data were excluded. The final sample
consisted of 1410 participants (853 female), ranging in age
from 18 to over 85 years (M = 41.13). As there is evidence
that judgments of deception are influenced by perceptions
of credibility (George et al., 2014; O’Sullivan, 2003), a
between-subjects design was used to avoid potential carryover effects: each participant was randomly allocated to
view only one of the stimulus videos and to complete only
one measure of either the target individual’s truthfulness
(n = 713) or credibility (n = 697). A priori sample size estimations using G*Power 3.1 (Faul et al., 2007) indicated that
a minimum sample of 725 participants would be required to
detect a small effect at an alpha level of 0.05 in a multiple
regression model with seven predictor variables. As participants completed only one measure of either deception or
credibility, a total sample of 1450 was targeted.
Materials
Stimulus Materials
Stimulus Development
Thirty-one autistic individuals (nine female) and 29 neurotypical individuals (15 female) ranging in age from 18 to
66 years (M = 29.62, SD = 11.57) were involved in the production of stimulus videos for this study. A breakdown of the
demographics of the target individuals can be found in Supplemental Materials (p. 1). Twenty-one autistic individuals
were recruited from a university autism database of individuals on the autism spectrum who reside in the same region
as the university and have indicated interest in participating in research projects (the majority of individuals on this
database were originally recruited through a mail-out by the
local autism association). The remaining ten autistic individuals were recruited through an advertisement at a local
psychology practice that specializes in working with people
493
on the autism spectrum. All 31 individuals reported receiving a formal diagnosis of autism from a registered diagnostician. To access support services from the local autism
association, individuals must have received a diagnosis of
autism from at least two independent registered diagnosticians (i.e., speech pathologist, psychologist, pediatrician,
or psychiatrist) or from a registered multidisciplinary team.
None of the individuals recruited were suspected to have an
intellectual disability; this was supported by records that all
individuals recruited from the university autism database
had a Wechsler Abbreviated Scale of Intelligence—Second
Edition (WASI-II; Wechsler, 2011) intelligence quotient
(IQ) above 85. Specific data on socioeconomic status and
educational attainment levels of the autistic sample were
not recorded. The 29 neurotypical individuals were all current university students who reported that they did not have
a diagnosis of autism and were also assumed to have IQ
levels above 85.
Given the heterogeneity of autism (Jeste & Geschwind,
2014; Lenroot & Yeung, 2013; Martinez-Murcia et al.,
2017), we recognized that there would likely be significant
variability in the demonstration of these behaviors. Although
a larger sample would more likely capture this variability
and provide a more powerful test of behavioral differences
between the two stimulus groups, the recruitment of autistic participants who are willing to attend a video-recorded
interview that will subsequently be used as stimulus material poses a considerable barrier. Nevertheless, in accordance with ASD diagnostic criteria, it was assumed that these
behaviors would be more prevalent in the autistic than the
neurotypical individuals.
Each individual attended a session at a psychology lab
and was informed that the purpose of the session was to create videos that would be used in a research study on deception. They were told that they would be asked to complete
a computer task and to then participate in a short videorecorded interview. Prior to the commencement of the task,
the individuals were shown an envelope and told that it contained $20. They were informed that: “People who watch
this video will be told that you may have taken this money
but are trying to convince the interviewer that you did not.
They will be told that (1) participants who took the money
but could successfully convince the interviewer that they
did not would receive $50 for participating in the study, (2)
participants who took the money but were caught by the
interviewer as lying would only receive $10 for participating in the study, and (3) participants who chose not to take
the money would receive $20 for participating in the study,
irrespective of whether they were judged as lying or being
truthful. After watching the video, they will be asked to indicate whether they think you were telling the truth. However,
in reality, you are not to take the money in the envelope.
In the interview, it is critical that you answer all questions
13
494
truthfully.” The individuals were then left to complete the
computer task, after which they were interviewed by a separate researcher. This interview was video recorded.
The individuals were asked questions about the envelope in the room (e.g., “In the room, there was an envelope.
Did you see this envelope?”), what they did (e.g., “Can you
describe what you did after the researcher left the room?”),
and whether they took the money (e.g., “Did you take the
money that was in the envelope?”). The video from one
autistic individual was excluded as the truthfulness of his
responses could not be ascertained, resulting in a final sample of 30 autistic individuals (nine female) and 29 neurotypical individuals (15 female). Fourteen individuals (seven
autistic individuals and seven neurotypical individuals)
provided one or more responses in the interview that were
factually incorrect (e.g., saying that they had not been told
about the different payment outcomes). However, as these
responses were likely the result of inattention, rather than the
desire to purposefully mislead the interviewer, they were not
considered to be deceptive for the purpose of this study, with
deception being defined as “intentionally, knowingly, and/
or purposely misleading another person” (Levine, 2014, p.
397). All individuals in the final sample correctly reported
that they had not taken the money. The videos ranged in
duration from 95 to 290 s (M = 132, SD = 33).
Behavioral Analysis
The videos were analyzed and coded for the presence of each
of the five target behaviors by a research assistant who was
not involved in any other aspect of this study and was blind
to both the purpose of the study and group membership of
the target individuals. The first author also independently
analyzed and coded all 59 videos. The coders were trained
on a set of six videos.
The coders watched each video five times, each time independently recording the presence of gaze aversion, repetitive
body movements (excluding communicative gestures), literal
interpretation of figurative language, poor reciprocity, or flat
affect using a behavioral coding scheme developed for this
study. The order of presentation of the videos was randomized for each behavior and coder. Inter-rater reliability
for the coding of gaze aversion, repetitive body movements,
and poor reciprocity was then assessed using three separate
two-way random, consistency, single-measures intraclass
correlations (ICC), while inter-rater reliability for the coding
of literal interpretation of figurative language and flat affect
were assessed using Gwet’s AC1, which is less influenced
by prevalence rates than Cohen’s kappa (Feinstein & Cicchetti, 1990; Wongpakaran et al., 2013). Inter-rater reliability was high for gaze aversion (ICC = 0.88), repetitive body
movements (ICC = 0.87), literal interpretation of figurative
language (AC1 = 0.96), and flat affect (AC1 = 0.88), but poor
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Journal of Autism and Developmental Disorders (2022) 52:490–507
for poor reciprocity (ICC = 0.51). To reconcile this discrepancy, a clinician with extensive experience working with
people on the autism spectrum also recorded the levels of
poor reciprocity displayed using the same operationalization
and coding guidelines. Disagreements in the coding of the
behavior were then discussed until a consensus was reached.
Clinical Impression
Given that the deficits in social communication and interaction, and restricted and repetitive behaviors and interests,
that characterize autism can manifest in a variety of ways,
it is likely that the behavioral analysis would fail to capture
the full range of behaviors displayed. Judgments of deception and credibility may be influenced by the presence of
other autistic behaviors that were not accounted for, or by
the overall presentation of the individual as having a condition or disability. To examine this possibility, a measure of
the general impression of each target individual was also
obtained.
Six trainee psychologists, who were enrolled in an accredited postgraduate clinical psychology program and blind to
both the purpose of the study and group membership of
the target individuals, were recruited. They independently
watched each stimulus video and indicated the likelihood
that the person in the video was on the autism spectrum from
1 (extremely unlikely) to 7 (extremely likely). To ensure that
the raters remained blind to the purpose of the study, they
also rated the target stimulus for nine other conditions (anxiety disorder, bipolar disorder, intellectual disability, language disorder, mood disorder, obsessive–compulsive disorder, personality disorder, post-traumatic stress disorder, and
schizophrenia); however, these ratings were not used as part
of the data analysis. Finally, raters provided an overall rating
of the likelihood that the person in the video had any mental
health or developmental disorder using the same scale. The
order of presentation of the videos was randomized for each
rater. Inter-rater reliability was then assessed using two-way
random, absolute agreement, average-measures intraclass
correlations (ICC). Inter-rater reliability was acceptable for
clinical impression of ASD (ICC = .70) and clinical impression of any condition (ICC = .76).
Perceived Deception
Perceived deception was measured with two questions: “Do
you think the person in the interview was telling the truth?”
rated on a 6-point Likert scale from 1 (deceptive) to 6 (truthful), and “Do you think the person in the interview took
the money?” rated from 1 (yes) to 6 (no). Responses were
reverse-scored and summed to obtain an overall score of
Journal of Autism and Developmental Disorders (2022) 52:490–507
perceived deception ranging from two to 12, with higher
scores indicating higher levels of perceived deception.
Perceived Credibility
Participants rated the perceived credibility of each target
individual using a modified version of McCroskey and
Teven’s (1999) source credibility measure. This measure
comprises three dimensions, Competence, Caring, and
Character, with higher scores indicating higher levels of perceived credibility. McCroskey and Teven’s (1999) validation
study revealed that each of the three dimensions accounted
for significant variance as unique predictors of believability and likableness, and the authors argue that the use of
all three individual dimensions in the operationalization of
source credibility would be more appropriate than a combined score. Further details on this measure can be found in
Supplemental Materials (pp. 2–3).
Procedure
The experiments were presented through an online survey
platform (Qualtrics, Provo, UT). To avoid response bias,
participants were not informed of the true purpose of the
study but were instead told that the study aimed to investigate deception detection accuracy. Participants were then
presented with a stimulus video and asked to provide a
judgment of deception or credibility. Participants were also
asked to briefly describe the reasons for their impression
(they were encouraged to state “unsure” if they were uncertain). Next, participants were told, “Now if you were to be
told that the person in the interview has Autism Spectrum
Disorder (ASD), please rate your impression of the person
in the interview again in light of this new information. Characteristics of ASD include difficulties with social interaction and nonverbal communication, repetitive behaviors,
and restricted interests.” They were then asked to rate their
impression of the individual a second time, using the same
measure they had previously completed. Finally, a funnel
debriefing procedure was used to identify any possible suspicion about the deception, and participants were debriefed
on the true purpose of the study.
Statistical Analysis
As it is unlikely that only one variable influences the
predictor-outcome relationship, Preacher and Hayes
(2008) advise that hypotheses involving multiple potential mediators should be considered. They recommend that
when multiple mediators are involved, a multiple mediation approach (in which all mediators are included in the
same model) is the most parsimonious and precise way
to analyze the data (Preacher & Hayes, 2008; however, to
495
illustrate how the strength of each mediator differed upon
the inclusion of other mediators in the model, multilevel
mediation analyses for each individual mediator are also
presented in Supplemental Materials, pp. 4–5). Because
the predictor (ASD diagnosis) and mediators (gaze aversion, repetitive body movements, literal interpretation of
figurative language, poor reciprocity, flat affect, clinical
impression of ASD, and clinical impression of any condition) were measured at the video level (level 2), while
the outcome variables (perceived deception, perceived
competence, perceived caring, and perceived character)
were measured at the participant level (level 1), 2–2–1
multilevel mediation analyses were used to test the model
shown in Fig. 1 using the multilevel structural equation
modelling (MSEM) framework outlined by Preacher et al.
(2010). Each outcome variable was examined separately.
The analyses were carried out using maximum likelihood
with robust standard errors estimators (MLR) in Mplus
version 8.
Results
Behavioral Analysis
The results of the behavioral analysis and ratings of clinical impression for each group are presented in Table 1.
Gaze aversion, repetitive body movements, and poor reciprocity were measured by percentage of time the behavior
was displayed. Clinical impression of ASD and clinical
impression of any condition were measured on 7-point
Likert scales ranging from 1 (extremely unlikely) to 7
(extremely likely). The number of individuals who were
coded as displaying literal interpretation of figurative language (binary scale) and flat affect (ordinal scale) are also
presented.
There was no significant difference in the demonstration of gaze aversion [t(57) = 1.85, p = .07, d = 0.48, 95%
CI − 0.04, 1.00], repetitive body movements [t(57) = 0.63,
p = .53, d = 0.16, 95% CI − 0.35, 0.67], poor reciprocity
[U = 345.00, p = .11, d = 0.36], literal interpretation of figurative language [χ2(1) = 2.82, p = .09, d = .45], and flat affect
[χ2(1) = 0.50, p = .48, d = 0.18] between the autistic and neurotypical groups. However, there was considerable variability within each group (as shown by the standard deviations)
and small effect sizes in the hypothesized direction were
detected in gaze aversion, poor reciprocity, and literal interpretation of figurative language. Autistic individuals were
rated significantly higher on clinical impression of ASD
[t(49.20) = 4.77, p < .001, d = 1.23, 95% CI 0.68, 1.79] and
clinical impression of any condition [t(57) = 6.00, p < .001,
d = 1.56, 95% CI 0.98, 2.15] than neurotypical individuals.
13
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Journal of Autism and Developmental Disorders (2022) 52:490–507
Fig. 1 2–2–1 Multilevel mediation model between ASD diagnosis, autistic behaviors, and perceived deception or credibility
Table 1 Behavioral coding of
autistic behaviors and ratings of
clinical impression
Autistic behavior/clinical impression
ASD (n = 30)
Gaze aversion
Repetitive body movements
Poor reciprocity
Clinical impression (ASD)
Clinical impression (any condition)
Autistic behavior
Literal interpretation of figurative language
Flat affect
The Relationship Between ASD Diagnosis, Autistic
Behaviors, and Perceived Deception and Credibility
When all mediators were included in the model, there
was high multicollinearity for clinical impression of ASD
(VIF = 4.11) and clinical impression of any condition
(VIF = 3.64). Thus, only clinical impression of any condition
was retained in the data analysis. The inter-item correlations
between clinical impression of any condition and each of the
five target behaviors were: .24 (gaze aversion), .04 (repetitive body movements), .08 (literal interpretation of figurative language), .30 (poor reciprocity), and .44 (flat affect).
None of the target individuals displayed high levels of flat
13
Neurotypical (n = 29)
M
SD
M
SD
35.39
23.51
1.79
3.15
4.21
ASD (n = 30)
No. of individuals
5
5
13.73
10.18
3.42
1.01
0.96
29.31
21.59
0.80
2.11
2.93
Neurotypical (n = 29)
No. of individuals
1
3
11.36
13.25
1.98
0.64
0.64
affect, only demonstrating either minimal emotional expression (n = 8) or appropriate emotional expression (n = 51).
This resulted in a highly disproportionate binary outcome.
Consequently, when flat affect was included in the model,
the standard errors of the model parameters could not be
reliably estimated. Therefore, for the purpose of hypothesis
testing, flat affect was excluded from the overall model (for
comparison, the results of the analysis when flat affect was
included as a mediator in the model are presented in Supplemental Materials, p. 6). The results of the analysis are
presented in Table 2.
The findings revealed that autistic individuals were rated
higher on perceived deception and lower on perceived
Journal of Autism and Developmental Disorders (2022) 52:490–507
497
Table 2 Multilevel mediation models between ASD diagnosis, autistic behaviors, and perceived deception and credibility
Perceived deception
Perceived competence
Perceived caring
Estimate
95% CI
Estimate
95% CI
Estimate
95% CI
Estimate
Perceived character
95% CI
0.39, 1.33
− 2.49***
− 3.54, − 1.44
− 0.59
− 1.18, 0.01
− 1.91*
− 3.33, − 0.48
0.14, 1.29
− 0.82
− 2.14, 0.50
0.01
− 0.70, 0.72
0.32
− 1.49, 2.13
6.07
0.77, 11.37
6.07
0.77, 11.37
6.07
0.77, 11.37
6.07
0.77, 11.37
Repetitive
body movements (a2)
1.92
− 3.06, 6.90
1.92
− 3.06, 6.90
1.92
− 3.06, 6.90
1.92
− 3.06, 6.90
0.13
0.01, 0.26
0.13
0.01, 0.26
0.13
0.01, 0.26
0.13
0.01, 0.26
Poor reciprocity (a4)
1.00
− 0.18, 2.17
1.00
− 0.18, 2.17
1.00
− 0.18, 2.17
1.00
− 0.18, 2.17
Clinical
impression
(a5)
1.29***
0.94, 1.63
1.29***
0.94, 1.63
1.29***
0.94, 1.63
1.29***
0.94, 1.63
Gaze aversion
(b1)
− 0.01
− 0.03, 0.02
− 0.004
− 0.05, 0.04
− 0.01
− 0.03, 0.01
− 0.01
− 0.06, 0.04
Repetitive
body movements (b2)
− 0.02
− 0.04, − 0.001
− 0.03
− 0.07, 0.003
0.02
− 0.002, 0.05
− 0.01
− 0.06, 0.04
− 0.07
− 0.60, 0.46
0.94
− 0.64, 2.53
0.69
− 0.07, 1.44
1.79
− 0.22, 3.80
Poor reciprocity (b4)
− 0.11**
− 0.17, − 0.04
− 0.07
− 0.25, 0.11
− 0.03
− 0.09, 0.04
− 0.12
− 0.26, 0.03
Clinical
impression
(b5)
0.27
− 0.02, 0.55
− 1.28**
− 2.01, − 0.54
− 0.51**
− 0.83, − 0.19
− 1.75***
− 2.58, − 0.92
Total indirect
effect (ab)
0.15
− 0.27, 0.57
− 1.67*
− 2.83, − 0.51
− 0.60
− 1.10, − 0.09
− 2.22**
− 3.52, − 0.93
− 0.16, 0.10
− 0.02
− 0.27, 0.23
− 0.05
− 0.18, 0.09
− 0.07
− 0.38, 0.23
Total effect
ASD diagnosis 0.86**
(c)
Direct effects
ASD diagnosis 0.71*
(c’)
Mediators on ASD diagnosis
Gaze aversion
(a1)
Literal interpretation
(a3)
Outcome on mediators
Literal interpretation
(b3)
Indirect effects
Gaze aver− 0.03
sion (a1b1)
Repetitive
body movements (a2b2)
− 0.04
− 0.16, 0.07
− 0.06
− 0.24, 0.12
0.04
− 0.09, 0.17
− 0.03
− 0.14, 0.09
− 0.01
− 0.08, 0.06
0.13
− 0.13, 0.38
0.09
− 0.06, 0.24
0.24
− 0.18, 0.66
Poor reciprocity (a4b4)
− 0.11
− 0.23, 0.02
− 0.07
− 0.26, 0.12
− 0.03
− 0.10, 0.04
− 0.12
− 0.30, 0.07
0.34
− 0.04, 0.73
− 1.64*
− 2.77, − 0.51
− 0.65*
− 1.10, − 0.21
− 2.25**
− 3.43, − 1.07
Indices of
model fit
RMSEA = .04, CFI = .81,
TLI = .60
Literal interpretation
(a3b3)
Clinical
impression
(a5b5)
RMSEA = .04, CFI = .85,
TLI = .68
RMSEA = .04, CFI = .82,
TLI = .63
RMSEA = .04, CFI = .84,
TLI = .66
Note. Estimates that are significant at the .05 level are indicated in bold
*p ≤ .05, **p ≤ .01, ***p ≤ .001
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498
competence and character compared to neurotypical individuals. As indicated by the regression coefficient of the
c pathway, the mean rating of perceived deception for the
autistic group was 0.86 points higher than that of the neurotypical group, on a scale ranging from 2 to 12. Likewise,
the mean rating of perceived competence was 2.49 points
lower for the autistic group than the neurotypical group
(scale = 5–35), and the mean rating of perceived character
was 1.91 points lower for the autistic group than the neurotypical group (scale = 6–42). To the best of our knowledge,
this study is among the first to provide empirical evidence
for the existence of such a relationship. There was no difference in ratings of perceived caring between the two groups.
The Relationship Between ASD Diagnosis
and Autistic Behaviors
As suggested by the earlier between-group comparison of
target behaviors, ASD diagnosis did not significantly predict
levels of gaze aversion, repetitive body movements, literal
interpretation of figurative language, or poor reciprocity
(a1–a4). However, ASD diagnosis was a significant predictor of overall clinical impression (a5).
The Relationship Between Autistic Behaviors
and Perceived Deception and Credibility
Poor reciprocity (b3) was a significant predictor of perceived
deception but not in the expected direction: the higher the
level of poor reciprocity displayed by the target individual,
the less deceptive they were perceived to be. Independent of
ASD diagnosis, there was no significant association between
any of the autistic behaviors and perceived competence, caring, or character (b1–b4), but overall clinical presentation
negatively predicted ratings of perceived competence, caring, and character (b5).
Mediation of the Relationship Between ASD
Diagnosis and Perceived Deception and Credibility
Contrary to the hypothesis, no statistically significant mediation pathways were found for the relationship between ASD
diagnosis and perceived deception, and only the mediation
analyses of the relationships between ASD diagnosis and
perceived competence, caring, and character through clinical
impression were statistically significant.
Participant‑Reported Cues Indicative of Deception
and Low Credibility
After indicating their impression of the target individual’s truthfulness or credibility, participants were asked to
provide a brief explanation of the reasons for their rating
13
Journal of Autism and Developmental Disorders (2022) 52:490–507
(participants were encouraged to state “unsure” if they were
uncertain). The participants’ qualitative responses were then
analyzed, and the number of participants who reported using
each of the target behaviors as a cue to deception or credibility was recorded. Given that participants provided openended responses, it was possible for participants to indicate
the use of more than one cue. Besides the target behaviors,
other commonly reported cues included hesitation, smiling,
and a change from baseline demeanor, and the number of
participants who reported using each of these cues was also
calculated. Responses that did not include any of the eight
listed behaviors were classified under “others” (see Table 3).
To ensure the reliability of the analysis, a research assistant also independently coded 20% of the data (n = 282).
Inter-rater reliability using Gwet’s AC1 ranged from .93 to
1 (Gwet, 2008; Wongpakaran et al., 2013).
Knowledge of ASD Diagnosis
Using the same multilevel mediation approach previously
described, we examined the relationship between ASD
diagnosis, autistic behaviors, and ratings of deception and
credibility after participants had been told that the target
individual may have autism. There was no longer a significant total effect of ASD diagnosis on perceived deception
or character; however, ASD diagnosis continued to affect
ratings of perceived competence, and this relationship was
influenced by overall clinical impression (see Table 4).
To examine the interaction effect of ASD diagnosis and
knowledge of ASD diagnosis on judgments of deception and
credibility, we aggregated ratings of perceived deception,
competence, caring, and character according to the target
individual in the stimulus video (N = 59). We then conducted
four two-way mixed ANOVAs, with ASD diagnosis and
Table 3 Participant-reported cues indicative of deception and low
credibility
Behavioral cue
Gaze aversion
Repetitive body movements
Literal interpretation of figurative language
Poor reciprocity
Flat affect
Hesitation
Smiling/ smirking/ laughing
Inconsistent demeanor
Others
Unsure
Deception
(n = 713)
Credibility
(n = 697)
n
%
n
%
202
108
2
28.33
15.15
0.28
111
67
2
15.93
9.61
0.29
33
3
57
85
98
237
56
4.63
0.42
7.99
11.92
13.74
33.24
7.85
13
4
37
48
38
375
93
1.87
0.57
5.31
6.89
5.45
53.80
13.34
Journal of Autism and Developmental Disorders (2022) 52:490–507
499
Table 4 Multilevel mediation models between ASD diagnosis, autistic behaviors, and perceived deception and credibility (with knowledge of
ASD)
Perceived deception
Perceived competence
Perceived caring
Perceived character
Estimate
95% CI
Estimate
95% CI
Estimate
95% CI
Estimate
95% CI
0.20
− 0.21, 0.60
− 1.57***
− 2.35, − 0.79
− 0.39
− 0.88, 0.10
− 0.48
− 1.41, 0.46
0.25
− 0.27, 0.76
− 0.37
− 1.36, 0.62
0.11
− 0.57, 0.79
0.20
− 1.10, 1.50
6.07
0.77, 11.37
6.07
0.77, 11.37
6.07
0.77, 11.37
6.07
0.77, 11.37
Repetitive
body movements (a2)
1.92
− 3.06, 6.90
1.92
− 3.06, 6.90
1.92
− 3.06, 6.90
1.92
− 3.06, 6.90
0.01, 0.26
0.13
0.01, 0.26
0.13
0.01, 0.26
0.13
0.01, 0.26
Poor reciprocity (a4)
1.00
− 0.18, 2.17
1.00
− 0.18, 2.17
1.00
− 0.18, 2.17
1.00
− 0.18, 2.17
Clinical
Impression
(a5)
1.29***
0.94, 1.63
1.29***
0.94, 1.63
1.29***
0.94, 1.63
1.29***
0.94, 1.63
Gaze aversion
(b1)
− 0.01
− 0.02, 0.01
0.01
− 0.02, 0.04
0.01
− 0.01, 0.03
0.02
− 0.02, 0.06
Repetitive
body movements (b2)
− 0.01
− 0.03, 0.01
− 0.01
− 0.04, 0.02
0.01
− 0.02, 0.03
− 0.03
− 0.07, 0.01
Literal inter− 0.39
pretation (b3)
− 0.81, 0.03
− 0.19
− 1.65, 1.27
0.70
− 0.24, 1.63
0.72
− 0.65, 2.08
Poor reciprocity (b4)
− 0.15***
− 0.21, − 0.08
− 0.09
− 0.21, 0.03
− 0.01
− 0.11, 0.09
− 0.15
− 0.32, 0.02
Clinical
impression
(b5)
0.16
− 0.09, 0.41
− 0.87**
− 1.36, − 0.38
− 0.50**
− 0.82, − 0.19
− 0.53
− 1.21, 0.15
Total indirect − 0.05
effect (ab)
− 0.49, 0.39
− 1.20**
− 1.94, − 0.45
− 0.50
− 0.96, − 0.04
− 0.68
− 1.51, 0.16
− 0.03
− 0.15, 0.08
0.05
− 0.14, 0.25
0.05
− 0.07, 0.16
0.11
− 0.14, 0.37
− 0.02
− 0.09, 0.05
− 0.02
− 0.09, 0.05
0.02
− 0.05, 0.08
− 0.06
− 0.20, 0.09
− 0.05
− 0.11, 0.01
− 0.03
− 0.22, 0.16
0.09
− 0.09, 0.27
0.10
− 0.12, 0.31
− 0.15
− 0.34, 0.05
− 0.09
− 0.23, 0.05
− 0.01
− 0.10, 0.09
− 0.15
− 0.30, 0.01
0.20
− 0.11, 0.52
− 1.12*
− 1.87, − 0.36
− 0.65**
− 1.06, − 0.24
− 0.68
− 1.55, 0.18
Total effect
ASD diagnosis
(c)
Direct effects
ASD diagnosis
(c’)
Mediators on ASD diagnosis:
Gaze aversion
(a1)
Literal inter0.13
pretation (a3)
Outcome on mediators
Indirect effects
Gaze aversion
(a1b1)
Repetitive
body movements (a2b2)
Literal interpretation (a3b3)
Poor reciprocity (a4b4)
Clinical
impression
(a5b5)
Indices of model RMSEA = .04, CFI = .78, TLI = .53 RMSEA = .04, CFI = .82, TLI = .63 RMSEA = .04, CFI = .77, TLI = .51 RMSEA = .04, CFI = .75,
TLI = .47
fit
RMSEA root mean square error of approximation, CFI comparison fit index; TLI Tucker-Lewis index. Estimates that are significant at the .05
level are indicated in bold
*p ≤ .05, **p ≤ .01, ***p ≤ .001
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500
knowledge of ASD diagnosis as the independent variables,
and ratings of perceived deception, competence, caring, and
character as the dependent variables, respectively (as the
overall multivariate effect was not of interest, three two-way
mixed ANOVAs with Bonferroni correction were used for
the analysis of perceived competence, caring, and character
instead of a two-way mixed MANOVA). The results revealed
a significant interaction effect of ASD diagnosis and knowledge of ASD diagnosis on ratings of perceived deception
[F(1, 57) = 9.12, p = .004, ηp2 = .14, 90% CI .03, .27] and
character [F(1, 57) = 6.15, p = .05, ηp2 = .10, 90% CI .01,
.23]. However, there was no significant interaction effect of
ASD diagnosis and knowledge of ASD diagnosis on ratings
of perceived competence [F(1, 57) = 5.19, p = .08, ηp2 = .08,
90% CI .01, .21] or caring [F(1, 57) = 0.34, p = 1, ηp2 = .01].
Discussion
We examined whether autistic individuals would be perceived as more deceptive and less credible than their neurotypical peers due to their demonstration of unexpected or
atypical behaviors that are commonly judged as indicative of
deception: specifically, gaze aversion, repetitive body movements, literal interpretation of figurative language, poor reciprocity, and flat affect.
Autistic individuals were indeed judged as more deceptive
and lower on perceived competence and character compared
to neurotypical individuals. To the best of our knowledge,
this study is among the first to provide empirical evidence
for the existence of such a relationship. Our findings contrast
sharply with those of Maras, Crane, et al. (2019), who found
that, when presented with video testimonies of witnesses
describing a past event, mock jurors perceived autistic witnesses to be equally as credible as neurotypical witnesses.
It is possible that this discrepancy may have been due to the
different contexts used in the stimulus videos. First, Maras
et al.’s targets were not involved in any two-way interactions.
Second, unlike the videos in the present study in which the
target individuals were interviewed about their involvement
in stealing money, the targets in the study by Maras, Crane,
et al. (2019) were asked to provide a free memory report of
a simulated first aid event. Thus, the observers were unlikely
to suspect the target individuals of being dishonest as there
was no apparent incentive or rationale for doing so.
The Relationship between ASD Diagnosis
and Autistic Behaviors
The expected significant behavioral differences between
autistic adults and neurotypical peers in gaze aversion, repetitive body movements, literal interpretation of figurative language, poor reciprocity, and flat affect were not detected.
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Several possible explanations exist for this unexpected finding. First, it is important to note that, while no statistically
significant differences were found between groups, small
effect sizes (in two instances, approaching moderate) in the
hypothesized direction were detected for certain behaviors
(e.g., gaze aversion, poor reciprocity, and literal interpretation of figurative language). Given the likely large variability
within both the autistic and neurotypical groups, it is possible our relatively small sample of targets did not capture the
“true” proportion of autistic individuals who prominently
display such behavioral characteristics. Thus, these findings
remain inconclusive and highlight the need for such comparisons to be repeated with a larger sample of target stimuli,
recruitment of which is likely to prove difficult.
Another possible explanation is that adults on the autism
spectrum may not exhibit these behaviors to either the
extent or as universally as is typically proposed. Given that
a large amount of research in this area has been conducted
among children, it is possible that these behavioral differences may not be reflected to the same degree in the adult
population (Fecteau et al., 2003; Georgiades et al., 2017).
Furthermore, there is also increasing evidence in the literature that social camouflaging is prevalent among people on
the autism spectrum (Cage & Troxell-Whitman, 2019; Livingston et al., 2019) and, thus, these behaviors may not be
displayed consistently across all situations. Although adults
on the autism spectrum are presumed to display higher levels
of gaze aversion, repetitive body movements, literal interpretation of figurative language, poor reciprocity, and flat
affect than the general population, there is little in the way
of rigorous behavioral data to support these assumptions.
Rather, these expectations are largely based on the broad
diagnostic criteria for autism outlined by the DSM-5 and
clinical observations of atypical behavior, which can lend
themselves to overgeneralizations about how people on the
autism spectrum behave.
Although ASD diagnosis did not significantly predict
the presence of any individual behavior, it was a significant
predictor of overall clinical impression. Though it is unclear
exactly what factors led to this impression, this provides
support that there was, in fact, some form of noticeable difference in the presentation of the autistic individuals that
distinguished them from neurotypical comparisons.
The Relationship between ASD Diagnosis, Autistic
Behaviors, and Perceived Deception and Credibility
Although it appears that autistic individuals are more vulnerable to being judged negatively than neurotypical individuals, it remains uncertain why this is the case. However, drawing on attribution theories (Kelley, 1971; Kelley & Michela,
1980) and the expectancy-violation model (Bond et al.,
1992; Burgoon, 1983), we surmise that there is something
Journal of Autism and Developmental Disorders (2022) 52:490–507
501
in the presentation of autistic individuals that contradicts
observers’ expectations of how a truthful and credible person should behave.
It may be that this relationship is explained by other
autistic behaviors that were not explored or captured in the
present study. Alternatively, perhaps subtle nuances or particular combinations of behaviors cause autistic individuals
to be judged negatively. This is consistent with the proposition by Sasson et al. (2017) that “negative first impressions
of ASD are not founded on any one feature of expression,
but rather represent an effect of subtle physical, dynamic,
and auditory cues of presentation” (p. 8). Either way, the fact
that participants were able to distinguish autistic individuals
from neurotypical individuals in their ratings of deception
and credibility suggests that there were noticeable differences between the two groups. However, further research
is necessary to identify exactly what these differences are.
available, there would be no need to attribute the behavior to
deception or low credibility. Informing participants that the
target individual was autistic negated the effect of ASD diagnosis on perceived deception and character. However, ASD
diagnosis continued to negatively affect ratings of perceived
competence. The description of autism provided to participants highlighted that “characteristics of ASD include difficulties with social interaction and nonverbal communication,
repetitive behaviors, and restricted interests.” Although this
reflects DSM-5 diagnostic criteria (American Psychiatric
Association, 2013), inherent in this statement is the fact
that autistic individuals experience impairments in certain
areas of functioning. It may be that defining autism in this
manner primed participants to think of autistic individuals in
terms of what they can’t do, thus resulting in lower ratings of
perceived competence relative to neurotypical individuals.
Participant‑Reported Cues Indicative of Deception
and Low Credibility
Practical Implications
Participants’ qualitative reasons for their impressions
revealed that gaze aversion was the most frequently cited
behavioral cue used in inferring both the truthfulness
(28.33%) and credibility (15.93%) of the target individuals.
However, despite this, gaze aversion was not a significant
predictor of perceived deception or credibility after controlling for other autistic behaviors. The large variability in
participant-reported cues (particularly within responses classified under “other”) suggests that there are many different
behaviors that people perceive to be indicative of deception
and low credibility. It was also noted that many participants
did not report the use of specific behavioral cues, but rather,
referred to broad presentations (e.g., “he seemed very nervous,” “he looked relaxed,” “she felt uncomfortable at times,”
“she carried a calm yet confident composure,” “he seemed
a little paranoid,” “body language seemed off”) or their gutfeeling about the individual (e.g., “she seems like a really
nice girl,” “she seemed honest,” “he seemed like a genuine
guy,” “she looked very simple and not very eloquent,” “she
seemed a bit cocky and self-interested”). These responses
suggest that participants may not have been fully aware of
the specific factors that influenced their judgments.
Knowledge of ASD Diagnosis
Attribution theories (Kelley & Michela, 1980) propose that,
while behaviors that are normative or expected are accepted
at face value, behaviors that are atypical or unexpected
demand an explanation. When no obvious explanations are
available, individuals begin to search for factors to which
the behavior could be attributed, such as deception and low
credibility. Thus, if an explanation for the behavior was
Our findings suggest that although people on the autism
spectrum are more likely to be viewed as deceptive and
lacking credibility when compared with neurotypical individuals, there may be more that contributes to these judgments than some very striking behaviors. Instead, it could
be that autistic people are more likely to display a unique
combination of behaviors and nuances that discriminate
them from neurotypical individuals and result in unfavorable impressions. Consequently, it is important for researchers and practitioners to recognize the complex network of
factors that may influence deception and credibility judgments when planning or evaluating possible interventions.
For example, prior to a court trial, attorneys often guide
witnesses and defendants on how to deliver a persuasive
testimony (Boccaccini et al., 2005). Witnesses and defendants are often made aware of common nonverbal behaviors
that may negatively affect evaluations of their testimony
and are taught to better control these behaviors (Boccaccini, 2002). For instance, defendants may be instructed to
maintain appropriate eye contact with the jury, as gaze aversion is commonly associated with lack of remorse (Corwin
et al., 2012). Similarly, research is emerging on the effectiveness of job interview training in improving interview
performance among people on the autism spectrum (e.g.,
Kumazaki, Muramatsu, Yoshikawa, Matsumoto, et al., 2019;
Kumazaki et al., 2017; Morgan et al., 2014). These training programs are designed to assist autistic individuals in
developing good communication skills, such as appropriate
eye contact and facial expressions (Kumazaki, Muramatsu,
Yoshikawa, Corbett, et al., 2019). Unfortunately, if autistic
individuals are perceived to lack credibility as a function of
their overall presentation, simply instructing them to display
certain overt behaviors may not be sufficient to negate this
13
502
effect (Sasson et al., 2017) unless the key behaviors can be
identified. It would thus be important for future research
to examine perceptions of deception and credibility toward
autistic individuals in a broad range of interpersonal situations, such as forensic investigations, job interviews, and
healthcare assessments, in order to better understand the factors that contribute to these impressions.
Consistent with the findings of Maras, Crane, et al.
(2019), the results of our study indicated that autistic individuals were rated as less deceptive and of higher character
when observers were provided with information about their
ASD diagnosis. This suggests that appropriate disclosure of
an ASD diagnosis, in conjunction with relevant education
on autistic behaviors, may be an effective way to reduce the
negative bias toward people on the autism spectrum (Maras,
Crane, et al., 2019; Maras, Marshall, et al., 2019; Sasson &
Morrison, 2019). However, Maras, Crane, et al. (2019) caution that research in this area is still preliminary and a better
understanding of the mechanisms through which credibility
judgments are formed would be necessary to determine how
these principles can be applied effectively by stakeholders.
Limitations
Our hypothesizing was stimulated by attribution theories
(Kelley & Michela, 1980), which propose that perceptions
of deception and low credibility arise due to the natural
tendency of observers to attribute a cause to unexpected or
atypical behaviors (Feldman & Chesley, 1984). The findings
of the study are consistent with this hypothesis, as there
was a significant interaction effect between ASD diagnosis
and observers’ knowledge of the target’s diagnosis on perceptions of deception and credibility. However, as we did
not directly measure attributions, no conclusions can yet be
drawn regarding the underlying cognitive processes that led
to participants’ judgments of deception and credibility.
Furthermore, the effect of the target behaviors on judgments of deception and credibility was only examined in one
context (i.e., being questioned about involvement in taking
money from a psychology lab). We speculated that if the
individual was in a situation in which it was expected that
the target behavior would be displayed, the presence of the
target behavior would have no effect, or perhaps even the
opposite effect, on judgments of deception and credibility.
However, as only one context was used for the stimulus videos, this conjecture was not empirically tested.
Another limitation of the study was that other possible
confounding variables were not investigated or controlled.
There is some evidence that variables such as age and gender
can also influence perceptions of credibility; however, these
13
Journal of Autism and Developmental Disorders (2022) 52:490–507
effects, in turn, appear to interact with a variety of additional
factors. For example, Neal et al. (2012) found that gender
differences in the perceived credibility of expert witnesses
only emerged when the witnesses lacked warmth and competence, while McKimmie et al. (2004) found that it was
the congruence between an expert witness’s gender and the
gender orientation of their domain of expertise that predicted
perceptions of credibility. Likewise, although studies have
shown that female mock jurors are more likely to perceive
victims of sexual abuse as more credible than male jurors
(Davies & Rogers, 2009; McCauley & Parker, 2001), male
jurors have been found to perceive defendants as more credible than female jurors (McCoy & Gray, 2007; Pozzulo et al.,
2010). It is thus possible that the relationship between ASD
diagnosis, autistic behaviors, and perceived deception and
credibility may differ according to various demographic and
situational factors; however, the present study’s design and
scope did not permit the examination of such interactions.
Future Research
A crucial question that still remains unanswered is: Why
were autistic individuals judged as more deceptive and
less credible than their neurotypical peers? We propose
four possible avenues for future research that could be
considered: examining groups of cues, examining nonlinear relationships, examining the “truthful prototype,” and
examining expectancy violation.
Examining Groups of Cues
Rather than examining individual behaviors in isolation, it
may be useful to examine whether certain types of cues are
more predictive of perceived deception and credibility than
others. For example, an interaction could be broken down
into its verbal content, paralinguistic cues, and nonverbal
cues. By presenting various combinations of these cues to
observers (e.g., using transcript only, audio only, video only,
audio and video, etc.), the degree to which each of these
groups of cues interact with ASD diagnosis to affect judgments of deception and credibility can be examined. Should
certain cues prove to be more predictive of perceived deception and credibility than others, this may assist researchers to
develop a more targeted approach in trying to identify possible behavioral mediators of the relationship between ASD
diagnosis and perceived deception and credibility. Considering groups of cues as a whole would also enable researchers
to better investigate the possibility that it is particular combinations of behaviors that affect perceived deception and
credibility, rather than individual behaviors on their own.
Journal of Autism and Developmental Disorders (2022) 52:490–507
503
Examining Nonlinear Relationships
Examining Expectancy Violation
The assumption of the present study was that the relationship
between autistic behavior and perceived deception and credibility is a linear one. However, this may not hold true for
all behaviors. For example, while high levels of gaze aversion may be unexpected or atypical, so too may extremely
low levels of gaze aversion. It is thus possible that there is a
particular range of the behavior that is perceived to be within
normal limits and that any demonstration that is above or
below this threshold is considered deviant. If so, further
research using nonlinear analyses may be better suited to
capture these relationships.
The expectancy-violation model proposes that it is not specific behaviors per se that lead to perceptions of deception
and low credibility, but rather, the fact that these behaviors
are unexpected (Bond et al., 1992; Burgoon et al., 1989).
Consequently, the extent to which a particular behavior is
considered to be indicative of deception or low credibility
may differ from observer to observer, depending on their
own unique expectations of how the target individual should
behave. To test this hypothesis, researchers have measured
observers’ expectations and examined whether the effect of
nonverbal behavior on perceived deception or credibility was
influenced by the degree to which their expectations were
violated (e.g., Ask & Landström, 2010; Bosma et al., 2018;
Hackett et al., 2008). The findings suggest that nonverbal
behaviors have stronger effects on judgments of deception
and credibility when they are more incongruent with the
observer’s expectations (Ask & Landström, 2010; Bosma
et al., 2018; Hackett et al., 2008). It is thus possible that
the reason why only small effects of autistic behaviors were
detected in the present study was that the specific cues that
were considered to be indicative of deception and low credibility varied for each observer and, therefore, these effects
were not detected when overall mean values for specific
behaviors were examined.
Examining the “Truthful Prototype”
According to the norm-violation model of veracity judgments, individuals infer deception from nonverbal behaviors that violate social standards of appropriate behavior
(Levine et al., 2000). This suggests that perceived deception may arise from a violation of the “truthful prototype”
as opposed to an activation of the “deceptive prototype.”
While the behaviors that constitute the deceptive prototype
have been fairly well documented (e.g., The Global Deception Research Team, 2006), much less is known about the
behaviors associated with perceived truthfulness. Though it
would be easy to assume that the truthful prototype is simply
the opposite of the deceptive prototype, this may not be the
case. Perhaps the way in which the truthful and deceptive
prototypes are construed are qualitatively different. Alternatively, it could be that the most salient features of the truthful
prototype may differ from those of the deceptive prototype
(e.g., the first thing that comes to mind when asked “How
can you tell when people are lying?” may not be the direct
opposite of the first thing that comes to mind when asked
“How can you tell when people are telling the truth?”).
The truthiness effect suggests that nonprobative information can influence the perceived truthfulness of a claim
by increasing the fluency with which the information is
processed: the more easily information is processed, the
more likely people are to believe it to be true (Newman
et al., 2012). This effect has been found with a variety of
types of nonprobative information; for example, claims
are more likely to be judged as trustworthy if they are presented alongside a decorative image (Newman et al., 2012),
attributed to a person with a name that is easy to pronounce
(Newman et al., 2014), or played with high audio quality
(Newman & Schwarz, 2018). Therefore, it could be that neurotypical individuals display a constellation of behaviors that
render their communication more easily processed than that
of autistic individuals. Examining deviations from the truthful prototype may thus shed light on possible behavioral cues
that have previously been overlooked.
Conclusions
To our knowledge, this is the first study to examine whether
the demonstration of stereotypical autistic behaviors causes
autistic individuals to be perceived as more deceptive and
less credible than their neurotypical peers. As predicted,
autistic individuals were perceived to be more deceptive
and less credible relative to neurotypical individuals when
telling the truth. However, limited support was found for
the hypothesized pathways by which this relationship was
thought to occur. No significant differences in levels of
gaze aversion, repetitive body movements, literal interpretation of figurative language, poor reciprocity, or flat affect
were detected between the autistic and neurotypical groups,
although some weak effects were apparent. Furthermore,
independent of ASD diagnosis, none of the target autistic
behaviors significantly predicted judgments of deception or
credibility. Instead, the results appear to suggest that an individual’s overall presentation as having a condition or disability had a stronger influence on the relationship between
ASD diagnosis and perceived deception and credibility than
individual behavioral cues. It is possible that this could be
due to the presence of other autistic behaviors that were
not accounted for in the present study, or to the complex
13
504
interaction of multiple different behaviors and idiosyncrasies. Nonetheless, the findings of this study seem to indicate
that there are noticeable differences in the presentation of
autistic individuals that distinguish them from neurotypical
individuals and result in less favorable impressions. Further
research is thus necessary to better understand the exact
mechanism by which this relationship occurs.
Supplementary Information The online version contains supplementary material available at https://d oi.o rg/1 0.1 007/s 10803-0 21-0 4963-4.
Acknowledgements This research was supported by Australian
Research Council (ARC) Grant DP190100162 and a Flinders University grant to N. Brewer and R. L. Young, and the Hamish Ramsay Fund.
This research was conducted as part of A. Lim’s doctoral dissertation
and part of the findings were presented at the 2020 conference of the
American Psychology-Law Society, New Orleans, LO, USA.
Author contribution AL, RLY, and NB developed the study concept
and design. AL collected and analyzed the data. AL drafted the paper,
and RLY and NB provided critical revisions. All authors approved the
final version of the paper for submission.
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/.
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PORTLAND CEMENT AND THE QUESTION OF ITS AERATION. (INCLUDING APPENDIX AND PLATES).
|
Minutes of proceedings
| 1,911
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public-domain
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FIGS. 1. rmhT
sCa2e of rmhes. a
3
e
S I n C h e s
BOILED AND UNBOILEO. UNSOUND CEMENT: FIGS. 2
BOILED AND UNBOILED. SOUND CEMENT: BOILED AND UNBOILED. SOUND CEMENT: BOILED AND UNBOILEO. UNSOUND CEMENT: BOILED AND UNBOILEO. UNSOUND CEMENT: BOILED AND UNBOILED. SOUND CEMENT: FIGS. 3. FIGS. 4. FIGS. 5. I m b o
7
2
. 3
e
S h l , ' % s
S
C
A
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SOUND AND UNSOUND CEMENT:
BOTH BOILED. COMPOSITE BRIQUETTES
:
BOILED AND UNBOILED. scale of Inches. .I&"O
I
2
3
9
SHOWING EXPANSIVE
FORCE OF UN
COMPOSITE BRIQUETTES
Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. FIGS. 4. FIGS. 5. 7
2
. 3
e
S h l , ' % s
S
C
A
of Imhes
ND AND UNSOUND CEMENT:
BOTH BOILED. COMPOSITE BRIQUETTES
:
BOILED AND UNBOILED. scale of Inches. .I&"O
I
2
3
9
S h d L e S
SHOWING EXPANSIVE
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niv Lib TSS] on [12/09/16] Copyright © ICE Publishing all rights reserved I m b o
7
2
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BOTH BOILED. FIGS. 4. FIGS. 5. o
7
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of Imhes
SOUND AND UNSOUND CEMENT:
BOTH BOILED. COMPOSITE BRIQUETTES
:
BOILED AND UNBOILED. scale of Inches. .I&"O
I
2
3
9
S h d L e S
SHOWING EXPANSIVE
FORCE OF UNSOUND CEMENT. COMPOSITE BRIQUETTES :
due Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. FIGS. 4. FIGS. 5. scale of Inches. .I&"O
I
2
3
9
S h d L e S
SHOWING EXPANSIVE
FORCE OF UNSOUND CEMENT. COMPOSITE BRIQUETTES : FIGS. 5. scale of Inches. .I&"O
I
2
3
9
S h d L e S
COMPOSITE BRIQUETTES : COMPOSITE BRIQUETTES
:
BOILED AND UNBOILED. scale of Inches. .I&"O
I
2
3
9
S h d L e S
SHOWING EXPANSIVE
FORCE OF UNSOUND CEMENT. COMPOSITE BRIQUETTES : COMPOSITE BRIQUETTES
:
BOILED AND UNBOILED. SHOWING EXPANSIVE
FORCE OF UNSOUND CEMENT. COMPOSITE BRIQUETTES : Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. By HENRY KELWAY
GWYER BAMBER,
Assoc. Inst. C.E. By HENRY KELWAY
GWYER BAMBER,
Assoc. Inst. C.E. MUCH has been said and written on the subject of Portland cement,
and to many there
may perhaps seem to
be little scope left for
further investigation or discussion, especially from the commercial
or the practical point of view, as distinct
from the chemical or
physical side. I n connection with the latter there is still a
wide
field for discovery and investigation; for
much uncertainty yet
exists on many of the complex problems which continue
to baffle
skilful investigators
in this
field of research. In the
present
communication, however, which
is concerned primarily with
the
question of aeration, it is not intended to refer to this part of the
subject more than may be necessary to explain the arguments and
conclusions put forward. HISTORIC I n its earlier years
the cement-trade, like many other
of the
old-established industries of this country, became stagnant, largely
owing to the
financial success which attended that stage
of its
history. The methods
of manuf:tcture as then
practised were
known to but a few, who, with well-established brands
of a
new and eminently useful materia,l, had practically a monopoly
of the English trade, and, for a considerable period,
of the
trade of the world. Such a
state of things was not conducive
to advancement on scientific and economical lines ; and for many
years after their establishment, the principal works were content to l-
G
R
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N
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Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. -. . . . . . . . . S E C T I O N A L
E L E V A T I O N . E L E V A T I O N . S E C T I O N A L E L E V A T I O N . .......... ~
. . . . . . . . . . . . ~. . ~
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K Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Proceedings.]
BAMBER ON PORTLAND CEMENT. 85 Proceedings.] BAMBER ON PORTLAND CEMENT. 22 November, 1910. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. BAMBER ON PORTLAND CEMENT AND 86 [Minutes of [Minutes of use primitive methods and
old-fashioned machinery, by means of
which, under conditions of plentiful trade and absence of competi-
tion, the manufacturers were able to amass considerable fortunes
without any pzrticular attention to reduction in the cost of manu-
facture, or to improvement in the quality of the product. Contrary to the practice during recent years, when the engineer
has prepared the specification detailing the properties of the material
he proposes to use, the manufacturer then held the engineer in the
hollow of his hand. He had a standard specification of his own,
which was not the lengthy document now in use, but which might
be summed up in the words, “ That’s my stuff; take it or leave it ” ;
and in the main, from the first introduction
of Portlmd cement
until well on in the latter
half of the nineteenth century, the
engineer generally took the material and did the best he could with
it, lest any hesitation on
his part or haggling with the monopolist
should enhance the cost of an article with which alone much of his
work could be constructed. Such a condition of things, and the adoption of this policy by the
pioneers of the industry, combined with the commercial success of
the early undertakings,
soon brought others into the
field, not
only in England, but also on the Continent, and the
competition
thus engendered had some effect in bringing about an improvement
in the quality of the material, although
the original and costly
methods of manufacture were still largely employed. The increase in the number of cement-works in various parts
of the country-frequently
through the influence of those who had
previously been subordinates in the older works-soon
caused the
supply to overtake the demand, with the inevitable result that the
handsome profits of the early days of the industry were diminished,
never to return to their former
level. Many of the works,
owing to financial difficulties, fell into the hands
of interested
capitalists, who, while having little or no knowledge of the cement-
industry, were reluctantly compelled, in order to save something, to
continue them in operation to the best of their ability. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. This brought new blood into the industry, which no doubt resulted
in some advance being
made, through the attempt
of individual
manufacturers to maintain their positions by improving the quality
of their product; but, although perhaps making
a better article,
they had to be content with reduced returns. It was not until towards the end of the nineteenth century that
development on
scientific and economic lines was undertaken by
many of the principal manufacturers
in all parts of the world. Their efforts met with the full appreciation of the members of the Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 87 THE QUESTION OF ITS AERATION. Proceedings.] engineering profession,
who, through years of trial and trouble,
success and failure, had by varied experience become fully acquainted
with the properties and eccentricities of the cements of the past. ledge thus gained, tried to impose specifications of varied character,
the value of some of which it would be unadvisable to enlarge upon :
strictly complied with, they would in many cases have left the
compiler of the specification, and his work, in a worse condition
than under the pioneer manufacturers. This was the period when the engineer, acting upon the know But by degrees a great improvement wits wrought ; and through
the initiative of Sir John Wolfe Barry, and with the able co-opera-,
tion of Sir William Matthews and other members
of The Institution
of Civil Engineers and of various other engineering societies, there
resulted the establishment of a scientific system of standardization,
which has had for Portland cement, as it has for other engineering
and chemical industries, a marked influence for good, as well on the
commercial as on the manufacturing side. The advantages to be derived from the great work of standardiza-
tion accomplished by the co-operati.on of engineers, manufacturers,
and consumers, have been fully described by Dr. Unwin in lectures
delivered to Students of The Institution,l and the Author
would only
briefly call attention to the great improvement in the quality
of
Portland cement manufactured
in the United Kingdom, as the
result of the almost unanimous adoption of the “British Standard
Specification for Portland Cement” as
now issued. Minutes of Proceedings Inst. C.E., vol. clxxvii, p. 93. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. The industry
as a whole, having accepted the conditions of that specification, has
improved the methods of manufacture by large expenditures
of
capital in the way best adapted to -meet local conditions and to suit
the character of the raw materials dealt with. Nevertheless, the advent of methods of standardization which place
on a recognized basis the requirements to be fulfilled has not stifled
healthy competition amongst manufacturers. Each
still seeks to
excel with his particular product, :and many have already improved
the quality of their cement far beyond the demands of the latest
issue of the British Standard
specification-a
point which
must
not be overlooked when considering
the arguments put forward
by the Author, who seeks to compare the finest products of to-day
with the cements of the latter half of the nineteenth century. y
Reference to Papers which have been read and discussed from
time to time before
The Institution,
more particularly those by
Mr. John Grant, who was one of the first to place the testing of Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 88 BAMBER
ON
PORTLAND
CEMENT
AND
[Minut of of cement on a comparatively satisfactory
basis, will show the difficulties
which the engineer had to contend with in early days when seeking
to satisfy himself that the cement he received was suitable for the
work to which it was to be applied. The following extracts from the discussion on Mr. Grant’s Paper
in 1865 indicate clearly the state of things prevailing in the cement-
industry at that period, and the costly and tedious methods which
it was generally necessary for
the engineer to adopt before
the
cement supplied to him could be safely used. Mr. Druce remarked : “The difficulty with Portlalld cement
was to obtain it of thoroughly good
quality, the demand *for it at the present time
being beyond the means of supply. With reference to the mode of testing, he had adopted the steelyard system, for
the last ten
years, but he did not think it sufficient. He had met with many
instances in which cement, after having stood the ordeal
of that machine, and
shown high results, had afterwards entirely failed from causes
for which the
cement-maker was chiefly to blame. Or, again, to quote a cement-manufacturer :- “Theoretically, the cement should be an impalpable powder
. . . but this
could scarcely be the case with a material, which it was so difficult to reduce to
powder . . . He might also add, that the coarsest ground cement being found
to weigh the heaviest, the exaction of &,very heavy specific weight, unaccompanied
by any other restriction, offered an inducement to imperfect pulverization.” Another authority writes about the same period :- ‘ . . . . the time has gone past for the ipse dixit of the manufacturer to impose
a cement of indifferent or doubtful quality, simply because
it has been made at
an old-established manufactory ; something more than this kind of reputation will
be reqaired for a maker to hold his ground amongst engineers
who have given the
question of Portland cement the necessary attention.” Minutes of Proceedings Inst. C.E., vol. xxv, p. 66. Minutes of Proceedings Inst. C.E., vol. xxv, p. 66. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. The surest way to guard against the effects
of inferior or ‘blowing ’ cement was t o have a sufficient stock on hand to enable
it to be thoroughly air-slaked before
it was used. Provided it was kept from
damp it could not have too much air. His practice
at Dover was to expose it to
the air, for at least one month, which reduced the imperfectly burnt portions to
an inert and harmless condition.” “The difficulty with Portlalld cement
was to obtain it of thoroughly good
quality, the demand *for it at the present time
being beyond the means of supply.
With reference to the mode of testing, he had adopted the steelyard system, for
the last ten
years, but he did not think it sufficient.
He had met with many
instances in which cement, after having stood the ordeal
of that machine, and
shown high results, had afterwards entirely failed from causes
for which the
cement-maker was chiefly to blame.
The surest way to guard against the effects
of inferior or ‘blowing ’ cement was t o have a sufficient stock on hand to enable
it to be thoroughly air-slaked before
it was used.
Provided it was kept from
damp it could not have too much air. His practice
at Dover was to expose it to
the air, for at least one month, which reduced the imperfectly burnt portions to
an inert and harmless condition.” Another speaker observed :- l ‘ The difficulty with Portland cement now was, that it could not be depended
upon . . . it was almost a matter of necessity that it should undergo a course
of tedious tests before assurance could be obtained
that the cement would not
blow’ in the work, and this bar to its use would remain, until the makers adopted
the course pursued in other manufactures, which
was, to aim at every possible
improvement in quality, with the certainty of a good article always commanding
its valuc on the market.” Proceedings.]
THE
QUESTION
OF ITS
AERATION. 89 Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Proceedings.]
THE
QUESTION
OF ITS
AERATION. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. REASONS FOR AERATIO Now what were the methods in the processes of manufacture then
practised which produced
the defects referred to in the foregoing
extracts ? There was at that time an almost entire absence of that
scientific chemical control which is an essential part of the methods
of to-day. I n consequence, the main factors governing the chemical
parts of the process were little understood, and hence points essential
to improvement and success received little or no attention. The importance of thorough amalgamation of the raw materials
and of chemical uniformity of the resulting mixture was not then
fully realized, or if it was understood, no adequate provision
was
made for producing so perfect a mixture as is now attained. The
Author well remembers, in the
eighties, when he first
became
interested in the cement-industry, finding the machinery for
the
amalgamation of the raw materials at some works such that, when
the operation was completed, the mixture often contained particles
of unreduced chalk equal in size to a split pea ; and the composition
of the slurry at the time of calcination sometimes ranged between
at least 5 per cent. above and 5 per cent. below the prescribed
narrow limit. No adequate arrangements
existed for mixing
the prepared
raw material
in large bulk
to ensure uniformity of composition,
and the subsequent
calcination was often carried
out in
a very
perfunctory manner, without any attempt being made afterwards
to separate the unburned portions from those which were thoroughly
calcined. The whole was ground together to produce the
powdered
material known
as Portland cement, which, notwithstanding its
many defects, was then, by reason of its great utility, making a
reputation for itself throughout the world. The defects being caused
chiefly by the presence of uncombined
lime, due either to faulty amalgamation of the raw materials or to
insufficient calcination, or to both, the engineer
wisely had recourse to
the simplest and most natural method for neutralizing the expansive
force of the cement, i.e., by convertsing the free lime into hydrated
carbonate of lime by absorption of moisture and carbonic anhydride
from the atmosphere, with
the object of rendering it inert. I n
treating cement in bulk this is a tedious and expensive process, but
with the knowledge then available perhaps no better system could
have been adopted to secure soundness and freedom from expansion. REASONS FOR AERATIO Att
t
t
t
t
l
lt di
th
ti cement in order to remove the principal source of danger, namely,
expansion and blowing, was that the percentage of material having
actual cementitious value was materially
decreased, and the com-
mercial value of the cement was thus greatly reduced. That this
tendency to expansion existed in the early Portland cement there
can be no doubt, and probably many engineers of long experience
could give numerous instances
of resulting failure in their work
from that
cause. I n order to guard against this
it bedame a
regular custom, particularly
in connection with large contracts,
to specify that the cement, after being delivered by the manu-
facturers on the site of the work, should be stored in bulk, turned
over several times at stated intervals, and aerated as completely as
was possible by such methods, during long periods of storage. In
cases where it was not convenient or possible thus to treat the
cement after
delivery, the manufacturer
was compelled by the
engineer to provide at the place of manufacture for such aeration,
the success of which, within a reasonable period and at heavy expense
for labour, depended largely upon the hygroscopic condition of the
atmosphere at the time the cement was being treated. When
the
air was heavily charged with moisture the free lime in the cement
rapidly absorbed it ; but in a dry atmosphere the treatment might
be continued for long periods without
appreciable improvement in
the soundness of the cement, because the absorption of carbonic
anhydride from the atmosphere, necessary to convert the calcium
oxide into calcium carbonate, cannot
take place except in the
presence of moisture: consequently these methods
were not alto-
gether satisfactory. Even in a moist atmosphere
a large bulk of
cement can absorb moisture only when, through the operation
of
turning over the powder, the particles of cement come in contact
with the air; and when after each turning over the heap again
closes up, the cement on the immediate surface absorbs after a time
an excess of moisture which seriously depreciates it, whilst at the
same time sealing up the interior against the admission of air and
consequently of moisture. REASONS FOR AERATIO A result, however, of the prolonged aeration of such unsatisfactory Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 90 BAMBER
ON
PORTLAND
CEMENT
AND [Minutes of cement in order to remove the principal source of danger, namely,
expansion and blowing, was that the percentage of material having
actual cementitious value was materially
decreased, and the com-
mercial value of the cement was thus greatly reduced. That this
tendency to expansion existed in the early Portland cement there
can be no doubt, and probably many engineers of long experience
could give numerous instances
of resulting failure in their work
from that
cause. I n order to guard against this
it bedame a
regular custom, particularly
in connection with large contracts,
to specify that the cement, after being delivered by the manu-
facturers on the site of the work, should be stored in bulk, turned
over several times at stated intervals, and aerated as completely as
was possible by such methods, during long periods of storage. In
cases where it was not convenient or possible thus to treat the
cement after
delivery, the manufacturer
was compelled by the
engineer to provide at the place of manufacture for such aeration,
the success of which, within a reasonable period and at heavy expense
for labour, depended largely upon the hygroscopic condition of the
atmosphere at the time the cement was being treated. When
the
air was heavily charged with moisture the free lime in the cement
rapidly absorbed it ; but in a dry atmosphere the treatment might
be continued for long periods without
appreciable improvement in
the soundness of the cement, because the absorption of carbonic
anhydride from the atmosphere, necessary to convert the calcium
oxide into calcium carbonate, cannot
take place except in the
presence of moisture: consequently these methods
were not alto-
gether satisfactory. Even in a moist atmosphere
a large bulk of
cement can absorb moisture only when, through the operation
of
turning over the powder, the particles of cement come in contact
with the air; and when after each turning over the heap again
closes up, the cement on the immediate surface absorbs after a time
an excess of moisture which seriously depreciates it, whilst at the
same time sealing up the interior against the admission of air and
consequently of moisture. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. REASONS FOR AERATIO Attempts to aerate cement at less expense resulted in the erection
of extensive and costly warehouses containing movable floors
in
series: in these stores
the cement, placed on the upper
floors in
comparatively thin layers, could be dropped when
desired, with
complete turning, on to the lower floors, until the operation was
complete. The Author has in mind a case in which the engineer
found, during a prolonged period of unusual drought, that cement
under treatment by this method was not improving. On the cause Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 91 THE QUESTION OF ITS AERATION. Proceedings.] being explained, his warehouses were supplied
with jets of steam
to keep the atmosphere moist-with
the desired result. Another method for securing efficiently the exposure of cement to
the action of moist air consisted in blowing compressed air through
the mass in the store. This arrangement, while saving some labour,
still necessitated the emptying of the delivered cement from
the
packages and its storage in
bulk, and there
was the additional
expense of providing compressed air and distributing it by means of
movable jets. But this process had at least the advantage
over
others of partially cooling the cement ; for the heat of compression
is lost during the transmission of the air, and its temperature falls
considerably on its release. By this method better access for the
penetration of air to the interior
of the bulk of the cement can
always be obtained. All these methods have no doubt been employed with more or less
success in the past, but it must be admitted that their adoption
presupposed the occurrence in the cement of some expansive force
which could not be counteracted otherwise. Many engineers-
especially those whose experience extends over a long period-have
become so accustomed to the use of some aerating process that,
perhaps by force of habit, they continue to require
it of their
contractors in connection with cements of even the highest class,
forgetting that cement which has already passed the stringent tests
of the British Standard specification has necessarily shown itself to
be entirely innocent of any expansive tendency. REASONS FOR AERATIO The object
of this communication 1s to convince the engineer
that although in the
past, in t,he absence
of such stringent
specifications, the methods described were
essential for safety,
the continuation
of the old practice is not
only wasteful and
unnecessary, but is actually detrimental in the
case of modern
high-class cement. The following
series of tests show the extent to
which the
strength may be reduced by aeration :- When fresh . . . . . . . . . . . . . After 7 days' aeration . . . . . . . . . . ,, 14 ,,
,,
. . . . . . . . . . ,, 28 ,,
,,
. . . . . . . . . . Tensile Strength at 7 Days,
in Lbs. per Square Inch. . ~ ~ _
~
I----
-- 1
/
2
(
a
790
670
640
642
567
640
760
782
717
332
605
490 Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 93 BAMBER ON PORTLAND CEMENT AND [Minutes of The crushing-resistance is also affected adversely, as is shown by
the following figures :- The crushing-resistance is also affected adversely, as is shown by
the following figures :- ~~
When fresh . . . After 6 weeks' aeration
Crnshtng-Resistance, in Pounds per Square Inch. Neat Cement. Sand and
Sand and
Cement :
Neat Cement. Cen,ent :
7 days. 28 days. 7 days. 28 days. 7 days. 28 days. 7 days. 28 days. -__-__
6,6501
2,515
2,475 5,983
5,625
4,515 3,275
8,007
6,425
2,580
6,128
6,000
4,450
3,550 7,870
6,200
2,451
5,838 5,250
4,580
3,000 8,144 ----
6,000
2,500
4,050
3,100
2,800
6,000
5,900
2,500
1,900
5,800 4,000 3,200
5,800
2,500
4,100
3,000
2,800
6,000
~ -----_ Such cements, when
kept in bulk
or enclosed in ordinary com-
mercial packages, retain their original strength unimpaired for very
long periods. DEFECTS
IN THE OLDER METHODS
OF MANUFACTU The constitution of Portland cement, both chemical and physical,
has received
the attention
of many scientists and
is still the
subject of considerable research; but the
possible explanations of
many of the intricate questions it involves are still without definite
confirmation and are indeed often contradictory one
of another. These problems, however,
are purely of a scientific and technical
character, and except from
the academic point
of view, do not
interest the engineer or other users of cement to the same extent
as does the behaviour of the material in practice. REASONS FOR AERATIO The efforts of engineers have
obliged the cement-industry to
revolutionize its methods-at very heavy expense
in the
recon-
struction of works-in
order to attain the high quality
demanded
by the British Standard
specification; and yet
many engineers
continue to stipulate for the expenditure
of further large
sums
on procedure which has been rendered entirely unnecessary
by the
almost universal adoption of their own creation. If this specifica-
tion is adhered to for
all their requirements, their aeration methods
have become as obsolete as the ancient machinery of the cement-
industry, which has found its way to the scrap-heap. DEFECTS
IN THE OLDER METHODS
OF MANUFACTUR DEFECTS
IN THE OLDER METHODS
OF MANUFACTUR Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 93 THE QUESTION OF ITS AERATION. Prooeedinge.] For instance, it is immaterial to the engineer in precisely what
manner the various ingredients constituting
a sound cement
are combined chemically : whether they consist of tri-calcic silicates
and aluminates (if such exist) or di-calcic silicates and aluminates
in separate form, or whether they consist
of a complex mixture
of these compounds in a solid solution of lime, or in any other
form. Again, the theory of the setting of Portland cement is of only
academic interest to the
engineer. It is of little consequence to
him whether
it is brought about
by the formation of complex
crystals of silicates and aluminates ol lime, or of crystals of hydrate
of lime only, binding the aggregate together;
or whether the
hardening is due to the effect of solidifying colloids or to some
other problematical cause. What he is greatly interested to know
is the period of time taken for
such reactions as occur to become
operative, and the points at which the setting commences and is
completed : these are much more important to him than the physical
or chemical explanation of the phenomena. It is also of real importance to him that the manufacturer, even
though not fully
cognizant of the scientific explanation of the
reactions that take place during the manufacture of cement, should
nevertheless conduct
the consecutive operations
in such uniform
and regular manner that he
always produces an article which will
comply with the recognized requirements as to strength, volume-
constancy, and other properties that have been found by experience
to be essential to stability. The engineer rightly expects the lnanufacturer to satisfy
himself
as to the suitability of the raw materials and to proportion
and incorporate them
so that, when submitted to the subsequent
process of calcination, they are in
:t condition to enter into
com-
bination in a uniform manner, without which the resulting product
cannot be of satisfactory quality. It was during the preliminary
.processes of the manufacture,
which were conducted
in a very rough-and-ready manner in the
early days of Portland cement, that the mistakes resulting in a
" blowing " cement were made, which it was difficult, if not impos-
sible, to rectify afterwards. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. In the absence of a chemist properly
qualified to control the mixing of the ingredients, this was generally
left to a foreman
or old attendant, who was
credited with
the
possession of some inherited or specially-acquired knowledge, not
to be imparted even to his own employer. Tests RS to the
correctness of the proportions were made by
calcining in small kilns samples of the mixed raw materials taken Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. BAMBER ON PORTLAND CEMENT AND 94 winutes of winutes of day by day, which operation took
2 or 3 days to perform. If,
as a result
of this examination, it was thought the proportion
of carbonate of lime was too high
or too low, then a quantity
of material containing an excess of clay or of chalk was added to
the faulty product; but no proper
mechanism was provided for
amalgamating the two incorrect mixtures. Even though the proportions might be found correct, the fineness
of reduction is
an all-important matter
which in the early days
received but scant attention. Samples of such mixtures and of the
mixtures of modern plants are exhibited for
comparison-which
will justify the engineer of the past, when using cement made with
such badly amalgamated material, for having
employed one or
other of the aeration methods already described. I n order that the
materials may enter into
proper chemical
combination on being submitted
in the kiln to the clinkering
temperature of about 2,800” F., it is essential that they should be
in the finest possible state of subdivision; and this
is even more
important where the raw material is calcined in stationary kilns in
large masses than when it is calcined in small particles in a rotary
kiln, for in the latter case the application of heat to the interior of
the particles is more nearly
perfect. The temperature in either
process is never raised
to a
degree sufficient to fuse the raw
materials. If fusion took place, the fineness of reduction of the
raw material
would be
of less importance, as
possibly a perfect
amalgamation of the raw materials
would occur
in the
molten
condition. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. As has been shown, then, the “blowing” cement of the
past was largely due to imperfection of the raw mixtures, which
was not even compensated by sufficient care to remove from the
kiln those unburned
or partially burned
pieces which
had only
reached a
temperature sufficient to expel the carbonic anhydride
from the chalk or limestone, and which had therefore left with the
clinker a quantity of free lime. The briquettes illustrated in Plate 3 indicate clearly the difference
in volume-constancy or soundness which has been shown from time
to time between many of the older cements and the more reliable
cements of to-day. I n Figs. 1 are shown two briquettes made in the 24-inch-section
form, now disused. They were made
with the same cement, and,
after setting for 24 hours, one was immersed in boiling water for
48 hours, while the other was kept in cold water. This
treatment
resulted in expansion of the boiled briquette in all directions, but
without altering its original proportions. The Figure shows the
difference in size before and after boiling. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 95 THE QUESTION OF ITS AERATION. I n Figs. 2 are shown two briquettes, one
boiled and the other
unboiled, which were made of a different cement from those depicted
in Figs. 1. These two Figures
(1 and 2) exemplify a cement that required
aeration and another that
did not. All the briquettes were the
same size before boiling. Two other briquettes made with these two cements are shown in
Figs. 3, both of which have been submitted to the boiling test. The two briquettes in Figs. 4 are of a compound character: the
upper half of each was made with s,ound cement and the lower half
with unsound cement, the two halves being moulded together
at
the same time in the same mould. The upper briquette has been
submitted to the
boiling test, the lower briquette has not. The
expansion of the unsound cement .has fractured the sound cement,
thus showing the enormous force exerted by the increase in volume,
at a high temperature, of unstable lime compounds. The briquettes
in Figs. 5 also illustrate the expansive force of
cement. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. PRESENT-DAY
METHODS
OF MANUFACTUR Nizing.-The
uniformity in the composition of the raw materials
found in the district
of the Thames and Medway, which
still
remains the chief seat of the industry in England, is such that, with
ordinary care, a fairly uniform cement
was produced without the
aid of any but the most elementary chemical knowledge. With the development of the industry, however, in other districts
in the United Kingdom and
on the Continent, where
the raw
materials were of a much more irregular character, the aid of the
trained chemist was soon found to be essential for securing homo-
geneity; and
mainly for this reason the more scientific methods
which now prevail were
developed earlier in Continental practice
than in England. With materials varying greatly
in composition
from day to day, and almost from hour
to hour, the difficulties of
proportioning of and thorough calcination, and the chances of faulty
manufacture, were much increased,
so that uniformity of the product
could be safeguarded only by the unremitting attention
of a competent
chemical staff. But the necessity for close chemical supervision became
apparent by degrees even where
the materials were
less difficult
to proportion. The numerous
specifications already referred to,
which at one period were showered upon
the industry, and which
frequently placed restrictions of varying character on
the ingredients,
necessitated accurate knowledge
of the composition of the raw
materials at earlier stages than had been customary
before, and
hastened the resort to scientific methods. Whereas,
in many cases
in the past, the first information obtainable as to the quality of the
cement being produced was after the clinkering stage, it now became
essential to know the chemical composition before
entering upon this,
the only chemical part-and a very important part-of
the manu-
facturing process. In the earlier days density, measured
by the
weight per bushel,
and tensile strength
were practically the only
requirements, and these could be obtained by rule of thumb ; but
when stipulations were made as to specific gravity, and limitations
were placed upon the percentage of the principal ingredients-
silica, lime, alumina, oxide
of iron-and
on impurities such as
magnesia, sulphuric anhydride, sulphur, etc., the aid of the chemist
became of paramount importance. BAMBER ON PORTLAND CEMENT AI?D
[Minutes of BAMBER ON PORTLAND CEMENT AI?D [Minutes of Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. The composite briquette in the centre was made with three
cements: it retained its original d-imensions until boiled, when it
assumed the shape seen in the Figure, the unsound cement in the
centre shearing itself completely from the sound cements at the
two ends. The briquettes shown standing on end were
also of a composite
character, the one on the right hand being made with the unsound
cement and with the sound cement on the right of the centre bri-
quette, while the one on the left wa13 made with the unsound cement
and with the
sound cement on
the left of the centre briquette. Upon boiling, the force of expansion of the unsound material
distorted the briquettes and fractured both the sound cements. q
The tensile strength of the unsound cement at the usual periods
in cold water was abnormally high, and no signs of disintegration
were apprent. It is perhaps not surprising
that engineers, who may have had
experience in the past with such uns,tnble cements as the typical one
illustrated, should be slow to discontinue any precaution that might
guard against possible disaster resulting from the use
of similar
cement. It should not be forgotten, however, that cement of this
description, although it might pass satisfactorily the requirements of
some obsolete specification, could not, possibly comply with the strin-
gent conditions of the British Standaxd specification ; and if the tests
of that specification be properly enforced, the engineer is absolutely
protected from inadvertently using ab cement of this faulty nature. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 96 BAMBER ON PORTLAND CEMENT AI?D
[Minutes of Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. PRESENT-DAY
METHODS
OF MANUFACTUR Methods and apparatus
were introduced
which enable rapid
quantitative determinations to be made during the early stages of
mixing and reducing the raw
materials, by either the wet
or the
dry process, SO that any irregularity due to carelessness in manipula- Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 97 THE
UESTION
OF ITS AEEATION. Proceedings.] Proceedings.] tion or diversity of composition is quickly discovered and rectified. Under the
old methods such defects remained through
the
subsequent process of calcination, and their ill effects were difficult
of removal from the finished cement,, even by aeration. Given efficient chemists-now
an important part of the staff of
every well-equipped works-no difficult,y is experienced in obtaining
continuously a well-proportioned
mixture of the raw materials,
whatever their character; and
by the use of specidly-designed
machinery these materials are so effectually reduced that it is usual
to find 95 per cent. (calculated on t8he water-free material) ozpable
of pa,ssing through a screen having 32,400 holes per square inch. This is as near perfection
as it is commercially possible to
attain, and, given proper
cxlcinntion, the clinker should contain
only traces
of free lime, by which. is meant amorphous calcium
oxide capable of hydration or slaking with expansion,
as distinct
from the crystalline form in
which it is said to exist, together
with the
silicates and aluminates
of lime, in solid solution. Crystallized oxide of lime in this form is thought by some of the
most recent investigators
of the subject to be relatively inert,
hydrating or slaking very slowly without expansion or evolution of
heat ; whereas the amorphous oxide of lime slakes immediately upon
the addition of water, giving off much heat and undergoing a large
increase in volume. It is the absence of lime in this latter condition
in modern cements which renders
futile any attempt to
improve
them by aeration. It is only during the last
10 years that the
improvement in
quality derived from
the storage in bulk of large quantities
of
prepared raw materials has been fully appreciated. The necessity
for this was originally brought about by the change in the system
of burning, from intermittent
fixed kilns to continuous revolving
kilns. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. PRESENT-DAY
METHODS
OF MANUFACTUR The custom of pumping the raw material direct
from the
wash-mills to the drying-floors of t,he intermittent kilns, in the
absence of any intermediate mixing and storage in bulk, resulted in
considerable irregularity in the
composition of the clinker. This
did not create any great difficulty, from the point of view of actual
burning, in the intermittent kiln; but with the rotary kiln, in
which
the heat is
applied directly to
the passing materials, an excess of
lime over the normal limits necessitated
an increase in tempera-
ture and consequent waste of fuel, while raw material containing
insufficient lime was too easily fused. This gave rise to mechanical
difficulties in the
kiln, and
the cement produced
was weak. It thus became necessary to provide the more modern kilns
with material
of uniform compositi.on, which is done by means
[THE INST. C.E. VOL. CLXXXIII.]
H H Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 98 RAMBER ON PORTLAND CEMENT AND [brinutes of of large btorage-
and mixing-tanks
or silos, holding in some
cases as much as a
week’s requirements (Figs. 6, Plate 4). The variation in
the composition is now confined
within very
narrow limits-less
than 0 * 25 per cent. of the theoretical quantities. Reference to the
Table in the Appendix, which is a
copy of n
monthly record of manufacturing particulars taken at random at a
large modern works,
will indicate the extreme regularity in the
average composition of the prepared raw material. Immense advan-
tage has resulted from
the introduction of this mixing-process,
not only at works where the rotary-kiln system prevails, but also
in those still using the intermittent kiln; and this additional
process has gone far to remove many of the difficulties experienced
in producing cement to
the British Standard
specification under
the older and more casual methods. Burning.-It
is not necessary to describe in detail the working of
the old intermittent kiln or
of the continuous fixed kiln; these
processes are conducted much as formerly, but with greater care as
to the selection of the product drawn from
the kilns. The whole
character of this fixed-kiln clinker is greatly improved, by reason of
the developments, already described, in the preceding processes of
mixing and amalgamating the raw materials. 1 W. H. Stanger and
B. Blount, “The Rotatory, Process of Cement Manu-
facture.” Minutes
of Proceedings Inst. C.E., vol. cxlv, p. 44. wnloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 1 W. H. Stanger and
B. Blount, “The Rotatory, Process of Cement Manu-
facture.” Minutes
of Proceedings Inst. C.E., vol. cxlv, p. 44.
Purdue Univ Lib TSS] on [12/09/16] Copyright © ICE Publishing all rights reserved PRESENT-DAY
METHODS
OF MANUFACTUR No difficulty is now
experienced, at works still using such kilns, in producing a cement
which, when fresh from the mill, is equal to British Standard require
ments. The most important improvement,
however, in this part of
the manufacture is the introduction on a practical scale, since 1900,
of the rotary kiln. This process 1 is now also so well known that it
is unnecessary to describe it here at length, but the illustration of a
typical rotary kiln in Fig. 7, Plate 4, may be of interest. yp
y
g
y
The advantage of this process is that the calcination is completely
under control. The flow of the raw material to the kiln is regulated
automatically ; the time of passage through the kiln is governed by
the speed of rotation, which is susceptible of wide variation to suit
all conditions ; and the quantity
of fuel consumed, and therefore
the temperature attained, is completely under control,
and can be
regulated to great nicety. The mixed wet raw material (“slurry ”),
containing about 40 per cent. of water, is dried by passing it through
the hot gases escaping from
the lower or furnace end of the kiln,
which position is eventually reached by the material in the condition
of small pieces, broken by continuous agitation during its
passage
down the kiln. These small piecea, in passing through the clinkering-
zone, or hottest part of the kiln, are more effectively calcined than THE QUESTIOX OF ITS AERATION. 99 Proceedings.] :ue the larger pieces of the intermittent processes. The chemical
combination is more regular, by reason of the uniformity of the
temperature to which each of the s:mall particles is subjected, ancl,
as a result, clinker of extremely stable character, well and uniformly
burned, is produced, there being an almost entire absence of partially
calcined pieces such as are frequently present in the product
of
intermittent kilns. That the chemical combinations resulting from
this process are
complete and stable
is shown by the fact that the
clinker so
produced may be kept for long periods exposed to a moist atmo-
sphere without any signs
of disintegration ; whereas clinker 'produce
from the same raw
material by older methods
is alw-ays subject
more or less to breaking-down of the pieces, when exposed to the
air for any length of time. PRESENT-DAY
METHODS
OF MANUFACTUR This phenomenon
is due largely to the
presence of some less stable lime compounds produced
at various
temperatures, through the difficulty of obtaining an equable distribu
tion of heat in the intermittent kiln, and to the greater variation
in size of the pieces calcined therein. The
defect has been largely
remedied by the finer condition of the raw materials now operated
upon, and entirely overcome by the introduction of the rotary-kiln
system of calcination. For example, some clinker made with badly reduced and amalga-
mated materials as in the past, became disintegrated after exposure
to the atmosphere under cover for 8 weeks, while clinker produced
by the rotary-kiln
system and stored in
the open air for 7 years
after manufacture, showed no disintegration under such adverse co
ditions, thereby indicating
the stable character
of the original
product. Nor did the long exposure seriously diminish,
as might
have been expected, its vitality, for when it was ground and tested
-mixed
with three parts
of standard sand-its
tensile strength
was found to be as follows :- Lb. per Square Inch. At 7 days . . . . . . . . . . . 180
,, 14 ,,
. . . . . . . . . . . . 240
,, 21 ,, . . . . . . . . . . . . 250
,, 3 months
320
,, 28 ,,
285
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7)
6
1 ,
400 These results, although not so high as when the cement xas tested
fresh, as shown in the Appendix, are still above the requirements
of the British Standard specification. Grinding.-The
next operation to which a large amount of atten-
tion has been given
of late years
if3 that of grinding. It is well Grinding.-The
next operation to which a large amount of atten-
tion has been given
of late years
if3 that of grinding. It is well
known that the gauge of the ccmcntitioas value or binding quality g
p
g
on has been given
of late years
if3 that of grinding. It is well
nown that the gauge of the ccmcntitioas value or binding quality
H 2 tion has been given
of late years
if3 that of grinding. PRESENT-DAY
METHODS
OF MANUFACTUR It is well
known that the gauge of the ccmcntitioas value or binding quality known that the gauge of the ccmcntitioas value or binding qualit H 2 H 2 Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. BAMBER ON PORTLAND CEMENT AND 100 [Minutes of of cement is the quantity of very fine or impalpable powder present,
anything retained on a sieve of 32,400 meshes (180 X 180) per square
inch being devoid of cementitious value. Up to the last
decade of the
nineteenth century, this question received little attention, especially
in England, and it was quite usual at thxt time for the manufacturer
to supply cement of which not more than 80 to
85 per cent. would pass
through a sieve having 2,500 holes per square inch. Later, in a Paper
by Mr. H. K. Bamber,l read at The Institution in November, 1891,
it was recommended that “ the whole of the cement should pzss
through a sieve having 2,500 meshes to the square inch, and not
less than 90 per cent. of it should pass through a sieve of about
5,625 meshes to the square inch.” In a specification proposed in a Paper by Mr. A. E. Carey,2 read
at the same time, the requirements as to fineness read : “ Thecement . . . shall all pass through a sieve of 1,600 meshes per square inch,
and leaye a residue by weight of not more than 35 per cent. on a sieve of 5,000
meshes per square centimetre
(32,257 per square inch). Should the proportion
of residue exceed 42 per cent.. on the latter mesh, the cement will be rejected ;
and if the residue be more
than 35 per cent., and less than 42 per cent., the
cement may be used,
but a proportionate increase in the
specified quantity of
cement per cubic yard of concret,e shall be made.” ’ The recommendations made
at tllat time indicate
a distinct
advance upon earlier practice, of which the Author had considerable
personal experience; but the intervening
period of 20 years has
been still more progressive, ancl the British Standard specification
now requires 82 per cent. of the cement to pass through a sieve
having 32,400 holes per square inch,
which greatly increases the
area covered by the cement in use, and therefore its cementitious
value. Minutes of Proceedings Inst. C.E., vol. cvii, p. 31.
Ibid, p. 40. Minutes of Proceedings Inst. C.E., vol. cvii, p. 31.
b d Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Ibid, p. 40. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Many manufacturers, with the object of further increasing
the value of their product, have gone
far ahead of the British
Standard requirements in this respect : they now produce, and supply
as their standard quality,
cement of which about 93 per cent. will
pass through a sieve having 32,400 holes
per square inch, which
is at least 10 per cent. better than the requirements
of the latest
specification. This also has the effect of reducing the risk of expansion due to
the slaking of caustic lime, if present. Hydmti,ng Process.-Perhaps
the greatest
improvement in the
production of sound cement, and the one which renders subsequent
aeration unnecessary, is the introduction of a new and additional
process carried out in conjunction wit11 the grinding. This
process, Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Proceedings.]
THE QUESTION OF ITS AERATION. THE QUESTION OF ITS AERATION. 101 which has now been in use for 5 years or more, consists in submitting
the cement, during its final reduction, to a saturated atmosphere at
a comparatively high temperature. It achieves in a scientific, rapid,
and highly efficient manner what the aerating operations
of the
engineer have only imperfectly effected in the past. Every particle of the cement is submitted to an atmosphere of
steam, which hydrates its surface
; and that surface is in turn
removed
by attrition in the
mill, exposing fresh surfaces for
successive
operations, until the whole leaves the mill in a condition which an
unsound cement would take months to reach if stored and turned. Indeed, it is doubtful whether any treatment by aeration in bulk
could produce the same effect as is obtained by this process. As has
been explained, aeration is ineffective in the absence of moisture;
this the process supplies, in an atomized form, equally to each par-
ticle of the cement, and at temperatures ranging between 200' and
300" F., at which the hydrating action is rapid and complete. I n the Author's
own experience many cements
which expand
20 or 30 millimetres under
the Le Chatelier
test of the British
Standard specification are so improved by the application of this
process during grinding that the
expansion is reduced to 1 millimetre,
and this without the total absorption
of moisture exceeding 1 per
cent., and without any depreciation
in the strength of the cement
so treated, but, on the contrary, with some improvement. The description of mill used for this process is illustrated in Figs. 8,
Plate 4, which indicate also details of the method of application. This process of hydration, in addition to neutralizing any slight
tendency to expansion which may have been present in the clinker
before grinding, has a markedly beneficial effect in regulating the
setting-time, and in rendering it more permanent, by preventing to
a large extent the return of the cement to a quick-setting condition,
a danger which exists when gypsum alone has been
used to retard
the setting-time. g
Cooling.-A further improvement recently introduced in some of
the largest works in England, and one which involves an additional
process previous to delivery of the cement in the warehouse or store,
is that of cooling. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. It is well known that during the grinding
of a refractory
material such
as Portland-cement clinker, especially when it is
ground to the extreme
fineness now customary, considerable heat
is generated in the powerful machinery required for
the purpose,
so that on leaving
the mill, especially in the
summer months,
the cement has
n, temperature ranging beheen 150" and 300' F. It was usual to convey the hot cement to the warehouse or silo, Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 102 BAMBER ON PORTLAND CEMENT AND [Minutes of where it is frequently stored
in large bulk ; and it is within the
Author’s experience that the inner portions of large heaps of cement
so deposited have remained hot for many weeks. These conditions of
storage have no doubt been responsible in the past for some of the
difficulties of the manufacturer in ensuring the stability
of the setting-
time of his cement, and the process of cooling has proved highly
beneficial in this
respect. On leaving the grinding-mill, the hot
cement is conveyed to revolving coolers, which
are kept
cold by
external water-jackets ; in these coolers the cement is agitated con-
tinuously, losing its sensible heat, and leaving the material ready for
storage at the normal temperature of the atmosphere. The opera-
tion is conducted in the presence of air, which, besides assisting the
cooling, adds the finishing touch to
the beneficial results of the
previous hydration. I n describing briefly these processes, the Author
desires to
bring to the notice of the profession facts with which they may not
be already acquainted, and which, when generally understood, may
go far to bring engineers
t o share his conclusion
that to aerate
British Standard cement is to waste time and money. It is not necessary, in this communication, to go in detail into the
question of the cost of aerating after delivery : engineers and con-
trttctors, especially those who have been in the habit of adopting this
practice, will know from experience the cost and the inconvenience. The Author is of opinion, however, that an average of about 9d. per ton is expended in preparation for, and in carrying out, aeration
Such a figure would involve the outlay of quite large sums on many
of the works now under construction and in prospect. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. The abolition
of the practice, under the safeguards of the British Standard specifi-
cation, might entail some change in the weights of the packages now
customary, in order to bring them into conformity with the volume
quantities used in actual gauging of the concrete ; but this presents
no serious difficulty. Another argument affecting the
cost that has often been
used
in support of aeration is, that by this operation the
bulk of the
cement is slightly increased, owing to presumed expansion due to the
slaking of any free lime present ; and it is urged that the cost of the
operation is thereby recovered. This
plea appears to the Author
to be based solely
on the custom of buying cement
by weight
and specifying its use by measure
; and he thinks that
if the
engineer were convinced that there was no free lime to slake, he
would not go through an unnecessary operation merely on
the
erroneous supposition that he was thereby getting more than he
had paid for. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 103 THE QUESTION OF ITS AERATION. Proceedings.] With the modern cements of British Standard quality it is not
the case that an appreciable increase in volume is obtainable by
aeration ; first, because, as already explained, the possibility of the
presence of amorphous free lime-whether through excess or faulty
manipulation--is carefully guarded
against, and secondly, because,
should any small percentage
be present, it will be
slaked in the
process of hydration in the
grinding-mill, during which process
any increase in bulk that might be obtainable from that source will
be realized. Subsequent attempts to produce a further increase in
bulk by the less effective operation of hand-turning would, there-
fore, be futile. There is another reason, however, for this apparent increase in
bulk when cement is aerated-one which applies with
greater force
to the very finely ground cement of to-day, containing as it does a
large percentage of impalpable powder, than to the coarsely ground
cements of the past-and
that is, the mixing of air with the fine
particles of cement, which are thus prevented from being
closely
compacted. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. This phenomenon was particularly noticeable in the days of the
weight-per-bushel test, when it was customary to judge the value
of cement by determining the weight of a given volume filled in a
definite manner. Repetition
of that test
on a sample
of fresh
cement ground through close-running millstones proved that, after
each turning and mixing with air, a
less quantity by weight than
before could, under precisely the same conditions, be accommodated
in the bushel measure, on account of the support given to the par-
ticles by the enclosed air. The increase in bulk thus obtained must
have been due to physical, and not chemical, causes, because, even
though the cement had contained some free lime, the time occupied
in a series
of such experiments
would be
totally inadequate to
produce appreciable chemical action between
the moisture in the
air and the lime. The action
of present systems
of grinding-machinery is quite
different from that of the old millstones, which ground the cement
in a confined space between two close-running surfaces
without
any free
passage of air. By
modern methods
air is continually
being mixed with the powder, particularly in the case of grinding in
a tube mill, and again in the cooling-tubes, in both of which the
finely ground cement is rolled over in the presence of air until it
leaves the mill in what, if one were dealing with a liquid, would be
described as a frothy condition. That the fine cements of to-day are not subject to any further
expansion of bulk after such methods of grinding is proved by the Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 104 BAMBER ON PORTLAND CEMENT AND
[Minutes of BAMBER ON PORTLAND CEMENT AND [Minutes of results of the following tests-made
with cement ground
to pass
93 per cent. through a 32,400-mesh sieve. results of the following tests-made
with cement ground
to pass
93 per cent. through a 32,400-mesh sieve. Weight per bushel as ground
. . . . . . 1032 lbs. ,,
,,
,,
after
1st
turning
. . . . l044 ,,
,,
,,
,,
,,
2nd
,,
. . . . 1042, ,,
,,
,,
,, 3rd
,,
. . . . lOl& ,,
,,
,,
,,
,, 4th
,,
. . . . Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 104& In each case the cement was turned over in a simi1a.r manner and
passed carefully through the standard cone hopper which was in use
when this test was in vogue. In conclusion, the Author would point to the particulars of tests
given in the Appendix, which is a record of the routine daily tests
in a large cement-factory over a period of :L month, as showing not
only the uniform composition of the raw material before cnlciuation,
but also the uniformity
of the results of tests as to strength,
soundness, and
fineness. The tensile strengths are all given in
pounds per square inch, the soundness is denoted by the number of
millimetres of expansion under the Le
Chatelier test, and the
fineness is expressed by the percentage of residue on 10,000- and
32,400-mesh sieves. In respect to the raw materials, the composit,ion is regulated by
the percentage of carbonate of lime, which it will be seen ranges
between 76.02
and 76.24 per
cent., showing
great regularity
and resulting
in clinker containing 64
to 65.5 per
cent. of
calcium oxide. The greater variation
in the clinker as compared with the ram
mzlterials is due to the variation in the percentage of ash in the fuel
used, which in a small degree only is absorbed by the red-hot clinker,
but is sufficient to cause the variation mentioned. The limits of expansion permissible under the British Standard
specification are 10 millimetres after 24 hours’ aeration, and it will
be seen that it is quite possible to come well below
this limit, the
maximum expansion for
the month when
fresh, in respect of an
output of over 25,000 tons, having been only 3 millimetres. When it is realized how grea,tly the manufacture of cement 11:~s
been improved of late years in all its methods, and how continuously
Portland cement can
be produced
of the character shown, it is
interesting to recall the words of a speaker in the discussion on
Mr. Grant’s Paper of 1871, who remarked that It was t o be regretted that, with one or two exceptions, the rule
of thunh
still so largely prevailed in the manufacture of Portland cement, and he believed
that by raising the standard of quality still higher than Mr. Grant had yet done,
the manufacture would be improved and cement more uniform in quality
be
produced,” A P P E N DAILY TESTS FROM WORKS 5
1
-
3
t
S
3 *
6
3
4
6
3
4
6
3
4
6
3
4
5
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
1
-
Tensile Strength : Lbs. per Square Inch. I
:val. 7 days. eat. Sand. ,010' 450
,020' 420
,020' 460
900' 460
850' 460
950' 430
800 430
800 440
850 440
800 460
870 470
910 470
960: 470
960' 450
910' 480
950' 460
900
430 930
480
850
470
950
480
450 880
380
950
_- --
, G o 4 i 0
,020
430
9801
440
440 900
430 930
750, 430
95OI 450
850) 460
960: 500
880, 440
870 440
9301 440
l
7 days. 28 days. ~~~~
~~
~~~
Neat. 1
Sand. ?P
Neat. ~
Sand. I---------
530-580 340-350 730-7701 450-430
.. 700-710: 390-400 310-330
610-570
.. 740-760
440-460
310-320
540-560
_. 440-450
740-700
320-370
590-550
.. 400-400
750-740
590-570 310-320
.. 410-430
700-700
550-560~ 340-330
.. 580-610 340-350
780-820' 440-460
.. 560-600 350-320 760-800'
430-450
.. 550-590, 330-330
700-710~ 440-450 .. 580-560
340
750-720'
450
.. 550-5301
330
760-800~
430
.. 530-550: 290-270
720-720' 400-410
_. 540-570 310-300
570-7301 400-420 .. 500-520' 300-290
760-780
440-450 .. 590-620' 350-340
770-730' 440-450 _. --
560-600
.. 710-750' 430-440
320-340
600-570'
350
.. 750-730
450
620-580
330-350
.. 760-730' 450-450
730-760' 420-410 .. 700-7001 400-410
_. .. 750-790, 400-420 .. 750-790' 400-420
770-750 440-450
.. 1
.. .. 600-5901 340-350
.. 730-7701 420-440
530-560
320-330
.. 740-760' 420-410
550-540~ 320-340
_. 7'00-f1O1 400-420
590-620' 320-340
.. 750-710' 430-450
540-560' 330-350
.. 750-710 420-440
540-560: 300-320
550-570,
330
.. 760-730;
430
300
550-530
.. 750-710,
400 310 540-520
.. 700-710 420-450
340-340
540-570
.. 700-730
370-390
540-570! 340
_. 710-700i
430
.. 750-720
430
l
~~
~-
00%
200:
1502
---
0.2
0.3
.. 5.7
.. 8'0
0.6
._
6.0
0'2
_ . 7'2
0-4
.. 6.2
0.3
_ . 6'0
0.3
.. 8 ' 0
0.5
.. 7.0
0.4
.. 6.5
0.3
,. 6.5
0.3
.. 6.5
0.4
, ,
6.0
0.2
,. 7 ' 5
0.5
.. 6.2
0.4 6.7 .. 0.3 6.0 ,. 0.4 7.7 .. 0.5 7.2
_ _
0.6 8.0 ,. 0.4 6.5
_ _
0.5 7.5 .. 0'2 5.5 .. 0.6 8'0 .. BAMBER
ON
PORTLAND
CEMENT AND
[Minutes of BAMBER
ON
PORTLAND
CEMENT AND
[Minutes of 106 Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 105 Proceedings.] THE QUESTION OF ITS AERATION. Contrasting the properties of the cement then under discussion
with those
of the best cement made to-day,
it should not be
difficult to convince engineers of the desirability of abandoning an
obsolete method of treatment. Areliable means of diagnosis is now
in the hands of the engineering profession in the form of the British
Standard specification, and there is no
necessity to, as it were,
impair the vitality of the patient, by treating him for a complaint
which can be proved
to be non-existent. What would be
said at
the present day
of port-authorities who, having satisfied them-
selves by recognized and unfailing methods that an arriving vessel
was entirely free from disease, nevertheless placed her in quarantine
for several weeks, at great inconvenience and expense to the owners,
and partial damage to the cargo. Why, then, treat British Standard
cement in this way 1 The Author desires to express his indebtedness to Mr. R. Gordon
Nicol, M. Inst. C.E., for photographs, to Mr. Fred. A. White,
Assoc. Inst. C.E., and to the Associated Portland Cement Manufac-
turers 1900, Ltd., for the preparation and loan of models. The Paper is accompmied
by tracings and
by a number of
photographs, from which Plates 3 and 4 have been prepared, [APPENDIX Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. A P P E N 0 - 4 6.0 l .,
_ _
0.4 6.0 .. 0.3 6'2 .. 0.4 7-7 _ . 0.4 6.5 _. 0.3 6.5 .. 0.4 6.0 _ . 0.6' 9'0. .. d:4 617
_ _
0.41 7.0 .. 0.4l 6 2
.. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. 107 Proceedings.]
THE QUESTION OF ITS AERATION. 107 Proceedings.] Proceedings.] D I X . FOR FEBRUARY,
1910. FOR FEBRUARY,
1910. Rotary works only. Used in
by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. BAMBEB ON PORTLAND CEMENT AND 108 BAMBEB ON PORTLAND CEMENT AND DAILY TESTS FROM WORKS -
a
CI e
c2
e,
c
d
21st
2;nd
23rd
,
2ith
25th
l
I
Tensile Strength : Lbs. per Sq. In. I
4 Deval. 7 days. 6
=: S
Neat. Sand. l-
--
3
950
470
5
3 1,100 470
4
980 430
6
950
440
3 1,030 440
4 1,010~ 450
6
800, 460
3 1,010, 410
4 1,100' 380
6 1,0301 450
3
'780' 450
4 1,000; 350
5
780! 450
6
790; 390
3
9301 410
4
910
450
5
910 460
6
940
420
3
880 440
4 1,020 430
6
Si0 45b
,,
4 1,020 380
5
8801 430
6
910 440
3 1,020 450
I 3
800 450
4 1,010 440
4
930 450
6
800 470
: 6 1,010 440
! 3
910 450
,,
4 1,010 440
1 6
950' 490
2;th
3 1,0301 460
,,
~ 4
950 380
,,
1 5
920, 460
c
Fineness. 1002 I 1802 1 ZOOS
570-610 330-350 820-790' 440-460
_ . 550-580 320-3401 740-7501 420-4401
,,
.. .. 550-580/ 3461330
72i1750~ 400-420 ::
580-600 310-330 800-840' 400-410
.. 540-570 310-300 760-730' 430-450
. _
610-570 320-320
730-700 410-430
, _
570-600: 330-350 710-700 410-430
. _
570-580! 350-330
750-720 440-420 .. 560-530! 330-340 750-780l 400-430
.. 540-530. 330-330
i00-740/ 430-430 .. 550-580; 310-330
730-750 410-430 .. 550-530, 280-290 750-710 400-380
_ . 550-5501 3-10-350 780-7501 380-400
._
530-500 300-300 750-790 400-410
540-570 300-280
760-810 400-420
550-520
330
750-720
430
610-580
310
770-740
400
520-500
330
780-750
410
570-580
320
780-700
400
550-560 300-300
750--800 460-440
560-540
330-340 750-730 410-430
500-620 340-360 900-850 430-430
550-530 340-320 760-780i 450-430
560-570 340-820 850-820 420-400
590-620 340-360 740-770' 440-450
540-580 340-350 750-710: 400-430
580-550 350-350 780-740 440-460
580-620 320-300 820-770l 440-420
510-530 320-330 740-790 440-450
550-580 340-350 880-830 470-450
540-570 340-340 760-760 430-410
600-560 330-350 740-760 440-450
590-550 350-340 750-800 430-450
600-570 340-320 790-820 400-410
530-560 310-330
750-710 440-450
570-550
320
870-840
440
610-594
590-.560,
320
350
800-820
780-750
450
450
550-610 340 360 850-800 420-450
620-590~ 340-340 850-890 440-450
570-5501 3501330 750-780 400-430 Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Proceedings.]
THE QUESTIOlr OF ITS AERATION Proceedings.]
THE QUESTIOlr OF ITS AERATION b
d
i
ib
SS
12/09/16
C
i h © C
bli hi
ll i h
d Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved. Downloaded by [ Purdue Univ Lib TSS] on [12/09/16]. Copyright © ICE Publishing, all rights reserved.
|
https://openalex.org/W2037252107
|
https://www.cambridge.org/core/services/aop-cambridge-core/content/view/7B9EB948CB254690BE5F832F1652DDC6/S1758320900006533a.pdf/div-class-title-hospital-treatment-and-management-in-relapse-of-schizophrenia-in-the-uk-associated-costs-div.pdf
|
English
| null |
Hospital treatment and management in relapse of schizophrenia in the UK: associated costs
|
The psychiatrist
| 2,011
|
cc-by
| 4,915
|
The Psychiatrist (2011), 35, 95-100, doi: 10.1192/pb.bp.109.027714 1Optimal Medicine, London; 2King’s
College London; 3Janssen-Cilag;
4Amaris Group
Correspondence to Janet Munro
(janet.munro@optimalmedicine.com)
First received 7 Aug 2009, final
revision 10 May 2010, accepted
28 Sep 2010 Aims and method
Relapse in schizophrenia carries a heavy burden. This study
aimed to describe the characteristics of patients admitted to hospital for relapse and
to explore the treatment, length of stay and associated in-patient costs. The sample
comprised individuals discharged from a London National Health Service trust
following a relapse of schizophrenia. The costs of hospital treatment were obtained by
applying referenced unit costs. Results
The cohort comprised 71 patients. Treatment non-adherence was implicated
in 76% of relapses. Mean length of hospital stay was 138.9 days (range 1 week-
1.8 years). Mean admission cost was £25 852 (range £1270-120 000). Over 97% of
costs were hospital costs and less than 3% drug costs. Clinical implications
Hospital treatment for relapse of schizophrenia carries a
considerable economic burden, as shown in this study. Treatment non-adherence is a
significant factor contributing to relapse. Declaration of interest
J.M. and S.O. were employed by King’s College London,
Institute of Psychiatry and funded by Janssen-Cilag for their work in this project. L.D., K.P. and M.P. are employees of Janssen-Cilag. financial burden, its prevention has become an important
goal. Schizophrenia progresses according to different phases.1
Wiersma et al2 reported that the first psychotic episode
lasts, on average, 20 months. Only 10-15% of individuals do
not experience further psychotic episodes.2,3 After recovery
from the first acute episode, most individuals have periods
of partial or complete remission interspersed with relapses
of varying length and severity.4 In 2002, the National Institute for Health and Clinical
Excellence (NICE) issued guidance on the use of atypical
antipsychotic drugs for the treatment of schizophrenia.10 It
recommended that individuals experiencing adverse effects
or
unsatisfactory
response
to
typical
medications
be
switched
to
atypical
antipsychotics. In
addition,
the
prescription of clozapine was advocated in those with
treatment-resistant schizophrenia. Implementation of this
guidance was expected to improve treatment adherence
and shift individuals from in-patient care.10 In an updated
guideline,11 NICE emphasises the importance of relapse
prevention and treatment adherence: ‘For people with
established schizophrenia, the chance of relapse while
receiving continuous antipsychotic medication appears to
be about a third of that on placebo’ (p. 98). ORIGINAL PAPERS ORIGINAL PAPERS Munro et al Relapse of schizophrenia in the UK Schizophrenia relapse: costs and risk Relapse in schizophrenia is a major clinical event and
commonly affects the personal and social functioning of the
person. Moreover, relapses are associated with a high
economic burden. A study conducted in the UK showed
that the 6-month direct cost of healthcare services for
individuals who experience a relapse is four times higher
than for those who do not (£8212 v. £1899), most of the
difference being explained by in-patient costs.5 Although the cost of schizophrenia in the UK is
relatively well documented, data related to the character-
istics of individuals who experience relapse and its cost are
scarce
since
the
implementation
of
the
2002
NICE
guidance. The only publication on this topic we identified
is the study conducted by Almond et al,5 which compared
patients’ characteristics
and
6-month
treatment
costs
according to relapse status. However, as the relapse may
last longer than 6 months, this estimate may not capture the
total cost. The risk of relapse for a person with schizophrenia has
been estimated at 3.5% per month6 and approximately 40%
experience a relapse within the year following hospital
admission.7 Risk factors include poor adherence, severe
residual psychopathology, poor insight, substance misuse
and poor interpersonal relationships.6 It is estimated that
40% of relapses are caused by poor treatment adherence,8 a
major problem in many service users.9 As relapse in
schizophrenia is associated with a heavy human and 95 https://doi.org/10.1192/pb.bp.109.027714 Published online by Cambridge University Press Method The study was approved by the local ethics committee and
research and development unit. The study was conducted
between 2004 and 2005. The SLAM trust provides mental
health and substance misuse services to a catchment
population of more than 1 million.12 The
study
team
(research
nurse
or
psychiatrist)
identified in-patients aged 18 or older, with a diagnosis of
schizophrenia or schizoaffective disorder. Diagnosis was
determined using the ICD-10 codes entered as standard
practice by the psychiatrist (responsible medical officer) on
admission. A relapse admission was defined as readmission
with
a
diagnosis
of
schizophrenia
or
schizoaffective
disorder, thus excluding individuals experiencing their
first in-patient episode. The circumstances precipitating
hospital admission assessed in the study were reviewed and
individuals admitted for reasons other than a schizophrenia
relapse were excluded. Patients provided written consent to
participate. Once the person was discharged from hospital, a
retrospective analysis of medical notes was undertaken. Patient characteristics Two-thirds of patients were men (n = 47; 66%) and mean age
on admission was 40.3 years (Table 1). More than half of the
cohort was Black and minority ethnic (n = 37; 52%) and a
quarter was White (n = 18; 25%). The majority of patients
were
single
(n = 61;
86%)
and
only
one
patient
was
employed. Information was obtained from the patients and
the trust database. The sociodemographic characteristics
documented
were:
age,
gender,
ethnic
origin,
marital
status, employment status and accommodation. Medical
history was recorded: age at diagnosis of schizophrenia,
substance misuse, prior prescription of clozapine and
number of in-patient stays for schizophrenia over the
5 years preceding the current admission. Last, the details
of in-patient stay were recorded: diagnosis on admission
(ICD-10), treatment at the time of admission and whether
or not the person was detained under the Mental Health
Act. Details of prescribed psychotropic medications were
recorded from admission to discharge. For each treatment
the drug name, dose, route, start date and stop date were
recorded. The case notes were reviewed to identify whether
non-adherence to treatment was documented to be a cause
of the relapse and were classified as: ‘Not known’; ‘No’; ‘Yes
suspected’; ‘Yes definite’; and ‘Yes, as one of a number of
factors’. No further information regarding other causes of
relapse was documented. ORIGINAL PAPERS ORIGINAL PAPERS Munro et al Relapse of schizophrenia in the UK Munro et al Relapse of schizophrenia in the UK Results A total of 112 in-patients met the eligibility criteria, of whom
71 consented to participate. Study aims Pensions (£2.30). The cost of prescribed medications was
estimated using unit costs from the British National
Formulary and treatment duration.14 Total cost of hospital
treatment was the cost of in-patient stay plus the cost of
prescribed in-patient medications. We aimed to assess the cost of a schizophrenia relapse
admission in South London and Maudsley NHS Foundation
Trust (SLAM trust). The first objective was to describe the
characteristics of individuals admitted to hospital for
relapse. Second, the study aimed to describe the pharma-
cological treatment of the relapse. The final objective was to
determine the length and cost of the relapse admission. Data analysis was undertaken using SAS version 9.1 for
Windows. Descriptive statistics were generated on patients’
characteristics, medical history, Mental Health Act status
and prescribed psychotropic medications. Analyses were
undertaken to evaluate the relationship between cost of
relapse and patients’ characteristics, medical history and
Mental Health Act status on admission. As healthcare costs
are generally positively skewed, this variable was log-
transformed. Analysis of variance (ANOVA) was used to
assess
the
effect
of
categorical
variables
and
linear
regression was used to test the influence of continuous
variables on the total cost of relapse. https://doi.org/10.1192/pb.bp.109.027714 Published online by Cambridge University Press ORIGINAL PAPERS
Munro et al Relapse of schizophrenia in the UK ORIGINAL PAPERS https://doi.org/10.1192/pb.bp.109.027714 Published online by Cambridge University Press In-patient length of stay and cost For one in-patient, information on length of stay was not
available. Generally, the duration of stay varied markedly
and ranged from 1 week to 1.8 years (658 days); median was
15 weeks (105 days) (Fig. 2). Mean length of stay was 138.9
days (s.d. = 133.6). In only 24% of in-patients (n = 17) length
of stay was 5 weeks or less and in 24% it was longer than
6 months (n = 17). Almost a third of patients (n = 23; 32%) had received
clozapine before the study admission (including those
receiving clozapine on admission). All had been admitted
at least once within the 5 years preceding the relapse
admission; merely 11% (n = 8) had been admitted only once,
whereas a third had been admitted at least four times
(n = 24; 34%). The analysis of costs used log-transformed costs; the
log-transformation resulted in a normal distribution of the
regression residuals. Medical history The mean age at diagnosis of schizophrenia/schizoaffective
disorder was 25.2 years (s.d. = 7.2; range 16-50). Psychiatric Table 1
Patient characteristics on admission (n = 71)
Gender, n (%)
Male
47 (66)
Female
24 (34)
Age, years
Mean
40.3
Range (s.d.)
20-69 (11.7)
Ethnicity, n (%)
White
18 (25)
Mixed
9 (13)
Black Caribbean
23 (32)
Black African
6 (8)
Black Other
8 (11)
Other
7 (9)
Marital status, n (%)
Married
5 (7)
Cohabiting
3 (4)
Single
61 (86)
Observations missing
2 (3)
Accommodation status, n (%)
Own home
6 (8)
Living with parents/siblings
6 (8)
Rented accommodation
34 (48)
Supported accommodation
17 (24)
No fixed abode
8 (11) Table 1
Patient characteristics on admission (n = 71) Cost per in-patient day was estimated using the
Personal Social Services Research Unit 2006 estimate.13
Daily cost of National Health Service mental health hospital
services is specific to the type of stay: acute stay (£201), long
stay (£181) and psychiatric intensive care (£528). To avoid
overestimating the cost of relapse, the daily cost of long stay
was applied to the whole length of stay in hospital. This
includes: capital costs (£25.30); salary, supplies and service
costs (£99); agency overheads (£54); and disability living
allowance, as calculated by the Department for Work and 96 https://doi.org/10.1192/pb.bp.109.027714 Published online by Cambridge University Press https://doi.org/10.1192/pb.bp.109.027714 Published online by Cambridge University Press ORIGINAL PAPERS Munro et al Relapse of schizophrenia in the UK Munro et al Relapse of schizophrenia in the UK oral atypical antipsychotic. Other switches occurred in
single cases only. comorbidities
were
identified
in
14%
of
individuals
(n = 10) on admission. Substance misuse was reported in
27 in-patients (38%): 21 reported using one substance
only (30%); two-thirds of these were using cannabis
(n = 14; 20% of the total) and fewer were using alcohol
(n = 6; 8% of the total) or crack cocaine (n = 1; 1% of the
total). Four
patients
(6%
of
the
total)
were
using
multiple substances. oral atypical antipsychotic. Other switches occurred in
single cases only. On admission At the time of admission, only a third of individuals (n = 24;
34%)
were
receiving
psychotropic
medications, which
always included at least one antipsychotic (Fig. 1). Oral
preparations were prescribed in 31% of individuals (n = 20)
and 10% were receiving depot medications (n = 7), including
three in-patients on both depot and oral medications. Olanzapine was prescribed most frequently (10%; n = 7)
and sodium valproate, clozapine, quetiapine, oral risper-
idone and risperidone depot injection were each used in 4%
(n = 3) of individuals on admission. Fig 1 Antipsychotic treatments on admission and at discharge. Admission for relapse The mean in-patient cost of relapse was £25 852 and
varied from £1270 to almost £120 000 (Table 2). More than
97% of the costs were attributable to hospital care costs and
less than 3% related to pharmacological treatment. The
hospital costs alone for the 24% of in-patients (n = 17) that
were admitted for 6 months or more ranged from £32 942 to
£119 098 and accounted for 57% of the hospital costs
incurred by the whole cohort. On admission, 87% of individuals were diagnosed with
schizophrenia and 13% with schizoaffective disorder. For a
substantial proportion of individuals (n = 50; 70%) the
admission was compulsory. Non-adherence to treatment
was at least suspected as having precipitated admission in
three-quarters of individuals (n = 54): for more than a half
(n = 38) it was recognised as a definite cause of relapse, in
14% it was a suspected factor (n = 10) and in 8% it was
considered one of a number of factors (n = 6). Factors significantly affecting the cost of relapse were
analysed via univariate analyses: individuals admitted under
the Mental Health Act incurred significantly higher costs
compared with those admitted voluntarily, with an observed
mean incremental cost of £12 650 per relapse. The cost of
relapse was negatively correlated with age on admission
and 8% of the variation in the log-transformed costs was
explained by age. Diagnosis, history of clozapine, ethnicity,
gender, age at diagnosis, and 5-year history of admission
and drug misuse were not significantly correlated with
the cost of relapse in this cohort. As length of stay is the
primary driver of cost, the relationships between in-patient
characteristics
and
length
of
stay
were
not
further
explored. https://doi.org/10.1192/pb.bp.109.027714 Published online by Cambridge University Press On discharge The majority of individuals (93%; n = 66) were discharged
with at least one psychotropic medication, which always
included at least one antipsychotic. The most common type
of antipsychotic was an oral atypical used as a single agent
in 21 in-patients (30%), followed by typical depot injections
as a single agent (n = 18; 25%). A combination of anti-
psychotics was prescribed in 13 in-patients (18%) (Fig. 1). The most often prescribed drugs on discharge were
sodium valproate (21%); clozapine (17%); oral olanzapine
(14%); flupentixol decanoate depot (13%); risperidone long-
acting injection (10%); pipotiazine palmitate (10%); and oral
risperidone (8%). Among the 12 individuals who were prescribed atypical
oral antipsychotics on admission, 6 were discharged on the
same treatment they were admitted on (50%), 4 switched to
or added depot medication (33%) and 2 stopped anti-
psychotic treatment (17%). The two individuals on a single
depot antipsychotic remained on the same treatment and
the two receiving clozapine on admission were added an Fig 1 Antipsychotic treatments on admission and at discharge. 97 Main findings This study demonstrates the considerable economic costs of
hospital admissions precipitated by a relapse in individuals
with schizophrenia. It provides further insight into the
extent and consequences of non-adherence to treatment,
which was at least suspected to have precipitated the
relapse admission in three-quarters of the cohort. Only a
third of individuals were receiving antipsychotic treatment
at the time of admission and of those, only a third received
an antipsychotic depot preparation. Use of depot anti-
psychotics was markedly more frequent on discharge, when
43% of in-patients received depot. Although the duration of
admission varied markedly between in-patients, the mean
length of stay was 4.6 months, leading to a mean cost of
hospital admission exceeding £25 000 per person. The mean
cost of medication was £711, corresponding to 3% of the
hospital care budget. Our study confirms and reinforces these findings, as
non-adherence was identified as a definite cause of relapse
in the majority of the sample and was at least suspected in
75%. These proportions are consistent with the study
conducted by Kamali et al,18 who reported non-adherence
in two-thirds of patients re-admitted for schizophrenia or
schizoaffective disorder. Treatment adherence is influenced by multiple factors. Person-related factors are often a consequence of the illness
itself, such as impaired insight into the need for medication
or cognitive impairment of memory or understanding. The
person’s relationship with the physician and factors relating
to the medication itself, such as tolerability, efficacy and
dosing schedules, also influence adherence. Although only
the overall contribution of non-adherence to the relapse
admission could be determined from the case notes for this
study, further research assessing the importance of the
various risk factors may guide targeted interventions to
minimise non-adherence. https://doi.org/10.1192/pb.bp.109.027714 Published online by Cambridge University Press ORIGINAL PAPERS Munro et al Relapse of schizophrenia in the UK Fig 2 Hospital length of stay in weeks (mean 19.8; median 15,
range 1-94). level (34% v. 8%). It is worth noting that the prevalence of
functional psychosis has been reported to be three times
higher in Black than in White people in the UK.16 Only one individual recruited in this study had part-
time employment (1.5% of the cohort under 65 years of age). The survey conducted by Singleton et al16 reports that 28%
of
individuals
with
probable
psychotic
disorder
are
employed compared with 67% of individuals without such
a disorder. The lower employment rate in our study may
partly be explained by the local demographics and partly by
the severity of illness in the enrolled population. In the 2002 guidelines,10 NICE reported that a third of
individuals
with
schizophrenia
were
unresponsive
or
intolerant to conventional antipsychotic drugs and should
be considered for treatment with clozapine, which is
consistent with the fact that 32% of the sample in our
study had previously been prescribed clozapine or were on
clozapine at admission. The high proportion of individuals
admitted under the Mental Health Act (70%) and the
frequent in-patient episodes before the study admission
suggest that individuals enrolled in this study experienced
severe schizophrenia. Table 2
Cost of relapse admission (n=70)
Total cost
In-patient cost
»
Mean (s.d.)
25 852 (24777)
25141 (24186)
Median
19 482
19 005
Range
1270-119 933
1267-119 098 Non-adherence to treatment Adherence to treatment has been identified as a main
challenge in the management of schizophrenia. Weiden &
Zygmunt9 estimated that the rate of out-patient non-
adherence to maintenance antipsychotic treatment was
50% within 1 year of discharge and 75% within 2 years. Furthermore, approximately 40% of relapses are because of
non-adherence. Robinson et al17 reported that individuals
who discontinue antipsychotic therapy after a first episode
of schizophrenia multiplied their risk of relapse by almost
five. Study cohort The majority of in-patients in the study were Black and
minority ethnic (52%). The Office for National Statistics
2001 census15 indicates that people of Black African and
Black Caribbean ethnicity are in the top three ethnic groups
for each borough in the SLAM trust catchment area. Moreover, the proportion of ethnic minorities in this
geographical area is much higher than at the national Strategies for improving treatment adherence may
reduce the rate of relapse.18,19 As reported in the NICE
guidelines:11 ‘There is also some evidence to suggest a better
global outcome with depot as compared with oral anti-
psychotics20 with a reduced risk of rehospitalisation.’21 98 https://doi.org/10.1192/pb.bp.109.027714 Published online by Cambridge University Press https://doi.org/10.1192/pb.bp.109.027714 Published online by Cambridge University Press ORIGINAL PAPERS
Munro et al Relapse of schizophrenia in the UK ORIGINAL PAPERS ORIGINAL PAPERS Munro et al Relapse of schizophrenia in the UK Treatment pathway of 130.2 days for 2004-2005 in individuals with schizo-
phrenia. However, the national median (41 days) is much
lower than the median observed in our study (105 days). This indicates that although hospital length of stay, and
hence cost, varies widely between patients from this
cohort (a quarter of patients accounting for 57% of the
in-patient costs), the distribution at the national level is
even more scattered, with few patients concentrating a
large proportion of the costs. This reflects the NICE
guideline update:11 On admission, only a third of individuals received any
psychotropic medication. The most commonly prescribed
were oral atypical antipsychotics. On discharge, almost all
in-patients (94%) received antipsychotic treatment and the
number receiving atypical oral antipsychotics had more
than doubled. Sodium valproate was among the most often prescribed
medications at discharge (21%). Although this treatment is
usually used as a mood stabiliser in individuals with
schizoaffective disorder, more than two-thirds of the in-
patients who received sodium valproate at discharge did not
have a diagnosis of schizoaffective disorder at admission. This finding reflects the diagnostic continuum of schizo-
phrenia and schizoaffective disorder and shows that a
person’s diagnosis, and hence treatment, may alter over
time. Hospitalisation costs have been shown to drive healthcare
costs incurred by people with schizophrenia, both in published
evidence and in the economic analysis carried out for this
guideline. It might be reasonable to argue that antipsychotic
drugs that reduce the rate and length of hospital admissions
(for example, drugs that reduce the rate of future relapses and/
or the length of acute episodes) are cost-saving options in the
long term, despite potentially high acquisition costs. This
hypothesis is supported by published evidence, which shows
that increased adherence to antipsychotic treatment is
associated with a significant decrease in healthcare costs
incurred by people with schizophrenia through a reduction in
the risk of relapse and subsequent need for hospitalisation. Last, the number of individuals receiving antipsychotic
depot medication more than quadrupled between admission
and discharge, from 10 to 44%, which suggests that
physicians recognise the potential of depot formulations
to improve outcome, possibly through better adherence. This study was undertaken in the period between the
publication of the two NICE schizophrenia care guidelines
(2002 and 2009). Subsequent research could, therefore,
focus on the treatment patterns following the publication of
the 2009 guideline. Cost of relapse Individuals re-admitted for a relapse of schizophrenia
incurred a mean admission cost exceeding £25 000, of
which only 3% was attributed to medications. This study did
not capture any costs associated with care after discharge,
such as community mental health visits or supported
accommodation, and it may therefore underestimate the
total cost of a relapse involving hospital admission. The high
cost of in-patient care has been documented by other
authors.5,22 Almond et al5 reported that individuals who
experienced relapse incurred 6-month treatment costs more
than four times higher than those who did not (£8212 v. £1899). The definition of relapse used in their study was
based
on
re-emergence
or
aggravation
of
psychotic
symptoms and included episodes that did not require
hospital treatment. The Almond study population had less
severe schizophrenia
than
our own study
cohort, as
confirmed by the lower rate of admission under the
Mental Health Act (20% as compared with 70% in our
study). In addition, our study captures the full in-patient
treatment
costs,
as
retrospective
data
capture
was
conducted after individuals were discharged, regardless of
length of stay, whereas Almond et al report cost at a 6-
month cut-off. Truncating the in-patient costs at 6 months
in our study resulted in a cost of approximately £18 900 per
patient. https://doi.org/10.1192/pb.bp.109.027714 Published online by Cambridge University Press Study limitations In this study the definition of a relapse admission was
re-admission of a person with a diagnosis of schizophrenia/
schizoaffective
disorder. Although
admissions
were
screened for precipitating events other than relapse (e.g. social crises), no prospective assessments of symptom
severity were available to confirm a deterioration of
symptoms and a relapse of schizophrenia. However, the
high level of compulsory admissions and the pressure on
beds within the trust suggest that these in-patients had
significant symptomatology and were not admitted for
‘social’ reasons. The study was based on a retrospective
analysis of case notes, which inherently leads to a number of
limitations. For example, the duration of historical data on
prior relapse may differ between individuals. Moreover,
some of the information, such as medication adherence
and the reasons for any non-adherence, may not be
systematically recorded during medical visits. For these
parameters, the level of information and definition used
may
also
differ
between
physicians. This
study
was
conducted in the SLAM trust, whose catchment area may
not be representative of all areas of the UK, as indicated by
the longer median length of stay in this cohort than
nationally. Therefore, the results from this cohort may
not be directly representative and transferrable to other
trusts in the UK, but they provide an indicative cost of an
in-patient relapse. Last, the results of the study are 4 years
old. However, it is important to note that this type of
detailed review of schizophrenia relapse admissions in the
UK setting has not been published before; the timing of the
study in the context of the two NICE guideline documents
is also of interest. Future research should focus on a broader Based on data from a 1994 survey report on adults
living in institutions that accommodate people with mental
illness, Knapp et al22 estimated the mean in-patient cost of
schizophrenia at £7487 per annum. Jones et al23 analysed
healthcare resource use (including hospital in-patient and
out-patient services, primary and community care services,
and prescribed medications) and estimated the direct cost
of schizophrenia at approximately £20 000 per annum. The mean length of stay observed for the study
cohort (138.9 days) was similar to the national mean;
hospital episodes statistics24 report a mean length of stay 99 About the authors 15 Office for National Statistics. Census 2001: Lewisham. ONS, 2003
(http://www.statistics.gov.uk/census2001/profiles/00az.asp). Janet Munro is chief medical officer, Optimal Medicine, London; Sarah
Osborne is clinical researcher and honorary specialty doctor in psychiatry,
Institute of Psychiatry, King’s College London; Lindsay Dearden is head of
outcomes research and Katie Pascoe is senior outcomes research manager,
both at Janssen-Cilag, High Wycombe; Aline Gauthier is director, Health
Economics and Outcomes Research, and Amaris Group, London; Martin
Price is external affairs director, Janssen-Cilag, High Wycombe. 16 Singleton N, Bumpstead R, O’Brien M, Lee A, Meltzer H. Psychiatric
Morbidity among Adults living in Private Households, 2000: Summary
Report. Office for National Statistics, 2001. 17 Robinson D, Woerner MG, Alvir JM, Bilder R, Goldman R, Geisler S, et al. Predictors of relapse following response from a first episode of
schizophrenia or schizoaffective disorder. Arch Gen Psychiatry 1999;
56: 241-7. 18 Kamali M, Kelly L, Gervin M, Browne S, Larkin C, O’Callaghan E. Psychopharmacology: insight and comorbid substance misuse and
medication compliance among patients with schizophrenia. Psychiatr
Serv 2001; 52: 161-6. Acknowledgements We wish to acknowledge the support and encouragement of the late
Professor Robert Kerwin, who was the principle investigator for this study. The study data analyses and write up were undertaken following his
untimely death in 2007. We thank Jayantha Ratnayake from Quanticate for
undertaking the statistical analysis and the clinical directorates and patients
of the SLAM trust who participated in the study. 12 South London and Maudsley NHS Foundation Trust. Annual report
2004/05. SLAM,
2005 (http://www.slam.nhs.uk/about-us/
publications/annual-report.aspx). 13 Curtis L, Netten A. Unit costs of Health and Social Care 2006. University of Kent, 2006. 14 British Medical Association, Royal Pharmaceutical Society of Great
Britain. British National Formulary (issue 52). BMJ Group and Pharm
Press, 2006. Funding The study was funded by Janssen-Cilag. A.G. was funded by Janssen-Cilag
for study analyses and preparation of study data. 11 National Collaborating Centre for Mental Health. Schizophrenia: The
NICE Guideline on Core Interventions in the Treatment and Management of
Schizophrenia in Primary and Secondary Care (Updated Edition) (National
Clinical Guideline Number 82). British Psychological Society, Royal
College of Psychiatrists, 2010. capture of costs beyond the in-patient treatment and should
assess these costs in several diverse catchment areas. capture of costs beyond the in-patient treatment and should
assess these costs in several diverse catchment areas. 9 Weiden PJ, Zygmunt A. Medication noncompliance in schizophrenia. Part I: Assessment. J Prac Psych Behav Health 1997; 3: 106-10. 10 National Institute for Clinical Excellence. Guidance on the Use of Newer
(Atypical) Antipsychotic Drugs for the Treatment of Schizophrenia
(Technology Appraisal No. 43). NICE, 2002. 24 Hospital Episode Statistics (HESonline) Primary Diagnosis: 3 Character.
HES, 2010 (http://www.hesonline.nhs.uk/Ease/servlet/ContentServer?
siteID=1937&categoryID=203). https://doi.org/10.1192/pb.bp.109.027714 Published online by Cambridge University Press ORIGINAL PAPERS Munro et al Relapse of schizophrenia in the UK Munro et al Relapse of schizophrenia in the UK the Treatment of Schizophrenia (eds CL Shriqui, HA Nasrallah): 411-34. American Psychiatric Publishing, 1995. https://doi.org/10.1192/pb.bp.109.027714 Published online by Cambridge University Press References 1
Lamberti JS. Seven keys to relapse prevention in schizophrenia. J Psychiatr Pract 2001; 7: 253-9. 19 Fenton WS, Blyler CR, Heinssen RK. Determinants of medication
compliance in schizophrenia: empirical and clinical findings. Schizophr
Bull 1997; 23: 637-51. 2
Wiersma D, Nienhuis FJ, Slooff CJ, Giel R. Natural course of
schizophrenic disorders: a 15-year follow up of a Dutch incidence
cohort. Schizophr Bull 1998; 24: 75-85. 20 Adams CE, Fenton MKP, Quraishi S, David AS. Systematic meta-review
of depot antipsychotic drugs for people with schizophrenia. Br J
Psychiatry 2001; 179: 290-9. 3
McGlashan TH. A selective review of recent North American long-term
follow-up studies of schizophrenia. Schizophr Bull 1988; 14: 515-42. 4
American Psychiatric Association. Diagnostic and Statistical Manual of
Mental Disorders (4th edn) (DSM-IV). APA, 1994. 21 Schooler NR. Relapse and rehospitalization: comparing oral and depot
antipsychotics. J Clin Psychiatry 2003; 64 (suppl 16): s14-7. 5
Almond S, Knapp M, Francois C, Toumi M, Brugha T. Relapse in
schizophrenia: costs, clinical outcomes and quality of life. Br J Psychiatry
2004; 184: 346-51. 22 Knapp M, King D, Pugner K, Lapuerta P. Non-adherence to antipsychotic
medication regimens: associations with resource use and costs. Br J
Psychiatry 2004; 184: 509–16. 6
Csernansky JG, Schuchart EK. Relapse and rehospitalisation rates in
patients with schizophrenia: effects of second generation
antipsychotics. CNS Drugs 2002; 16: 473-84. 23 Jones PB, Barnes TR, Davies L, Dunn G, Lloyd H, Hayhurst KP, et al. Randomized controlled trial of the effect on quality of life of second- vs
first-generation antipsychotic drugs in schizophrenia: Cost Utility of the
Latest Antipsychotic Drugs in Schizophrenia Study (CUtLASS 1). Arch
Gen Psychiatry 2006; 63: 1079-87. 7
Hogarty GE, Ulrich RF. The limitations of antipsychotic medication on
schizophrenia relapse and adjustment and the contributions of
psychosocial treatment. J Psychiatr Res 1998; 32: 243-50. 24 Hospital Episode Statistics (HESonline) Primary Diagnosis: 3 Character. HES, 2010 (http://www.hesonline.nhs.uk/Ease/servlet/ContentServer? siteID=1937&categoryID=203). 8
Weiden P, Mott T, Curcio N. Recognition and management of
neuroleptic noncompliance in schizophrenia. In Contemporary Issues in 100 https://doi.org/10.1192/pb.bp.109.027714 Published online by Cambridge University Press
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Latin America revisited The Human Face of Two Success Stories in Economic Development
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** Dr. David Morawetz taught economics at Boston University, the Hebrew University of Jerusalem,
Monash University, Harvard University and MIT during the 1970s and 1980s, and he was a consultant to
the World Bank and many other international organisations during that period. He is the author of several
books on economic development, including Twenty-five Years of Economic Development 1950-1975 (written
at the request of the then President of theWorld Bank Robert McNamara, and published by Johns Hopkins
University Press, 1977), Why the Emperor’s New Clothes Are Not Made in Colombia (New York: Oxford
University Press, 1981), and The Andean Group: A Case Study in Economic Integration among Developing
Countries (Cambridge, Mass.: MIT Press, 1974). He is now a clinical and counselling psychologist in
private practice in Melbourne Australia, but he still maintains a keen interest in the economics of developing
countries. (morawetz@bigpond.com). Economía Vol. XXXV, N° 69, semestre enero-junio 2012, pp. 233-243 / ISSN 0254-4415 Economía Vol. XXXV, N° 69, semestre enero-junio 2012, pp. 233-243 / ISSN 0254-4415 * This paper was written in 2001, but it has not been previously published. Latin America revisited*
The Human Face of Two Success Stories
in Economic Development David Morawetz** Some people argue that poverty in developing countries is getting worse. Half the people
in the world live on less than $1 a day, these people say, and a billion people will go to
bed hungry tonight. Nevertheless, according to a recent World Bank report, the number
of people living in absolute poverty has fallen by about 200 million in the last 20 years,
while in the same period the world’s population has risen by 1.6 billion. That is, more
people have been lifted out of poverty in the past 20 years than in any other period in
human history (Dollar and Kraay, 2001). Economic development is a long slow process, measured in generations rather than
decades. True, it is important always to be aware of how much remains to be done, and
to learn from the failures, that we may not repeat them in future. Yet it is important,
too, to celebrate and learn from the successes, for this helps to maintain the enthusiasm
that is needed to overcome the challenges that lie ahead. It is in this spirit – the spirit
of celebrating and learning from the successes – that this brief paper has been written.h The paper presents the human face of two success stories in economic development,
one in Bolivia, the second in Chile. Both are examples with which I have a personal
connection. The reason I am writing the paper now is that I have recently made my first
return visit to Latin America in 15 years, so I have been privileged to see at first hand
these economic successes and their human dimensions. Economía Vol. XXXV, N° 69, 2012 / ISSN 0254-4415 234 1. SOYBEANS IN BOLIVIA It is fashionable in some circles to be critical of the World Bank and the work that it
does. So in the spirit of giving credit where credit is due, it may be worth relating a
remarkable World Bank success story. The story-in-brief begins in Bolivia in the mid-1980s. In 1985 two commodities,
tin and natural gas, accounted for fully 87% of the value of Bolivia’s exports (Table 1). Unfortunately for Bolivia, the international tin cartel soon collapsed, and the price of
tin fell sharply. In addition, the prospects for the country’s second major export, natural
gas, appeared to be deteriorating. «Either we export or we die,» said Bolivia’s President
Paz Estenssoro at the time. And indeed, the dollar value of exports of tin and natural gas combined was to fall by
40% by 1990, and by 80% by 2000. (All dollar figures in this paper are in US dollars). Similarly, the share of tin and natural gas in Bolivia’s total value of exports was to fall
from 87% in 1985 to a mere 10% by 1999 (before new discoveries caused natural gas
exports to begin a major comeback in 2000). The question in the mid-1980s was: what could Bolivia export to take the place of tin
and natural gas? The World Bank sent me to Bolivia as an economic consultant for three
weeks in May 1986 to try to answer that question. For the first two and a half weeks, I
found nothing remotely approaching the required magnitude. Then, in the middle of
the third and final week, someone said: «You must talk to Joaquin Aguirre». David Morawetz Latin America revisited. The Human Face of Two Success Stories in Economic Development 2 235 David Morawetz Latin America revisited. The Human Face of Two Success Stories in Economic Development In 1989, Sr. Aguirre was instrumental in facilitating an agreement among the
presidents of five countries (Argentina, Bolivia, Brazil, Paraguay and Uruguay) to
improve navigation and security of transit on the Paraguay-Parana River. He has received
a «plaque of distinction» from the five nations’ Comisión Permanente de Transporte de
la Cuenca del Plata in acknowledgement of his «pioneering work in the development of
the Paraguay-Parana River.» More recently, Sr. Aguirre has been named Bolivian Man of the Year by four separate
organisations, and he was voted one of the eight living «Bolivians of the 20th Century»
in a public opinion survey carried out by Enfoque magazine, sharing that honour with
former presidents Paz Estenssoro, Sánchez de Lozada and Banzer. All in all, he is a remarkable man. Sr. Aguirre said to me in 1986: Joaquin Aguirre, Bolivian visionary and entrepreneur Joaquin Aguirre Lavayen is a central character in this story, so I need to say just a few
words about him. Sr. Aguirre is a Renaissance Man: an entrepreneur, an inventor, a
philosopher, a lateral thinker, an author who has published six books (including three
historical novels), a playwright who has had his works performed on stage, and a Bolivian
patriot. He is a man of vision, passion, insatiable curiosity, reflection and action. l
Sr. Aguirre studied philosophy, literature, playwriting and business management at
Dartmouth College, Stanford University, the University of Paris (France) and Harvard
University. He was the secretary to the Bolivian Delegation at the 1945 founding conference of
the United Nations. He has been a Bolivian Senator, he has been CEO of the Bolivian
Development Corporation, and he has been Adviser on Economic Development to the
President of Bolivia. When he lived in Colombia in the early 1950s, Sr. Aguirre established the first
supermarkets in that country. While working in the food business in Colombia, he
invented a method of drying bananas. As a result he became the first Bolivian to obtain
a US patent, and his method was used commercially in the United States. Role of the World Bank The initial reaction at the World Bank was, understandably and appropriately, sceptical. But to its great credit, the Bank decided to send a mission of agriculture and transport
experts to see if they could find fault with this unlikely-sounding scheme. They, too,
came away impressed. i After the Bank had satisfied itself that the scheme seemed feasible and worthwhile, the
International Finance Corporation took a small percentage of equity in Central Aguirre
Portuaria (the company that was building the river port). At least as important, the World
Bank allocated an IDA loan of US$ 35 million to a project titled Eastern Lowlands of
Bolivia: Natural Resource Management and Agricultural Production Project. An additional
US$ 7.5 million was contributed by the Bolivian Government and German aid. Sr. Aguirre assured me when we met again recently that the fact that the World Bank
put in this money, and the way that the money was used to help expand agricultural
production for export, was crucial in enabling the economic success that followed. In
particular, if the World Bank had not invested its money, he said, he certainly would
not have risked repatriating to Bolivia more than a million dollars from his life savings
to build a port on a river. This was at a time when most wealthy Bolivians were sending
their money out of the country, only a couple of years after Bolivia had experienced an
annual inflation rate of over 20,000%. To cut a long story short, the end result of the World Bank’s interventions has been
that, coming from nowhere, Bolivia now ranks seventh in the world in exports of
soybeans, behind countries like the USA, Brazil, and Argentina. The value of Bolivia’s exports of soybeans and soybean products increased sixty-fold
in fifteen years, from $5m in 1985 to $300m in 2000. Over the same fifteen year period,
the World Bank’s investment of $35 million has generated total Bolivian exports of
soybeans and soybean products of more than $2 billion (Table 1), which is more than
50 times the Bank’s initial outlay. The World Bank’s project has been so successful that, during 1996-2000, exports of
soybeans and soybean products were by far the largest single item in Bolivia’s exports. In
1999, soybean and soybean product exports amounted to more than double the value
of exports of tin and natural gas combined. Sr. Aguirre said to me in 1986: One hundred years ago, Bolivia lost its access to the sea in the Guano War with Chile. For the last hundred years, we have been dreaming about reclaiming our access to the
sea – but we were looking in the wrong direction. What we need to do to get access to the
sea is to look east to the Atlantic, not west to the Pacific. The Paraguay-Parana River runs from the eastern border of Bolivia 2700 km to Buenos
Aires and the Atlantic. I plan to build a port on that river. What Bolivia needs to do now
to replace its exports of tin and natural gas is to produce soybeans, and ship them out to
the Atlantic on barges on the Paraguay-Parana River for export to the world. The soybeans can be grown in Bolivia’s eastern Department of Santa Cruz, which has
excellent land for soybeans, and is larger in area than Iowa and Kansas combined. The
2700 km that the goods will have to travel on the river is about the same distance as
from Minneapolis to New Orleans on the Mississippi River. The Paraguay-Parana River
will be our Mississippi. This all seemed too good to be true, and of course when things seem too good to be
true, they often are. So I spent many, many hours with Sr. Aguirre trying to find out why
his ideas would not work. But I could not fault them. So I stuck my neck out and wrote a detailed 203 page consultant’s report for the
World Bank titled «After Tin and Natural Gas, What? Bolivia’s Exports and Medium-
Term Economic Strategy: Prospects, Problems and Policy Options.» In this report I
indicated my firm belief that, even though Bolivia had not tried it before, Sr. Aguirre’s
idea (that soybeans could be grown in the Department of Santa Cruz and transported
on barges along the Paraguay-Parana river to the Atlantic for export) looked like the best
option by far to increase Bolivia’s exports significantly after the fall of tin and natural gas. Economía Vol. XXXV, N° 69, 2012 / ISSN 0254-4415 236 2. CASTRO, A FISHING TOWN IN CHILE The second example of success in economic development is in many ways totally
different, yet there are important similarities as well. Castro is a small fishing town on the island of Chiloe in southern Chile. In 1976,
I spent six weeks in Castro studying the human side of economic life in the town. The
paper that was published in World Development as a result of that research project was
subtitled «Slices of Economic Life in A Poor Chilean Fishing Town» (Morawetz, 1978). If I was writing the paper today, the word «Poor» would have to be omitted. Per capita
income in Castro in 1976 was about $300. Today, in constant inflation-adjusted dollars,
it would probably be at least double that figure. i
In the late 1970s, the centrepiece of the town of Castro was the fish and vegetable
market, located down by the waterfront. Here, a small number of fishermen sold their
catch, a couple of dozen people sold a limited range of vegetables and seaweed, and a
few others sold used clothing, live hens, and other odds and ends. Elsewhere in the town
there were several rows of half-empty glass-front shops, a shoeshine stand, and a couple
of barefoot boys selling the previous day’s Santiago newspapers. Because of the high rate
of unemployment and underemployment, many adult males were forced to migrate for
much of the year to Argentine Patagonia, far from family and friends, to seek work. In
those days Castro had just one place offering accommodation to tourists (an expensive
hotel), and a few small cafes and restaurants. Returning to Castro a quarter of a century later, it was amazing to see the changes. In 25 years the population has approximately doubled, to about 34,000. There are now
(in round figures) more than 30 well-stocked clothing stores, 15 shoe shops, six large
supermarkets and 20 smaller ones, 40 cafes and restaurants, and 30 places offering board
and lodging to backpackers and other tourists. There are also 20 hardware stores, more
than a dozen giftshops, seven furniture manufacturers and sellers, seven hairdressers,
four pharmacies, four accountants, several plumbers, a number of auto workshops and
service stations, four bookshops, two driving schools, six telephone and fax centres,
and three internet centres. Role of the World Bank And in 2000, soybeans and their products
accounted for almost a quarter of Bolivia’s total export value, and for almost as much as
all of the rest of Bolivia’s non-traditional exports combined (Table 1).h The World Bank’s project has also generated exports (and substituted imports) of
a number of other agricultural products from the lowlands of Santa Cruz, including
wheat. And of course, in addition to foreign exchange earned and saved, there are
also many other direct and indirect benefits (creation of thousands of additional jobs,
reduction of poverty, raising of per capita incomes, and so forth) that are clearly visible
when visiting the city and the Department of Santa Cruz, and comparing them with
how they looked fifteen years earlier. 237 David Morawetz Latin America revisited. The Human Face of Two Success Stories in Economic Development 237 Indeed, I imagine that this may rank among the most effective and cost-effective
projects that the World Bank has undertaken. Indeed, I imagine that this may rank among the most effective and cost-effective
projects that the World Bank has undertaken. 2. CASTRO, A FISHING TOWN IN CHILE There are also a number of stores selling electrical goods,
including one which has more than 50 TV sets on display, as well as washing machines,
stoves and other white goods. I do not recall any TV sets or other electrical goods on
display in Castro 25 years ago. The town also has a new hospital, two new campuses of established Chilean
universities, and a new sports stadium. Down by the water where the fish and vegetable
market used to be, a large handicraft market now exists, mostly for tourists, with more
than 50 stalls. Economía Vol. XXXV, N° 69, 2012 / ISSN 0254-4415 238 The Castro fish and vegetable market itself has moved a couple of kilometres up
the hill to be closer to the new geographic centre of the expanded town. It, too, has
expanded in quantity and quality so as to be almost unrecognisable. The market now occupies a building constructed especially for the purpose, and
covers 2,400 square metres, more than ten times the area that it occupied 25 years
earlier. More than ten times the quantity of produce is now available, and the quality
of the produce and the professional way it is displayed on shelving and racks is much
improved as well. Indeed, the way it looks today, it could be a fish and vegetable market
in any small town anywhere in the world. On a weekday, more than one hundred people (locals and people from neighbouring
islands) were selling vegetables and fruit, with most sellers offering a number of different
items. There were also 32 sellers of fish, compared to only a handful 25 years earlier. hi
In vegetables and fruits, more than 50 people were selling potatoes and garlic, more
than 40 carried beans and lettuce, and more than 30 offered peas and herbs. Over
20 people were selling tomatoes, capsicums, onions, plums, lemons, limes and wheat
flour, and more than ten offered beetroot, cucumbers, zucchinis, cabbages, pumpkins,
chillies, watermelons, oranges, nectarines, bananas, apples, locally made liquor, and cut
flowers. More than half a dozen stallholders sold sweetcorn, silverbeet, pears, peaches,
eggs, honey, pork cuts, pork fat and dry goods. Others offered cauliflower, artichokes,
radishes, rhubarb, dried peppers, avocados, cantaloupes, honeydew melons, raspberries,
grapes, cakes, natural remedies and charcoal. Two stallholders sold cheese from a glass
case, replacing the suitcase from which it was sold 25 years earlier. What has caused the rapid rise in living standards in Castro? There have been two main drivers of the rapid increase in standards of living in Castro
and surrounds over the past quarter of a century: the cultivation of salmon, and tourism. Twenty-five years ago, salmon was unknown in Castro and in Chile. Then, in about
1980, a group of Chileans in the government institution Fundación Chile dared to
dream. Chile has sheltered waters that are not too different from those in Norway, they
thought, so is there any reason why Chile could not cultivate salmon as Norway does? These officials did the research, and found that salmon cultivation did indeed seem
feasible. Understandably, Chilean businesspeople were wary of investing in such an
untried venture initially, so it was a couple of foreign firms who invested first, bringing
with them their technical expertise on how to farm salmon and how to sell it on the
world market. Once the foreign firms had demonstrated that salmon could be farmed
profitably in Chile and exported, local entrepreneurs followed. i
To cut a long story short, coming from nowhere, Chile, now ranks second in the world
in exports of salmon and trout, accounting for a remarkable 34% of world exports, behind only
Norway (42%). Incredibly, Chile’s exports of salmon and trout recently reached a billion
dollars a year, placing it fourth on the list of Chile’s exports, behind only copper, forest
products and fruits, and ahead of wine. Most of the salmon produced in Chile comes from
the 10th region, of which Castro (and the island of Chiloe on which it is located) is part. How has this increase in exports of salmon helped to reduce poverty and raise the
standards of living of people living in Castro and surrounds? The salmon are farmed in
sheltered waters near the shore. Workers are needed to build the infrastructure for fish
farming, to grow, feed, transport, fillet, process and pack the fish, and also to repair and
maintain the nets. Land with access to the water near the salmon fisheries has increased
more than tenfold in value. Some salmon escape from the nets, and it is these fish that
are caught by individuals and sold at the Castro fish and vegetable market. 2. CASTRO, A FISHING TOWN IN CHILE Most of the vegetables on display were grown in the rural areas and small islands around
Castro, while most of the fruit and some of the vegetables, especially the more exotic ones,
were brought by truck from Santiago and other central and south-central regions of Chile. All of the above figures relate to a weekday; because of the large influx of vendors from
neighbouring islands on Saturdays, the figures for that day are more than 50% greater.i i
In the fish market area, a dozen or more people were selling fresh salmon ($1 per
kilogram!), smoked salmon, fresh and dried mussels, and seaweed. At least half a dozen
people were selling fresh sea bass and fresh clams, and others offered fresh and/or smoked
hake, perch, sea urchins, eels, crabs, king crab, oysters and a range of other local fish
and shellfish. One fish seller and a number of vegetable sellers could be seen talking on
their cell phones. Outside the fish and vegetable market, people sold baskets, mats and other items
woven from cane or straw, as well as hand-knitted woollen socks, hats, gloves, scarves,
ponchos and sweaters. There were also more than 40 vendors selling new and used
clothing and shoes from trestle tables, or from car roof-racks, or from pieces of plastic laid
out neatly on the ground. This contrasts with the handful of sellers of used clothing with
minimal wares 25 years earlier. Upstairs in the market building there was a restaurant
catering to shoppers who needed a break. David Morawetz Latin America revisited. The Human Face of Two Success Stories in Economic Development 23 239 There is still much absolute poverty in Castro. Some people still shine shoes, others
still sell lottery tickets. But as the above picture implies, there has also been a noticeable
rise in incomes of many in the working class, and a significant increase in the size of the
middle class, which hardly existed 25 years ago. Maria, proprietor of a backpackers hostel in Castro Maria, aged 46 but looking ten years younger, is the owner and manager of one of the
many Castro backpacker hostels. She lives in the hostel with her husband, four sons and
mother-in-law. During the busiest two summer months each year, she employs an 18
year-old, Nancy («she could be my daughter»), to help her with the work. Maria and
Nancy work hard, from morning to night. Several times each day, Maria and Nancy walk from the hostel to the bus station to
meet the latest bus from the mainland, including even the bus that arrives on Sunday
night at 10 pm, to offer accommodation to any newly arrived tourists. There is much
competition at the bus station from others offering similar lodgings. Maria’s trump card
is that her hostel is listed in the Lonely Planet guide to Chile, where it is described
(accurately) as «exceptionally friendly». Nevertheless, even in summer, her hostel is by
no means always full. Maria walks to the bus station because she has no car – when she
goes to the market to do her shopping, she takes the bus. Until the age of 15, Maria grew up as one of the campesino kids. «It used to be
difficult growing up in the countryside,» she said. «There was no electricity and no
running water. I had to carry water in buckets.» Maria went to elementary school until
age 12, up to grade 5. «I went to school without any shoes, except when it was very cold. Now it’s easy growing up in the countryside. They all have electricity, water, TVs and
refrigerators, and now almost everyone has shoes – they are born with shoes on now.» About 20 years ago, Maria was fortunate enough to inherit a share in a small hostel
in Castro. At first she offered full board, but she quickly moved to offering bed and
breakfast only. After modest renovations, the hostel is now a two-storey timber house,
about the size of a very small and very basic Swiss chalet. It has 32 beds crammed into it,
with a total of two bathrooms for all guests to share. The cost is just $8 per night for bed
and breakfast. This price includes kitchen privileges and cable TV in the family’s living
room, and there is also inexpensive access to broadband internet. Economía Vol. XXXV, N° 69, 2012 / ISSN 0254-4415 Economía Vol. XXXV, N° 69, 2012 / ISSN 0254-4415 240 A rise in rural incomes around Castro means that campesinos have more money to
spend in town. So more shops have opened up to service them, and existing shops have
prospered. Whereas campesino children often went barefoot 25 years ago, their parents
now have enough money to purchase shoes from the many shoe shops in town. One
woman from such a campesino family is Maria. («Nobody knows me,» she said, «so you
can use my real name.») What has caused the rapid rise in living standards in Castro? In addition,
because of the needs of the salmon industry, rural roads and electricity networks have
been expanded greatly outside Castro, which in turn enables rural people (or campesinos)
to grow more vegetables, transport them to town, and sell them at the market. For all these reasons, standards of living in Castro and surrounds have risen sharply,
and poverty has been reduced significantly, over the past 25 years. (By contrast, Ancud,
which is also on the island of Chiloe, but which does not have waters that are suitable for
salmon cultivation, is not doing nearly as well economically. Puerto Montt, on the other
hand, a nearby town on the Chilean mainland where much of the salmon processing
occurs, is booming as well, with unemployment less than half the rate in Santiago). Maria, proprietor of a backpackers hostel in Castro Over the years, Maria’s business has prospered with much hard work, and with
the world-wide rise in tourism that was enabled by globally and locally improved
communications and transport. Maria prefers foreign guests to Chileans from Santiago
(«they are too fussy»), and a large proportion of her guests tend to come from Argentina
and Europe. In the past couple of years there have been fewer Argentine tourists because David Morawetz Latin America revisited. The Human Face of Two Success Stories in Economic Development 241 of the economic problems in that country, and tourism from the northern hemisphere
dipped sharply after the events of September 11, 2001. So even though she is more
secure economically than she was 20 years earlier, her situation remains precarious. Two of Maria’s sons studied until 8th grade and 11th grade respectively, and are currently
employed as musicians in the Chilean army band. A third son is about to start studying
graphic design at a regional university in a small town on the Chilean mainland. The
youngest son, aged 14, is still at school; he is a whiz on the internet, to which he has access
when guests do not need it. Maria’s employee Nancy, who is intelligent, cheerful and
highly efficient, is about to finish her qualification as an assistant kindergarten teacher, and
would like to continue studying to be a kindergarten teacher in her own right. In summary, whereas Maria grew up very poor, she is now a little more secure
economically – and in these terms her children enjoy a much better start in life than she had. 3. COMMON FACTORS Although the differences are many, there are also significant common factors in the
Bolivian and Chilean success stories. Local initiative. In each case, the economic success was instigated by local initiative. In Bolivia, the local initiative was the dream (or lateral thinking) of Bolivia’s Sr. Aguirre,
and his risk-taking in being prepared to risk a million dollars of his life savings to build
a port on a river in the hope that it would be used for exports of soybeans, which were
yet to be grown. In Chile, the local initiative was the dream (or lateral thinking) of some
Chileans in the Fundación Chile who wondered why Chile could not produce salmon,
which it had never done before. Foreign capital. In each case, foreign capital was crucial too. In Bolivia, the bulk of
the foreign capital was provided by the World Bank, which took a small stake in the port
company, and lent the Bolivian government $35 million to help grow the soybeans. In
Chile, the foreign capital was provided by foreign firms who invested in the salmon industry,
bringing technical and export marketing know-how and contacts, while demonstrating to
Chilean entrepreneurs that the country could indeed produce and export salmon.ii National benefits. In each case, the benefits to the population as a whole have been
significant. Both Bolivian and Chilean exports have been increased by billions of dollars,
enabling additional imports of consumer and capital goods to this value, and creating
multiplied linkage effects on domestic production and employment, thereby raising
living standards and reducing poverty. Local benefits. In each case, the benefits to the local people have been widespread, and
are clearly visible to the eye of the visitor. In Bolivia, standards of living in the city of Santa
Cruz and in the small towns and rural areas of the Department of Santa Cruz have risen
significantly in the past 15 years, more so than in most other parts of Bolivia. In Chile,
standards of living in and around the fishing town of Castro (and in the salmon-processing Economía Vol. XXXV, N° 69, 2012 / ISSN 0254-4415 242 town of Puerto Montt), have also increased dramatically in the past 25 years, outpacing
most other parts of Chile. Unpredictability. The precise pattern that development will take is often difficult to
predict. 4. CONCLUSION In psychology, satisfaction or happiness is sometimes described as the difference between
expectations and outcomes/performance. One factor that reduces satisfaction with the
progress of economic development is that, too often, as soon as any economic progress
is made, this progress is taken for granted, and expectations are ratcheted upwards. For
those who despair at the apparently slow rate of progress in economic development, the
following words may be as relevant today as they were a quarter of a century ago: The historical experience illustrates a tendency for rapid interaction to occur between
development performance and expectations. ‘Upper-limit’ projections have been surpassed,
‘almost unmeetable challenges’ have been met; yet everywhere there is dissatisfaction. It
might be useful to bear in mind that… even in the best of cases, development is a long
slow process measured in generations rather than decades. (Morawetz, 1977, pp. 72-73). The historical experience illustrates a tendency for rapid interaction to occur between
development performance and expectations. ‘Upper-limit’ projections have been surpassed,
‘almost unmeetable challenges’ have been met; yet everywhere there is dissatisfaction. It
might be useful to bear in mind that… even in the best of cases, development is a long
slow process measured in generations rather than decades. (Morawetz, 1977, pp. 72-73). It may help to maintain enthusiasm for development (and to reduce despair and
compassion fatigue) if we could adopt a balanced perspective, not only focussing quite
properly on what is yet to be done (seeing the glass half-empty), but also celebrating the
successes that have been achieved (seeing the glass half-full). It is to this end, and in this
spirit, that the present paper has been written. 3. COMMON FACTORS Thirty-five years ago, when Bolivia’s exports of soybeans were zero, and Chile’s
production and exports of salmon were zero, who would have predicted that, by the
turn of the century, Bolivia would have exported billions of dollars worth of soybeans
by barge along a river to the Atlantic, and that Chile would have exported billions
of dollars worth of salmon? (In similar vein, who would have predicted that Europe’s
largest producer of false teeth and sausage skins would be… Liechtenstein?) Morawetz, David (1977). Twenty-five Years of Economic Development 1950-1975. Baltimore
and London: Johns Hopkins University Press for the World Bank. p
y
Morawetz, David (1978). «Castro Market: Slices of Economic Life in a Poor Chilean Fishing
Town». World Development, 6, pp. 861-880. Bojanic, Alan (2001). Bolivia’s Participation in International Trade Negotiations. London:
Overseas Development Institute. Bojanic, Alan (2001). Bolivia’s Participation in International Trade Negotiations. London:
Overseas Development Institute.
Dollar, David and Aart Kraay (2001). Growth is Good for the Poor. Washington D.C.: World
Bank.
Morawetz, David (1977). Twenty-five Years of Economic Development 1950-1975. Baltimore
and London: Johns Hopkins University Press for the World Bank.
Morawetz, David (1978). «Castro Market: Slices of Economic Life in a Poor Chilean Fishing
Town». World Development, 6, pp. 861-880. Ove seas Deve op e t
st tute.
Dollar, David and Aart Kraay (2001). Growth is Good for the Poor. Washington D.C.: World
Bank.i Bojanic, Alan (2001). Bolivia’s Participation in International Trade Negotiations. London:
Overseas Development Institute.
Dollar, David and Aart Kraay (2001). Growth is Good for the Poor. Washington D.C.: World REFERENCES Bojanic, Alan (2001). Bolivia’s Participation in International Trade Negotiations. London:
Overseas Development Institute. p
Dollar, David and Aart Kraay (2001). Growth is Good for the Poor. Washington D.C.: World
Bank.i Morawetz, David (1977). Twenty-five Years of Economic Development 1950-1975. Baltimore
and London: Johns Hopkins University Press for the World Bank. Morawetz, David (1978). «Castro Market: Slices of Economic Life in a Poor Chilean Fishing
Town». World Development, 6, pp. 861-880. David Morawetz Latin America revisited. The Human Face of Two Success Stories in Economic Development 24 243 Table 1. Value of Bolivian exports 1980-2000 (in millions of US dollars)
DETAIL
1980
1985
1990
1995
1996
1997
1998
1999
2000
a) Traditional Products
891.4 638.3 634.4
663.2
19.3
606.6
531.8
472.2 602.4
i. Minerals
646.3 263.8 407.7
510.5 478.0
499.6
435.2
397.1 424.9
Tin
340.9 176.9 107.6
89.6
83.5
81.6
66.1
69.4
76.5
Silver
111.2
9.5
33.6
70.8
64.0
59.3
73.1
68.1
74.0
Zinc
41.1
29.5 146.8
151.3 151.7
200.0
158.2
154.3 170.6
Tungsten
47.4
10.3
4.6
4.8
3.5
2.7
2.4
1.4
1.9
Antimonium
26.5
15.9
12.7
12.4
9.6
8.9
6.5
3.6
1.7
Lead
14.2
0.5
15.3
12.6
12.1
11.1
9.1
4.8
4.8
Gold
-
1.2
65.3
130.8 119.6
110.5
112.7
89.1
88.0
Other
64.9
20.0
21.8
38.1
34.0
25.4
7.0
6.4
7.4
ii. REFERENCES Hydrocarbons
245.1 374.5 226.7
152.6 141.3
107.0
96.7
75.1 177.5
Natural Gas
220.9 372.6 225.3
92.4
94.5
69.9
55.5
35.5 121.4
Other
24.3
1.9
1.4
60.2
46.8
37.1
41.2
39.6
56.1
b) Non Traditional products
145.5
34.2 288.3
474.5 595.2
647.2
576.3
570.0 642.9
Soybeans & derivatives
6.9
5.3
40.2
142.1 200.6
242.5
231.7
222.8 299.2
Coffee
20.7
13.9
14.1
16.9
16.5
26.0
15.0
13.8
10.4
Cacao
1.8
1.2
3.7
0.6
0.6
0.8
0.8
0.8
1.1
Sugar
47.6
1.6
31.6
16.8
27.9
22.1
23.6
9.2
7.2
Beverages
1.6
0.3
2.7
7.3
9.3
6.9
9.5
9.6
6.3
Wood & wood manufactures
32.3
5.9
49.8
75.9
82.6
87.6
67.5
72.6
75.3
Leather and leather goods
5.8
1.4 2
7.1
12.4
12.2
14.8
11.3
12.7
23.2
Rubber
4.6
0.5
2.0
0.1
0.0
0.0
0.0
0.0
0.0
Brazilian Nut
1.8
1.5
13.0
18.7
28.6
31.1
30.9
30.9
33.8
Cotton
1.0
0.6
6.4
30.1
32.8
40.7
16.9
19.8
10.6
Jewellery
78.5
39.8
20.0
3.4
15.4
31.9
Jewellery, with imported gold
11.6
49.5
54.3
44.8
32.0
13.7
Textiles & apparel
41.3
46.1
Food Products
47.8
52.1
Other
21.5
1.8
97.5
63.5
94.9
100.6
121.1
41.2
32.0
Sub-total
1,036.9 672.5 922.7 1,137.6 1,214 1,253.9 1,108.2 1,042.3 1,245
c) Re-Exports (2)
6.1
2.5
32.7
42.1
79.2
16.5
214.7
360.6 211.6
d) Personal Belongings
0.3
0.3
0.2
1.5
1.7
1.8
1.9
2.5
2.6
e) Electrical Goods
0.1
0.1
Official Value
1,043.3 675.3 955.7 1,181.2 1,295 1,272.1 1,324.8 1,405.4 1,459
F.O.B. Value
942.2 628.4 845.2 1,041.5 1,132 1,166.5 1,104.0 1,051.1 1,229
S
I
i
N i
l d E
dí i
F
B j
i (2001) Table 1. Value of Bolivian exports 1980-2000 (in millions of US dollars)
|
https://openalex.org/W4379052121
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https://zenodo.org/record/8034641/files/pathogens-12-00792.pdf
|
English
| null |
Molecular Investigations of Two First Brucella suis Biovar 2 Infections Cases in French Dogs
|
Pathogens
| 2,023
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cc-by
| 9,765
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Citation: Girault, G.; Djokic, V.;
Petot-Bottin, F.; Perrot, L.; Thibaut, B.;
Sébastien, H.; Vicente, A.F.; Ponsart,
C.; Freddi, L. Molecular
Investigations of Two First Brucella
suis Biovar 2 Infections Cases in
French Dogs. Pathogens 2023, 12, 792. https://doi.org/10.3390/
pathogens12060792 Keywords: Brucella suis biovar 2; molecular epidemiology; dog infection; wildlife; molecular typing Article
Molecular Investigations of Two First Brucella suis Biovar 2
Infections Cases in French Dogs Guillaume Girault 1
, Vitomir Djokic 1, Fathia Petot-Bottin 1, Ludivine Perrot 1, Bourgoin Thibaut 2,
Hoffmann Sébastien 3, Acacia Ferreira Vicente 1
, Claire Ponsart 1
and Luca Freddi 1,* e Girault 1
, Vitomir Djokic 1, Fathia Petot-Bottin 1, Ludivine Perrot 1, Bourgoin Thibaut 2,
n Sébastien 3, Acacia Ferreira Vicente 1
, Claire Ponsart 1
and Luca Freddi 1,* 1
EU/WOAH & National Reference Laboratory for Brucellosis, Animal Health Laboratory, 1
EU/WOAH & National Reference Laboratory for Brucellosis, Animal Health Laboratory,
Anses/Paris-Est University, 94700 Maisons-Alfort, France
2
Clinique Vétérinaire de la Ville Haute, 40400 Tartas, France
3
Clinique Vétérinaire des Genêts, 30300 Beaucaire, France
*
Correspondence: luca freddi@anses fr EU/WOAH & National Reference Laboratory for Brucellosis, Animal H
Anses/Paris-Est University, 94700 Maisons-Alfort, France Anses/Paris-Est University, 94700 Maisons-Alfort, France q
3
Clinique Vétérinaire des Genêts, 30300 Beaucaire, France *
Correspondence: luca.freddi@anses.fr Abstract: Despite Brucella suis biovar 2’s (BSB2) active circulation in wildlife, no canine infections
have been reported. The present paper is the first to describe two cases of BSB2 infections in French
dogs. The first case occurred in 2020 and concerned a 13-year-old male neutered Border Collie with
clinical signs of prostatitis. The urine culture revealed the excretion of significant levels of Brucella
in the sample. The second case concerned a German Shepherd with bilateral orchitis, in which
it was possible to detect Brucella colonies following neutering. HRM-PCR and classical biotyping
methods classified both isolated strains as BSB2, in contrast to expected B. canis, which is usually
the etiological agent of canine brucellosis in Europe. The wgSNP and MLVA analyses highlighted
the genetic proximity of two isolates to BSB2 strains originating from wildlife. No pig farms were
present in the proximity of either dog’s residence, ruling out potential spill over from infected pigs. Nevertheless, the dogs used to take walks in the surrounding forests, where contact with wildlife
(i.e., wild boars or hares, or their excrements) was possible. These cases highlight the importance of
adopting a One Health approach to control the presence of zoonotic bacteria in wild animals and
avoid spillovers into domestic animals and, potentially, humans. Citation: Girault, G.; Djokic, V.;
Petot-Bottin, F.; Perrot, L.; Thibaut, B.;
Sébastien, H.; Vicente, A.F.; Ponsart,
C.; Freddi, L. Molecular
Investigations of Two First Brucella
suis Biovar 2 Infections Cases in
French Dogs. Pathogens 2023, 12, 792.
https://doi.org/10.3390/
pathogens12060792 pathogens pathogens pathogens pathogens pathogens 1. Introduction Brucellosis is a worldwide zoonotic disease caused by Brucella spp. Their pathobiology
in mammalians has been extensively studied [1,2] due to the public health importance of
minimizing infections [3,4]. In France, like in other EU countries, the prevention, control
and eradication of brucellosis is regulated by Animal Health Law (AHL; regulation EU
No 2018/1882; EUR-Lex-32018R1882-EN-EUR-Lex (europa.eu)). The AHL includes the
surveillance and management of B. abortus, B. melitensis and B. suis in production animals
and wildlife. However, the AHL only considers dogs as potential reservoirs for B. abortus,
B. melitensis and B. suis. In France, surveillance is organised through departmental vet-
erinary diagnostic laboratories, which perform first-line analyses. When the suspected
Brucella colonies are detected, the isolated strains are sent to National Referent Laboratory
(LNR) for animal brucellosis, genetic confirmation and further species characterisation. Received: 21 April 2023
Revised: 22 May 2023
Accepted: 30 May 2023
Published: 1 June 2023 Received: 21 April 2023
Revised: 22 May 2023
Accepted: 30 May 2023
Published: 1 June 2023 g
p
Swine brucellosis caused by Brucella suis biovar 2 (BSB2) re-emerged in France in the
1990s following the development of free-ranged (outdoor) farms, causing major economic
losses to the industry [5]. Domestic pigs acquire B. suis through contact with wildlife
reservoirs (hares and wild boars) [6]. Occasionally, infection can be imported into intensive
farms through contaminated breeding animals (boars) and/or their semen [7]. From 1993
to 2014, the number of outbreaks in France ranged from 0 to 12 annually, with a total
of 94 cases reported in this period [8]. Although outbreaks principally occurred in the
western part of the country, where pig breeding and, more specifically, outdoor farms are Copyright:
© 2023 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/pathogens Pathogens 2023, 12, 792. https://doi.org/10.3390/pathogens12060792 Pathogens 2023, 12, 792 2 of 14 the most concentrated, several incidents were also reported in the south-east after 2012. One
epidemiological study of BSB2 causing brucellosis in French pork production identified
that the likelihood of transmission of infection among free-range farms is low due to lack
of commercial exchange in animals [5], suggesting random contacts with infected wildlife
as a major source of infection. The BSB2 is widely present in the wild boar population,
which has increased enormously since the 1990s due to a restricted hunting [9,10]. Since
1993, between 20 and 35% of positive Brucella serological reactions (determined via the
Rose Bengal Test and/or the Complement Fixation Test) were observed in wild boars that
were hunted or found dead in several departments throughout the country [5]. However,
the proportion of positive serological reactions in the tests carried out on commercially
reared pigs in quarantine stations and semen collection centers dropped from 4% in 2012
to 0.6% in 2014 [8]. Hares are an equally important reservoir for BSB2, with 28 strains
isolated between 1980s and 2000s, although no investigation of the prevalence of infection
in this species has yet been conducted. Due to the increasing number of outbreaks and
differentiation of livestock-breeding practices, a study commissioned by French authorities
sought to assess the risks of BSB2 transmission to humans exposed to naturally infected
pigs [11]. This study found that none of the breeders, nor their families, reported brucellosis
cases or related symptoms, suggesting low pathogenicity of BSB2 to humans. Seven human
cases of brucellosis caused by BSB2 were reported in the country between 2004 and 2016,
all of whom had direct contact with wild boars while hunting or preparing their meat for
consumption, highlighting how this species might be an emerging pathogen in people with
exposure to contaminated carcasses and specific immune statuses [12]. Outside of Europe,
B. suis biovars 1, 3 and 5 are identified as more pathogenic to humans, as well as other
mammals, compared to biovar 2; these biovars actively circulate among feral and domestic
animals [13–16]. Recently, canine brucellosis became primarily associated with B. canis. In Western
Europe, infections occurred only sporadically with B. melitensis, B. abortus or B. suis in
dogs in close contact with infected livestock or wild animals [17–19]. Dogs can be infected
through consumption of, contact with or inhalation of contaminated secretions, raw milk,
aborted fetuses or placentas [17,20–22]. Additionally, consumption of contaminated meat
is a possible source of canine outbreaks [23]. To date, infections in dogs with Brucella spp. have been detected in different parts of North and South America, as well as some European
and Asian countries [18,19,23–29]. In particular, canine infections with B. suis following
exposure to feral pigs through hunting or contact with their excrements have been reported
worldwide [22,29,30]. A cross-sectional study in eastern Australian dogs documented how
B. suis exposure is relatively common in our canine companions (seroprevalence 6.6%) if
they have contact with feral pigs [31]. To date, all reported cases of infections in dogs in
which B. suis biovar analysis was carried out concern only biovars 1 and 5 [21,23,28,30,32]. However, no study has systematically analysed the circulation of B. suis infection in dogs
(including serological surveys in European countries), suggesting that canine brucellosis
due to this Brucella species is potentially under-reported, probably due to the limitations of
accurate diagnosis, the lack of regulation and the low probability of human infection [24,33]. Our results demonstrate, for the first time, BSB2 infection of exposed dogs, highlighting
the high survival rate of BSB2 in the environment, the potential for infection in canids and
how even domestic pets without evident contact with wild animals or pig industries could
be sources for human infections. 2. Materials and Methods 2.1. Bacterial Cultivation and Strains Isolation 2.3. Molecular Analyses Genomic DNA was extracted using the commercially available QIAGEN QIAamp
DNA minikit (QIAGEN, Germany) following the manufacturer’s instructions. The Real-
Time PCR (RT-PCR) [35], Multiple Locus Variable-number Tandem Repeat Analysis (MLVA)-
16 [36] and High-Resolution Melting (HRM) PCR [37] analyses were performed as previ-
ously described. The individual DNA samples of 2 isolates were typed with the MLVA-16
panel single-plex PCR and agarose gel method. Clustering and congruence analyses were
conducted with BioNumerics 7.6.3 (BioMérieux), using data as a character dataset via the
categorical distance coefficient and MST (Minimum Spanning Tree) method. A total of 561
MLVA-16 profiles originating from different countries (29 Belgium, 1 Bulgaria, 4 Croatia,
2 Czech Republic, 9 Denmark, 214 France, 36 Germany, 69 Hungary, 10 Italy, 2 Poland,
96 Portugal, 77 Spain, 2 Switzerland, 5 United Kingdom and 5 not reported) and hosts
(8 bovine, 1 caprine, 2 dog, 92 hare, 5 human, 4 ovine, 212 swine and 227 wild boar) available
in MLVA database of our strain collection (https://microbesgenotyping.i2bc.paris-saclay.fr/
(accessed on 1 September 2022)) were used in the analysis (Supplementary Table S1). p
y
pp
y
The whole genome sequencing (WGS) of isolated strains was performed using Illumina
Nextera XT kit (Illumina). The sequencing run was performed on MiSeq equipment
(Illumina) at the Identypath core facility (ANSES, Maisons-Alfort). A de novo assembly
was performed using Spades 3.11 [38]. In total, 37 and 49 contigs larger than 1000 bp were
obtained for isolates 22-3912-5948 (second case isolate) and 20-02069-2828 (first case isolate),
with total genome sizes of 3,303,001 and 3,302,055 bp, respectively. The whole genome
SNP (wgSNP) analysis was performed using BioNumerics version 7.6.3 (BioMérieux) to
trace back the source of infection. The genome of B. melitensis strain 16M was used as the
reference genome for all the analyses. A total of 139 available B. suis genome sequences,
originating from different continents (2 Africa, 9 Asia, 100 Europe, 2 Eurasia, 10 North
America and 2 South America and 14 not reported) and various countries (2 Argentina,
3 Belgium, 8 China, 7 Croatia, 1 Czech Republic, 2 Denmark, 29 France, 5 Germany, 1 India,
18 Italy, 1 Netherlands, 3 Portugal, 2 Russia, 1 Romania, 4 Slovenia, 21 Spain, 3 Switzerland,
2 United Kingdom, 10 United States, 2 Zimbabwe), were used in this study for comparison
purposes (Supplementary Table S1). Chimeric genomes of chromosomes 1 and 2 were
generated to compare complete and draft genomes [39]. 2.2. Phenotypic Identification and Characterisation Isolates were characterized using standard procedures, in accordance with World
Organisation for Animal Health (WOAH) guidelines, in BSL-3 facilities [34]. The strains
were classically biotyped based on colonial morphology, gram staining, CO2 requirement,
H2S production, oxidase and urease activity, growth on dyes (basic fuchsin and thionin),
lysis by phages (Tb, Wb, Iz, R/C) and agglutination with monospecific sera (anti-A, anti-M
and anti-R). 2.1. Bacterial Cultivation and Strains Isolation All samples collected for bacteriological analysis were analyzed using routine bacte-
riological procedures in the local veterinary diagnostic laboratories, in accordance with
the safety regulation in force. In brief, the urine samples collected from the 13-year-old
Border Collie (first reported case) were directly cultivated using Chocolate PolyViteX
(BioMerieux, Craponne, France) and chromogenic CPS media (BioMerieux, Craponne, Pathogens 2023, 12, 792 3 of 14 3 of 14 France). In contrast, tissue biopsies of the scrotal region collected from the 5-year-old Ger-
man Shepherd (second reported case) were cut with a surgical single-use scalpel, and 10 g
was homogenised and diluted in 1/2 to 1/5 ratios in phosphate-buffered saline (PBS) solu-
tion (0.9%). The homogenate was then plated onto Chocolate PolyViteX and Columbia agar
(Sigma-Aldrich, Saint-Quentin-Fallavier, France) with sheep’s blood (5%) media. The re-
sulting colonies of the two cases were purified via replating and analyzed via MALDI-TOF
for first-line identification purposes. The two isolates (20-02069-2828 and 22-03912-5948)
suspected to be Brucella were transferred to the French National Reference Laboratory for
Animal Brucellosis (ANSES, Maisons-Alfort, France) to confirm the Brucella genus and
determine the species, in accordance with the safety regulations in force. 3.1. Presentation of Cases 3.1. Presentation of Cases In April 2020, a 13-year-old male neutered Border Collie presented with thickening of
the bladder, as well as urethral duct and prostate pain related to prostatic palpation, was
brought to a veterinary clinic. Urine was cloudy, and all other clinical signs were associated
with cystitis and/or prostatitis, suggesting an infection of the urogenital tract. The animal
presented no evident signs of brucellosis. This dog was acquired through e-commerce at the
age of 4 years and originated from South West France (Pyrennées Atlantiques department). Since then, the dog lived in the Landes department (also South West) as the only pet in its
household, and was regularly followed by a veterinarian. The dog used to take long walks
in the surrounding forests, where it could have been in contact with other domestic and/or
wild animals and their excrements. In order to exclude bacterial infection and/or prostatic
cancer, the dog was examined in the local veterinary clinic, where routine cytological and
bacteriological analyses of urine were performed. Based on initial anamnesis, brucellosis
did not fit the differential diagnosis. Therefore, no serological analysis was advised to detect
the potential presence of specific antibodies against Brucella sp. since the beginning of the
infection, thus preventing serological follow-up of the animal. The ultrasound analysis did
not show any evidence of tumoral tissues. The cytological results highlighted high levels
of leucocytes, confirming an ongoing infection of the urogenital tract; otherwise, negative
results were obtained from urine bacteriology. Considering the persistence of clinical signs,
a new urine sample was obtained 7 days later, together with ultrasound. The urine culture
identified colonies potentially belonging to Brucella genus with an amount of 104 colony
forming units (CFU) after three days of incubation, confirming the ongoing infection. The
dog was treated with Doxycycline for 20 days, without recovery, which led to decision to
put it to sleep, considering the dog’s age and deteriorated health condition. p
p
g
g
g
Another case of an infected dog occurred in February 2022. The 5-years-old male
German Shepherd presented high fever with an abnormal enlargement of the genital
apparatus, suggesting a bilateral orchitis. The dog was born in the Gard department
(South of France), where it was adopted by a family living in the countryside of Beaucaire,
an area where wild boars are prevalent. 2.3. Molecular Analyses A minimum set of position filters
were applied on the SNP matrix: (i) contiguous SNPs were removed (if found in a 10 bp-
window), (ii) with non-informative SNPs, (ii) a required minimum of 15-fold coverage for
each SNP, and (iv) ambiguous (i.e., non-ACGT bases) and unreliable bases (i.e., Ns) were Pathogens 2023, 12, 792 4 of 14 discarded. The refined SNP matrix was used to generate a maximum parsimony tree using
a maximum parsimony algorithm, allowing phylogenetic analyses. discarded. The refined SNP matrix was used to generate a maximum parsimony tree using
a maximum parsimony algorithm, allowing phylogenetic analyses. 3.1. Presentation of Cases The dog was a household pet and was never
used for hunting or had contact with animal breeding facilities. As first complementary
investigations, a blood analysis was performed, showing leucocytosis and lymphopenia,
indicating an ongoing infection. At the local veterinary diagnostic laboratory, indirect
serology was performed that targeted only rough Brucella sp., with a negative result. Since
the dog had no history of brucellosis, the local veterinary clinic initially decided to treat it
with a combination of amoxicillin, clavulanic acid and a non-steroidal anti-inflammatory
drug. As there was no improvement, a deterioration of the reproductive organs was
instead detected via echography. A neutering with scrotectomy was performed to avoid
any potential risk of spread and to identify the cause of the infection. Following surgery,
treatment was strengthened with the addition of enrofloxacin for 7 days as a second line-
antibiotic, after which dog completely recovered. The retrieved testicles were sent to a
diagnostic laboratory to perform a general bacterial culture, the result of which showed
the presence of colonies suspected to belong to the Brucella genus. After the clinical signs
retracted, no additional sampling was performed to follow up on the presence of specific
BSB2 antibodies, and no shedding of live bacteria occurred. In both presented cases, no livestock farms were present in the proximity of their
residences, ruling out potential contamination from infected domestic animals. 3. Results
3.1. Presentation of Cases 3.1. Presentation of Cases 3.2. Genomic and Bacteriological Identification 3.2. Genomic and Bacteriological Identification To confirm the Brucella genus identification and determine the species, both iso-
lated strains were transferred to the French National Reference Laboratory for Brucellosis Pathogens 2023, 12, 792 5 of 14 5 of 14 (ANSES, Maisons-Alfort). After total DNA extraction, the real-time PCR analysis targeting
IS711, bcsp31 and per Brucella genes was performed. From both strains, all three targeted
genes were amplified, yielding a positive signal. The DNAs were subsequently tested
for rapid identification and differentiation of Brucella genus using High-Resolution Melt-
ing (HRM) PCR assay. Surprisingly, the melting curve profiles matched with classical
BSB2 species, instead of B. canis, which was the first expected diagnosis, regarding the
host species. The standard bacteriological phenotypic identification of isolates (code num-
bers 20-02069-2828 and 22-03912-5948, respectively) confirmed the Brucella genus and the
biotyping traits were consistent with the BSB2 (Table 1). Table 1. Classical phenotypic characterization of two isolated strains (20-02069-2828, 22-03912-5948),
compared to referent BSB2 and B. canis strains. B. canis RM6/66
B. suis bv 2
Thomsen
20-02069-2828
22-03912-5948
Morphology
R
S
S
S
CO2
-
-
-
-
H2S
-
-
-
-
Oxidase
+
+
+
+
Urease
+ a
+ a
+
+
A
-
+
+
+
M
-
-
-
-
R
+
-
-
-
Thionin
+
+
+
+
Fuchsin
(−)
-
-
-
Tb RTD
-
-
-
-
Tb 104 RTD
-
+
+
+
Wb RTD
-
(+) b
+
+
Iz RTD
-
(+) b
PL
+
R/C RTD
+
-
-
-
R/S, colony morphology (rough/smooth), CO2 requirement, H2S production; agglutination with monospecific A,
M and R (rough) antisera; dye (thionin and basic fuchsin) concentration 20 µg/mL in serum dextrose medium
(1/50,000); + = growth or lysis by phages; - = no growth or lysis; PL, partial lysis; (+)/(−), most isolates
positive/negative. a Rapid rate. b Some isolates are not or only partially lysed by phage. Table 1. Classical phenotypic characterization of two isolated strains (20-02069-2828, 22-03912-5948),
compared to referent BSB2 and B. canis strains. R/S, colony morphology (rough/smooth), CO2 requirement, H2S production; agglutination with monospecific A,
M and R (rough) antisera; dye (thionin and basic fuchsin) concentration 20 µg/mL in serum dextrose medium
(1/50,000); + = growth or lysis by phages; - = no growth or lysis; PL, partial lysis; (+)/(−), most isolates
positive/negative. a Rapid rate. b Some isolates are not or only partially lysed by phage. 3.2. Genomic and Bacteriological Identification R/S, colony morphology (rough/smooth), CO2 requirement, H2S production; agglutination with monospecific A,
M and R (rough) antisera; dye (thionin and basic fuchsin) concentration 20 µg/mL in serum dextrose medium
(1/50,000); + = growth or lysis by phages; - = no growth or lysis; PL, partial lysis; (+)/(−), most isolates
positive/negative. a Rapid rate. b Some isolates are not or only partially lysed by phage. In order to identify the strains and find the origins of infection, phylogenetic investiga-
tions were performed. The MLVA analysis confirmed the BSB2 identity of two isolates, and
a strong proximity with strains isolated in French suids and human cases was determined
(Figure 1). The two isolated strains were clustered only with French isolates. Interestingly,
the first strain (20-02069-2828) was grouped into a branch that mainly contains isolates from
pigs and wild boars, while the second isolate (22-03912-5948) is grouped into a subgroup
that mostly includes strains isolated from cattle and humans. The wgSNP analysis was conducted on available strains with B. suis species (140 iso-
lates for the comparison, in addition to single B. canis and B. melitensis isolates used as
an outlier group) from various geographical regions of the world (Africa, Asia, Europe,
North America and South America). A phylogenetic tree using the maximum parsimony
algorithm was generated from the SNP matrix, yielding 8849 SNPs after filtering (Figure 2). The two strains were clustered with all BSB2 isolates, which were available from public
databases. The two analyzed strains are classified within a subclade from France, Spain and
Belgium. Finally, to better correlate the two isolates from dogs compared to BSB2 strains
circulating in French pigs and wildlife, a wgSNP analysis of 33 sequences available in Pathogens 2023, 12, 792
and Belgium
strains circu 6 of 14
SB2
ble our laboratory (returning 9500 SNPs after filtering) highlighted the presence of a subclade
encompassing another 15 strains isolated from pigs, wild boars and hares (Figure 3). ng another 15 strains isolated from pigs, wild boars and hares (Figure our laboratory (returning 9500 SNPs after filtering) highlighted the presence of a subclade
encompassing another 15 strains isolated from pigs, wild boars and hares (Figure 3). g another 15 strains isolated from pigs, wild boars and hares (Figure p
g
p g ,
(
g
)
3). Figure 1. MLVA-16 minimum spanning tree describing relationships of 561 B. suis isolates originat-
ing from different European countries. 3.2. Genomic and Bacteriological Identification Clustering and partitioning were generated with BioNumer-
ics, using data as a character dataset with categorical distance coefficient and Minimum Spanning
Tree method. Circles represent MLVA-16 genotypes, which are colored with respect to host-species,
and size of circle indicates number of strains with that genotype. Two canine isolates presented in
this work were marked with blue arrows. Figure 1. MLVA-16 minimum spanning tree describing relationships of 561 B. suis isolates originating
from different European countries. Clustering and partitioning were generated with BioNumerics,
using data as a character dataset with categorical distance coefficient and Minimum Spanning Tree
method. Circles represent MLVA-16 genotypes, which are colored with respect to host-species, and
size of circle indicates number of strains with that genotype. Two canine isolates presented in this
work were marked with blue arrows. minimum spanning tree describing relationships of 561 B. suis isolates originat-
uropean countries. Clustering and partitioning were generated with BioNumer-
haracter dataset with categorical distance coefficient and Minimum Spanning
represent MLVA-16 genotypes, which are colored with respect to host-species,
dicates number of strains with that genotype. Two canine isolates presented in
ked with blue arrows. Figure 1. MLVA-16 minimum spanning tree describing relationships of 561 B. suis isolates originating
from different European countries. Clustering and partitioning were generated with BioNumerics,
using data as a character dataset with categorical distance coefficient and Minimum Spanning Tree
method. Circles represent MLVA-16 genotypes, which are colored with respect to host-species, and
size of circle indicates number of strains with that genotype. Two canine isolates presented in this
work were marked with blue arrows. 7 of 14
7 of 14 Pathogens 2023, 12, 792
Pathogens 2023, 12, x FO Figure 2. Maximum parsimony tree of B. suis isolates from various geographical regions of world
(Africa, Asia, Europe, North America and South America). Parsimony tree was generated with Bi-
oNumerics using maximum parsimony algorithm from 8849 SNPs identified from complete ge-
nomes of 140 B. suis strains, using reference strain B. melitensis biovar 1 16M and B. canis reference
strains RM6/66, as two outliers. Phylogenetic tree was visualized with EMBL online toll “Interactive
Tree of Life” (iTOL v6) and annotated with four concatenated datasets separated based on under-
scores (strain name, year of isolation, isolation country and host) colored in black and blue for all
i
d
i
l
f
d
i
l
N
d d
k d
i h “NR” All
Figure 2. 3.2. Genomic and Bacteriological Identification Phylogenetic tree was annotated with four
concatenated datasets separated based on underscores (strain name, year of isolation, isolation
country and host). Not reported data are marked with “NR”. B. suis isolates are represented in pink,
with two canine isolates represented by blue arrows. B. canis RM6/66 strain is represented in red,
and referent B. melitensis 16M strain is represented in magenta. 3.2. Genomic and Bacteriological Identification Parsimony tr
was generated with BioNumerics using maximum parsimony algorithm from 9500 SNPs identifi
from complete genomes of 31 French B. suis strains, using reference strain B. melitensis biovar 1 16
and B. canis reference strains RM6/66 as two outliers. Phylogenetic tree was annotated with fo
concatenated datasets separated based on underscores (strain name, year of isolation, isolati
country and host). Not reported data are marked with “NR”. B. suis isolates are represented in pin
with two canine isolates represented by blue arrows. B. canis RM6/66 strain is represented in re
and referent B. melitensis 16M strain is represented in magenta. i
i
Figure 3. Maximum parsimony tree of B. suis biovar 2 isolates circulating in France. Parsimony tre
was generated with BioNumerics using maximum parsimony algorithm from 9500 SNPs identified
from complete genomes of 31 French B. suis strains, using reference strain B. melitensis biovar 1 16 M
and B. canis reference strains RM6/66 as two outliers. Phylogenetic tree was annotated with fou
concatenated datasets separated based on underscores (strain name, year of isolation, isolation
country and host). Not reported data are marked with “NR”. B. suis isolates are represented in pink
with two canine isolates represented by blue arrows. B. canis RM6/66 strain is represented in red
and referent B. melitensis 16M strain is represented in magenta. Figure 3. Maximum parsimony tree of B. suis biovar 2 isolates circulating in France. Parsimony tree
was generated with BioNumerics using maximum parsimony algorithm from 9500 SNPs identified
from complete genomes of 31 French B. suis strains, using reference strain B. melitensis biovar 1 16M
and B. canis reference strains RM6/66 as two outliers. Phylogenetic tree was annotated with four
concatenated datasets separated based on underscores (strain name, year of isolation, isolation
country and host). Not reported data are marked with “NR”. B. suis isolates are represented in pink,
with two canine isolates represented by blue arrows. B. canis RM6/66 strain is represented in red,
and referent B. melitensis 16M strain is represented in magenta. Di
i
Figure 3. Maximum parsimony tree of B. suis biovar 2 isolates circulating in France. Parsimony tree
was generated with BioNumerics using maximum parsimony algorithm from 9500 SNPs identified
from complete genomes of 31 French B. suis strains, using reference strain B. melitensis biovar 1 16 M
and B. canis reference strains RM6/66 as two outliers. 3.2. Genomic and Bacteriological Identification Maximum parsimony tree of B. suis isolates from various geographical regions of world
(Africa, Asia, Europe, North America and South America). Parsimony tree was generated with BioN
umerics using maximum parsimony algorithm from 8849 SNPs identified from complete genome
of 140 B. suis strains, using reference strain B. melitensis biovar 1 16M and B. canis reference strain
RM6/66, as two outliers. Phylogenetic tree was visualized with EMBL online toll “Interactive Tree
of Life” (iTOL v6) and annotated with four concatenated datasets separated based on underscore Figure 2. Maximum parsimony tree of B. suis isolates from various geographical regions of world
(Africa, Asia, Europe, North America and South America). Parsimony tree was generated with Bi-
oNumerics using maximum parsimony algorithm from 8849 SNPs identified from complete ge-
nomes of 140 B. suis strains, using reference strain B. melitensis biovar 1 16M and B. canis reference
strains RM6/66, as two outliers. Phylogenetic tree was visualized with EMBL online toll “Interactive
Tree of Life” (iTOL v6) and annotated with four concatenated datasets separated based on under-
scores (strain name, year of isolation, isolation country and host) colored in black and blue for all
species and two isolates from dogs, respectively. Not reported data are marked with “NR”. All spe-
cies are color-coded for branches: pink for B. suis, red for B. canis RM6/66 and magenta for B. melitensis 16M. Figure 2. Maximum parsimony tree of B. suis isolates from various geographical regions of world
(Africa, Asia, Europe, North America and South America). Parsimony tree was generated with BioN-
umerics using maximum parsimony algorithm from 8849 SNPs identified from complete genomes
of 140 B. suis strains, using reference strain B. melitensis biovar 1 16M and B. canis reference strains
RM6/66, as two outliers. Phylogenetic tree was visualized with EMBL online toll “Interactive Tree
of Life” (iTOL v6) and annotated with four concatenated datasets separated based on underscores
(strain name, year of isolation, isolation country and host) colored in black and blue for all species
and two isolates from dogs, respectively. Not reported data are marked with “NR”. All species are
color-coded for branches: pink for B. suis, red for B. canis RM6/66 and magenta for B. melitensis 16 M. 8 of 14
8 of 14 Pathogens 2023, 12, 792
Pathogens 2023, 12, x FO Figure 3. Maximum parsimony tree of B. suis biovar 2 isolates circulating in France. 4. Discussion
Brucellos
4. Discussion Reported clinical signs (prostatitis, orchitis) were
different between the two cases, though they were compatible with brucellosis infection. First-line treatments related to diagnostic hypotheses were not oriented against Brucella
infections, leading to a delayed diagnosis of brucellosis and misuse of antibiotics. The
lack of brucellosis awareness can probably be explained by the fact that France, like many
European countries, is free of brucellosis in ruminants and is not considered as an endemic
location for canine brucellosis. Therefore, vets do not consider Brucella spp. in differential
diagnosis of clinically ill dogs. The preferred hosts of BSB2 strains are domestic and wild pigs, as well as hares
and other wildlife; however, it had never been isolated from either domestic nor wild
canids, compared to serotypes 1 and 5 [21,23,28,30,32]. Continuous monitoring and genetic
characterisation of newly isolated strains in France shows the emergence of two distinct
clades with the preferences either for pigs or hares. At the same time, canine brucellosis in
Europe was only reported sporadically, being mainly related to B. melitensis, B. abortus and,
more recently, B. canis. Canine brucellosis caused by B. canis causes miscarriages, infertility,
orchitis, epididymitis, endocarditis, uveitis and discospondylitis. Interestingly, B. canis
is yet to be isolated from pigs, wild boar, hares or other wildlife; therefore, dogs can be
considered as the preference host species. At the same time, in the last decade, there was an
increase in the detection of B. suis in dogs, particularly in Australia and the United States. A recent seroprevalence study, for example, found that nearly one in 10 (9.3%) dogs in
eastern Australia, being exposed to feral pigs or their meat products, are seropositive to
smooth Brucella spp. (including B. suis) [44–46]. In Australia, B. suis biovar 1 is responsible
for canine infections. Humans and dogs being in contact with body fluids and/or tissues
from infected feral pigs, through hunting, butchering or consumption of uncooked pork, is
considered as the most common risk factor for infection [45]. Surprisingly, the two described
dogs were found to be infected with BSB2, a species usually isolated from pigs, hares and
wild boars [34]. The BSB2 infection of dogs was not previously documented. In France,
BSB2 is mainly encountered in hares and wild boars (Sus scrofa), and is known to have very
low pathogenicity to humans, with only seven human cases confirmed in a period spanning
of 12 years (between 2004 and 2016). 4. Discussion
Brucellos
4. Discussion Brucellosis is a highly contagious bacterial zoonosis mainly caused by ingestion of
contaminated unpasteurized milk, dairy produce or insufficiently thermically treated
meat or close contact with secretions of infected animals (semen, aborted or parturition
tissues and fluids). Etiological agents of brucellosis are bacteria from Brucella genus, with
12 species currently described. Considering that the whole genus shares ~97% genetic sim-
ilarity [40], it is unclear how host preferences are developed. However, the plasticity of
the genus continues to be discovered. In 2017, for example, European amphibians were
found to be infected by one such recently identified species (B. microti) [41,42], which was
already reported in rodents, foxes and wild boars, thus confirming the broad host range
of emerging Brucella sp [43]. However, no Brucella sp. are characterised by the develop-
ment of specific clinical symptoms, either in humans or in animal hosts. At the same time,
the emergence of new reservoirs for Brucella sp could be linked to a new consumption
Brucellosis is a highly contagious bacterial zoonosis mainly caused by ingestion of
contaminated unpasteurized milk, dairy produce or insufficiently thermically treated meat
or close contact with secretions of infected animals (semen, aborted or parturition tissues
and fluids). Etiological agents of brucellosis are bacteria from Brucella genus, with 12 species
currently described. Considering that the whole genus shares ~97% genetic similarity [40],
it is unclear how host preferences are developed. However, the plasticity of the genus
continues to be discovered. In 2017, for example, European amphibians were found to
be infected by one such recently identified species (B. microti) [41,42], which was already
reported in rodents, foxes and wild boars, thus confirming the broad host range of emerging
Brucella sp. [43]. However, no Brucella sp. are characterised by the development of specific
clinical symptoms, either in humans or in animal hosts. At the same time, the emergence of Pathogens 2023, 12, 792 9 of 14 new reservoirs for Brucella sp. could be linked to a new consumption pattern, especially
practices of organic and raw products, higher globalisation of food or the creation of
produce markets with consecutively augmented importation rates, as well as increased
animal movement. Therefore, the host range of the whole genus continues to be discovered. In France, the 13- and 5-years old dogs were confirmed as brucellosis infected in
2020 and 2022, respectively, following different persistent symptoms and the detection of
positive bacterial culture for Brucella. 4. Discussion
Brucellos
4. Discussion All patients had direct contact with wild boars while
hunting or preparing wild boar meat [12]. The absence of BSB2-reported cases in dogs may
be related to the low level of pathogenicity, as well as potential asymptomatic or latent
infections in dogs. It may be relevant to highlight that only the German Shepherd dog
was tested for B. canis (rough species) antibodies, excluding the possibility of employing
serological tests targeting smooth Brucella species. As there is no standardised brucellosis
testing scheme, no financial compensation and no compulsory policy measures for dogs in
France exist, while systematic serological testing through the course of clinical symptoms
cannot be imposed, especially when an isolated strain was already identified. This issue
highlights the difficulty of collecting repeated and standardized samples in dogs. Finding a cure for Brucella infected dogs remains a major issue due to the lack of
an effective treatment [33]. Current options include neutering together with a long-term
treatment with associations of antibiotics, including molecules that present potential toxic
side-effects [47]. Although no guidelines are available for the treatment of B. suis infection in
dogs, combination therapy with rifampicin and doxycycline would appear to be a possible
effective therapy [19,46]. However, the use of rifampicin in dogs can instigate severe
side-effects, such as vomiting, anorexia, lethargy and elevated levels of serum alanine
aminotransferase (ALT; important transferase for liver functions) [48]. At the same time,
rifampicin is guarded for treatment of human brucellosis and its use in animals may cause Pathogens 2023, 12, 792 10 of 14 10 of 14 the development of antibiotic-resistant B. suis strains, threatening public health protection. Moreover, recurrence and/or persistence of the disease cannot be excluded, which leads us
to consider a humane end to suffering as an alternative decision. Regarding the 13-year-old
dog, neutering was performed before appearance of symptoms. It may be hypothesised
that the dog presented a latent infection that reappeared with advanced age and/or a
depressed immune system. Antibiotics were not sufficient to restore the overall health
status of the dog. g
The broad-spectrum wgSNP analysis, despite the large number of sequences, made
it possible to locate these strains in a subclade including seven other French isolates
(Figure 2), which were essentially isolated from wild animals (hares and wild boars but
also from domestic pigs) between 1997 and 2022. 4. Discussion
Brucellos
4. Discussion This number increased to eight isolates
through targeted analysis of BSB2 circulating in French territory (Figure 3), with cases
mainly derived (seven out of eight strains) from infected pig herds isolated in France from
1996 to 2022. Although the 15 BSB2 French strains clustering with the two dog isolates
(Figure 3) could not be related to a specific region, three distinct regions can be identified
(the northwest, southwest and center of France; Figure 4). Interestingly, strains originating
from the northwest and southwest regions of France were mainly isolated from outdoor
breeding pigs, while isolates from central parts of country were found in wildlife (wild
boars and hares) and pigs. The southwest region of France regroups many outdoor pig
farms with local breeds, implying that pigs can be in direct contacts with wild animals. Based on the history of the strain isolated from the dog in this region (20-02069-2828),
no livestock farms were found in the proximity of the dog’s residence, which reaffirms
the wildlife origin hypothesis. The history of the second dog (22-3912-5948) is also not
correlated with proximity to any type of pig breeding facility or direct contacts with wildlife
or hunting. Assuming that porcine brucellosis outbreaks in France are related to brucellosis
in wildlife, the main hypothesis seems to be an infection through contacts with body fluids
and/or tissues from sylvatic reservoirs, such as hares and wild boars. Contacts may include
sniffing, licking or eating these tissues. Currently, BSB2 is not considered as a pathogenic
strain of Brucella and has never been isolated from dogs. In particular, in the case of the
13-year-old dog, Brucella infection could have occurred a long time before appearance of
symptoms, and the clinical symptoms could emerge once animal started to suffer from other
comorbidities. This possible latent infection complicates epidemiological investigations
and the search for sources of contamination. Furthermore, the inability to determine with
certainty the cause and time of infection may explain how this case occurred in an area
with no previous cases of BSB2 in either pigs or wild animals. Human cases of B. suis biovar 1, 3 and 5 have been reported all over the world [49–52]. So far, BSB2 strains are shown to be of low pathogenicity for humans, with only two cases
reported in the literature until 2017, when in France, seven new cases were identified
through national mandatory notification for brucellosis [12]. 4. Discussion
Brucellos
4. Discussion All patients were in contact
with wild boars and their meat, and five of them also suffered from chronic medical
conditions that could exacerbate disease or help B. suis infections. From all seven described
cases, BSB2 strains were isolated. These cases also show that even with Brucellae of low
pathogenic potential for humans, risks to public health augment with the increased numbers
of infected animals and host species. These risks are even higher when we take into
account that dogs are present in both rural and urban environments, their numbers are
rising constantly, and they are in close contact with human populations of all ages and
immune categories. This study highlights both the role of wildlife in transmission of diseases such as
brucellosis and the need to establish surveillance plans that include wildlife and envi-
ronment, in order to better characterize the epidemiological situation and to limit risk of
spill overs to domestic animals. Adopting a “One Health” approach would improve early
detection of new outbreaks and minimize the risks of transmission to humans. This study
also underlines the relevance of serological tests for classical smooth Brucella species in Pathogens 2023, 12, 792 11 of 14 11 of 14 diagnoses of cases in dogs, since canine brucellosis is frequently described as concerning
only rough B. canis strains. VIEW
11 of 14 diagnoses of cases in dogs, since canine brucellosis is frequently described as concerning
only rough B. canis strains. VIEW
11 of 14 diagnoses of cases in dogs, since canine brucellosis is frequently described as concerning
only rough B. canis strains. VIEW
11 of 14 only rough B. canis strains. Figure 4. Spatial representation of French subclade related to two dog isolates. Previous isolations
of BSB2 from wildlife (wild boars or hare) and pigs are presented with green and blue stars, respec-
tively, while two canine isolates are presented with red stars. Figure 4. Spatial representation of French subclade related to two dog isolates. Previous isolations of
BSB2 from wildlife (wild boars or hare) and pigs are presented with green and blue stars, respectively,
while two canine isolates are presented with red stars. Figure 4. Spatial representation of French subclade related to two dog isolates. Previous isolations
of BSB2 from wildlife (wild boars or hare) and pigs are presented with green and blue stars, respec-
tively, while two canine isolates are presented with red stars. Figure 4. 4. Discussion
Brucellos
4. Discussion Spatial representation of French subclade related to two dog isolates. Previous isolations of
BSB2 from wildlife (wild boars or hare) and pigs are presented with green and blue stars, respectively,
while two canine isolates are presented with red stars. ,
p
[
52]. So far, BSB2 strains are shown to be of low pathogenicity for humans, with only two
cases reported in the literature until 2017, when in France, seven new cases were identified
through national mandatory notification for brucellosis [12]. All patients were in contact
Supplementary Materials:
The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/pathogens12060792/s1, Table S1: List of genomes se-
quences and MLVA-16 profiles used in this study. with wild boars and their meat, and five of them also suffered from chronic medical con-
ditions that could exacerbate disease or help B. suis infections. From all seven described
cases, BSB2 strains were isolated. These cases also show that even with Brucellae of low
pathogenic potential for humans, risks to public health augment with the increased num-
bers of infected animals and host species. These risks are even higher when we take into
Author Contributions: Conceptualization, G.G., L.F., A.F.V., V.D. and C.P.; methodology, G.G., L.F.,
A.F.V., V.D. and C.P.; formal analysis, L.P., F.P.-B., G.G. and L.F.; investigation, G.G., L.F., B.T., H.S. and
C.P.; data curation, G.G. and L.F.; writing—original draft preparation, G.G. and L.F.; writing—review
and editing, G.G., V.D., A.F.V., C.P. and L.F.; supervision and grant submission, C.P., L.F., G.G. and
V.D. All authors have read and agreed to the published version of the manuscript. account that dogs are present in both rural and urban environments, their numbers are
rising constantly, and they are in close contact with human populations of all ages and
immune categories. Funding: This work was supported by the European Union Reference Laboratory for Brucellosis
(grant agreement no. SI2.801943) and the One Health EJP project (JRP-17–IDEMBRU: Identification of
emerging Brucella species: new threats for human and animals; grant agreement no. 773830). Pathogens 2023, 12, 792 12 of 14 12 of 14 Institutional Review Board Statement: The Statement of Ethics does not apply to this set of samples
taken for diagnostic purposes. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data generated during this study were deposited in the European
Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB61443 (https://www.ebi.ac. uk/ena/browser/view/PRJEB61443 (accessed on 29 May 2023)). 1.
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Industrial Activity Land Suitability Assessment Using Delphi and AHP to Control Land Consumption¨: The Case Study of Bordj Bouarreridj, Algeria
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Industrial Activity Land Suitability Assessment Using
Delphi and AHP to Control Land Consumption
The Case Study of Bordj Bouarreridj, Algeria Fateh Toufik Benaissa
Department of Urban Engineering
Institute of Urban Techniques Management
University of Msila
Msila, Algeria
fatehtoufik.benaissa@univ-msila.dz Boudjemaa Khalfallah
Department of Urban Engineering
Institute of Urban Techniques Management
University of Msila
Msila, Algeria
boudjemaa.khalfalllah@univ-msila.dz arrange land usage of the studied areas. Studying and
identifying land-use needs for each land-use type is critical and
fundamental for evaluating land use suitability for each land
mapping unit [3]. Land use suitability analyses are of
considerable use in the planning of mega-cities [4]. As a result,
it is critical to use a technique that allows weight estimation. One such strategy is the Analytical Hierarchy Process (AHP),
which is a multi-criteria decision-making method. Abstract-Studies and research on land suitability for the
localization of economic and industrial activities in cities are
attracting academics and local policies concerned with urban
planning and organization in order to make urban areas more
accommodating. The current paper aims to demonstrate the
ability of visualization and simulation techniques to assist
planners in preserving urban real estate by combining the Delphi
method with one of the multi-criteria hierarchical analysis
decision-making techniques. The city of Bordj Bouarreridj in
Algeria was chosen as the case study and, with the participation
of experts, a set of criteria were adopted to determine land
suitability. Three criteria were selected in the first phase with the
use of the Delphi method. In the second phase, we applied the
Analytical Hierarchy Process (AH, a hierarchical analysis
method. During the third stage, the obtained results were
translated into the GIS program to derive the spatial suitability
map. The following are among the most important achieved
results: 13.38% (521.793 km2) of the area was regarded to be of
high suitability, 67.54% (2,634.758 km2), achieved moderate
suitability, 12.94% (504,794 km2) attained low suitability, and
6.14% (239,685 km2) were found to be not suitable at all. The author in [5] used several variables that were related to
the environmental aspect. He focused his studies on
environmental indicators. In this study, the answers were
obtained by experts and institutions, and the obtained data were
analyzed with the AHP method, after which a map of spatial
suitability was derived by the Geographic Information Systems
(GIS). Authors in [6] used AHP to determine the GIS weights
based on 12 variables. Their study focused on social, economic,
and environmental indicators. Vol. 11, No. 5, 2021, 7738-7744 Vol. 11, No. 5, 2021, 7738-7744 Engineering, Technology & Applied Science Research 7738 I.
INTRODUCTION Both private and public sectors contribute to the increment
of the area of cities. In Algeria, we find that most of the
construction of industrial zones is usually at the expense of
agricultural lands, which affects the land uses of cities by
covering the increasing housing demand [1]. The economic
transformations of cities have created factors, represented in
factories and workshops, that contribute to the emergence of
management and organizational problems of the territory,
especially about the allocation of lands and their suitability. The planners confront a dual problem in this situation. They
must create initiatives and programs that maintain ecological
balance while promoting economic growth, while they must act
as mediators, attempting to prevent conflict and reduce
objections. Land use planning is a means of optimal allocation
of land resources [2]. It is frequently utilized in the spatial
allocation and characteristics of land size to scheme and This paper presents the results obtained by integrating the
Delphi approach, AHP, and GIS in analyzing the relevance of
land use to economic activity to control the land consumption
in urban areas. The case study area was the city of Bordj
Bouarerridj. Benaissa & Khalfallah: Industrial Activity Land Suitability Assessment Using Delphi and AHP … Corresponding author: Fateh Toufik Benaissa Industrial Activity Land Suitability Assessment Using
Delphi and AHP to Control Land Consumption
The Case Study of Bordj Bouarreridj, Algeria Such methods have the largest
number of variables but the participation of experts and
sometimes residents is often necessary. Authors in [7] used
GIS and AHP to select the best sites for urban growth in
Seremban,
Malaysia. Many
social,
economic,
and
environmental factors were considered to create a final land
suitability map, but the notable thing in this study is that the
researchers relied on the selection of factors and variables on
local literature and general observations only without relying
on experts or residents. Keywords-industrial
activity;
Bordj
Bouarerridj;
land
suitability; control consumption; Delphi method; AHP A. The Study Area Bordj Bouarerridj is an Algerian city in the high eastern
highlands. It is located in the Algerian east in the middle of two
Atlas Mountains series, the Beban mountain range and the Al-
Hodna mountain range with a population density of 186.4
inhabitants/hectare in 2014, according to the NSB (National
Statistics Bureau). Its total area is estimated at 3901.02km2, i.e. www.etasr.com www.etasr.com Vol. 11, No. 5, 2021, 7738-7744 Engineering, Technology & Applied Science Research 7739 the 1/600 of the total area of Algeria. The forms of land usage
vary, the land appropriateness is broad, and the degree of use is
high (Figure 1). 1) Suitability Factors for Land Use for Industrial Activities The criteria defined by the Delphi method to study the
suitability of lands for economic and industrial activities, and
their weights were appreciated by using the expert choice v11
program, which is an application for multi-criteria analysis
through AHP. After collecting the geographical data, layers of
determinant criteria, and sub-criteria were set in the open-
source QGIS program. The results are displayed graphically in
Figures 2-9. Fig. 2. Geological nature of the study area. Prepared by the researchers on
the QGIS program. Fig. 4. Principal roads of the study area. Prepared by the researchers on
QGIS. 2) Nature and Environment For this factor, forest areas, hydrographical networks, and
watershed basins have been identified, through the Bordj
Bouarerridj city Reconstruction Plan. The suitability sub-
factors were as follows (Figure 2): Forest areas: (0-5 Km), (5-9
Km), (9-13 Km), and (> 13 Km). Hydrographical network: (0-5
Km), (5-10 Km), (10-15 Km), and (> 15 Km). Watershed
basins: (0-5 Km), (5-10 Km), (10-15 Km), and (>15 Km). Fig. 1. The study area. Prepared by the researchers on the QGIS program,
2021. Fig. 3. Railways of the study area. Prepared by the researchers on QGIS. Fig. 4. Principal roads of the study area. Prepared by the researchers on
QGIS. Fig. 3. Railways of the study area. Prepared by the researchers on QGIS. Fig. 3. Railways of the study area. Prepared by the researchers on QGIS. Fig. 3. Railways of the study area. Prepared by the researchers on QGIS. Fig. 4. Principal roads of the study area. Prepared by the researchers on
QGIS. Fig. 1. The study area. Prepared by the researchers on the QGIS program,
2021. 3) Security and Protection Prepared by the researchers on QGIS. Fig. 9. Slopes of the study area. Prepared by the researchers on QGIS. Fig. 7. Urban areas of the study area. Prepared by the researchers on
QGIS. C. Methods Industrial site selection is a spatial decision problem. Such
problems typically involve a large set of feasible alternatives
[8]. Land suitability for industrial activities is a somewhat
complicated process considering the data and characteristics
that distinguish them. Therefore many possible alternatives
must take as well as appropriate multi-faceted and often
conflicting standards, such as economic, environmental,
climate, social, etc. The Delphi method is often applied to
define the evaluation criteria as a way of structuring the method
of collective communication to affect the complex problem of
extracting and studying the influential factors to seek out the
most appropriate sites. The suitability of lands for industrial
activities in urban areas is determined by calculating the degree
of importance of the percentage of significant value [9]. After,
we used the AHP method which is used to solve complex
decision problems [10]. That decision-making tool depends on
reliable measurement and thorough evaluation to present
suitable lands for economic activities [11]. It also serves as a
helpful tool for capturing both subjective and objective
assessment measures for checking the consistency of the
evaluations and thus reducing bias in decision making [12]. This study is based on the AHP and through interactive
computer programs (expert choice v11) [13]. The computer Fig. 7. Urban areas of the study area. Prepared by the researchers on
QGIS. 3) Security and Protection The relevant information was collected from the planning
and reconstruction plans of the Bordj Bouarerridj state. Layers
were determined in the ARC GIS program in urban tissues,
archaeological areas, slopes, and geological sites according to
the opinion of experts and technicians. The range of suitability
for the distance from residential areas was as follows: (< 4
Km), (4-10 Km), (10-15 Km), and (> 15 Km). For slope, the
range of suitability was as follows: (< 4%), (4%-8%), (8%-
12%), and (> 12%). The geological nature was determined
according to the permeability and type of soil. Fig. 2. Geological nature of the study area. Prepared by the researchers on
the QGIS program. Benaissa & Khalfallah: Industrial Activity Land Suitability Assessment Using Delphi and AHP … www.etasr.com Vol. 11, No. 5, 2021, 7738-7744 Engineering, Technology & Applied Science Research 7740 Fig. 5. Watershed basins of the study area. Prepared by the researchers on
QGIS. Fig. 6. Forests on the study area. Prepared by the researchers on QGIS. Fig. 8. Water ways of the study area. Prepared by the researchers on
QGIS. Fig. 9. Slopes of the study area. Prepared by the researchers on QGIS. C. Methods
Industrial site selection is a spatial decision problem. Such
problems typically involve a large set of feasible alternatives
[8]. Land suitability for industrial activities is a somewhat
complicated process considering the data and characteristics
that distinguish them. Therefore many possible alternatives
must take as well as appropriate multi-faceted and often
conflicting standards, such as economic, environmental,
climate, social, etc. The Delphi method is often applied to
define the evaluation criteria as a way of structuring the method
of collective communication to affect the complex problem of Fig. 5. Watershed basins of the study area. Prepared by the researchers on
QGIS. Fig. 8. Water ways of the study area. Prepared by the researchers on
QGIS. Fig. 5. Watershed basins of the study area. Prepared by the researchers on
QGIS. Fig. 8. Water ways of the study area. Prepared by the researchers on
QGIS. Fig. 8. Water ways of the study area. Prepared by the researchers on
QGIS. Fig. 6. Forests on the study area. Prepared by the researchers on QGIS. Fig. 9. Slopes of the study area. Prepared by the researchers on QGIS. Fig. 6. Forests on the study area. Prepared by the researchers on QGIS. Fig. 6. Forests on the study area. www.etasr.com Benaissa & Khalfallah: Industrial Activity Land Suitability Assessment Using Delphi and AHP … 4) Economic Determinant For this factor, we considered all the main roads and
structured railway lines of the case study. The data were
collected from the planning and reconstruction plans of Bordj
Bouarerridj in 2018. The range of suitability was determined by
the experts as follows: (0.5-4 Km), (4-10 Km), and (> 10 Km)
(Figures 3, 4). www.etasr.com Vol. 11, No. 5, 2021, 7738-7744 Engineering, Technology & Applied Science Research 7741 uses a hierarchical framework of criteria and comparisons
between them to establish the criterion weight. The additive
transformation function of the weighted summation technique
is applied to get a final score for each choice. Expert Choice
Network accepts no restriction on the size of the decision
issued in this scenario. The DM's preferences on criteria are
represented by cardinal weights [14]. measurement of evaluation inconsistency. This measurement is
among the most essential parts of the priority determination
process. The higher the consistency ratio, the assessment result
becomes more inconsistent [24]. After the hierarchy creation,
the relative importance of all resolution elements is captured
and detected through binary comparisons. The Expert Choice is
a decision-oriented program that provides support to decision-
makers by organizing information with regarding to the
complexity of a multi-solution problem into a hierarchical
model consisting of objective, potential scenarios, agents, and
alternatives. The priorities are determined along with the
comparison matrix. We begin with a matrix that is consistent
and has known priority pi. pi/pj is used to compare alternatives i
and j which is multiplied with the priority vector p results [25]: Fig. 10. The analysis used for this research.
⁄
⁄
⋯
⁄
⁄
⁄
⋯
⁄
⋮
⋮
⋱
⋮
⁄
⁄
⋯
⁄
⋮
⋮
(1)
Or grouped:
⁄
⁄
⋯
⁄
⁄
⁄
⋯
⁄
⋮
⋮
⋱
⋮
⁄
⁄
⋯
⁄
⋮
⋮
(1)
Or grouped: (2) where is the prioritization vector, n the matrix dimension,
and A the matrix of comparison. 4) Economic Determinant The author in [13] proposed a Consistency Index (CI)
because priorities are only relevant if they are generated from
coherent or close-to-consistent matrices, which is related to the
eigenvalue method [26]: Fig. 10. The analysis used for this research. F. Geographic Information System (GIS) GIS is a process of mapping and integrating computer-
founded information with the ability of data layer management
to make appropriate decisions by combining geographical
information layers [29]. Data combined with an application of
GIS provide alternative, flexible and scalable means [30]. The
availability of digital data archives since 1987 enables the
quantitative and qualitative assessment of land use patterns
[31]. Accordingly, several studies have been conducted
emphasizing the adoption of Delphi, AHP, or a combination of
these methods, with GIS for modeling and analyzing spatial TABLE I.
RANDOM CONSISTENCY INDEX.
n
1
2
3
4
5
6
7
8
9
10
RI
0
0
0.58
0.90
1.12
1.24
1.32
1.41
1.45
1.49 If CR ≥ 10% the data are considered inconsistent. If
CR < 10% we have data consistency and the matrix is
considered as having an acceptable consistency [25]. www.etasr.com Benaissa & Khalfallah: Industrial Activity Land Suitability Assessment Using Delphi and AHP … !"#! $ %&! ∑(".*" (4) The Consistency Ratio (CR) is calculated to measure the
consistency of the solution [27], and it is obtained by (5). The
Random Consistency Index (RI) is shown in Table I [28]. CR
,-
.- (5) D. Delphi Method CI
(3)
!"#! $ %&! ∑(".*" (4 CI
(3) The Delphi method is a consensus method that aims to find
general agreement among an expert panel on a specific
research topic [15]. It is considered a way for organizing the
group communication process to facilitate group issue solving
and model building. Its principal area of application has
remained that of technical forecasting [16]. In this research
paper, we relied on 15 experts of various disciplines, namely
geology, urbanization, economics, and law, who are familiar
with the research topic and close to the decision-making
authorities. Our questions were presented to them in four
stages. After the first stage, we sent an anonymous summary of
the resume of the experts' outlook of each previous stage and
its reasoning [17]. This gradually led to the convergence of
viewpoints and after the questionnaire's results were obtained. Through the questions posed to the experts and based on their
opinions, criteria of approval for each standard set of sub-
criteria were set. After this, the AHP series pyramid was
analyzed to determine the weights of the main indicators and
the sub-criterions in Expert Choice v11 software. !"#! $ %&! ∑(".*" (4) III.
RESULTS AND DISCUSSION This research relied on a MCDA using the AHP analysis to
study the suitability of land use for economic activity in order
to control the consumption of urban land involving the
opinions of experts in the field. Eight criteria were used to
quantify the suitability for economic and industrial land use,
namely urban areas, slopes, forest areas, hydrographical
network, watershed basins, geology, roads, and railways. A
comparison matrix was implemented between the main factors
and subfactors and paired-wise comparison was conducted
between the sub-criteria of the same level [34]. Afterwards, the
weight of each determinant was extracted (Figure 11). Fig. 13. Priorities with respect to goal. Fig. 13. Priorities with respect to goal. Based on the previous hierarchical analysis of the suitability
of land use for economic and industrial activity, the role of GIS
comes in the form of a model that summarizes all stages of
work in the ARC GIS. We compiled all the pre-defined
standard maps in GIS via Raster calculators with each standard
multiplied by the weight generated by the AHP and Expert
Choice to produce a suitable land use map. (Figure 14). Fig. 11. Results of AHP among main and sub criteria. Fig. 11. Results of AHP among main and sub criteria. Fig. 12. Performance sensitivity for nodes. ,00
,10
,20
,30
,40
,50
,60
,70
,80
,90
,00
,10
,20
,30
,40
,50
,60
,70
Obj%
Alt%
CL ha
CL pt
5 km - 9 km
> 10 %
9 km - 13 km
LP li
0 km - 5 km
CL haye
8 % - 10 %
5 km - 10 km
2 km - 4 km
> 13 km
> 12 km
4 % - 8 %
7 km - 12 km
4 km - 10 km
0 % - 4 %
3 km - 7 km
10 km - 15 km
0 km - 3 km
> 15 km
Watershed ba
slope (L: ,1
urban area (
forest (L: ,
roads (L: ,2
railway (L:
geologie (L:
waterway (L:
OVERALL Fig. 14. The produced land suitability map for for economic and industrial
activity regarding the Bordj Bouarerridj area. Prepared by the researchers on
the QGIS program. Fig. 11. Results of AHP among main and sub criteria. Fig. 11. Results of AHP among main and sub criteria. Fig. 12. Performance sensitivity for nodes. E. Hierarchical Analysis Process AHP is a Multiple-Criteria Decision-Analysis (MCDA)
technique used in solving various decision-making problems
[18-20]. It is suitable for the evaluation of complex multi-
attribute alternatives [21], because it is considered one of the
most effective and easy procedures [22]. The AHP model has
been applied to determine weights and ranks of all the
subcategories [23]. The AHP methodology may tolerate
inconsistency according to pair-wise comparison by given a Vol. 11, No. 5, 2021, 7738-7744 Engineering, Technology & Applied Science Research 7742 Fig. 13. Priorities with respect to goal. suitability [32]. It assists decision-makers to assign priority
weights to decision criteria, evaluate the suitable alternatives,
and visualize the results [33]. Benaissa & Khalfallah: Industrial Activity Land Suitability Assessment Using Delphi and AHP … www.etasr.com III.
RESULTS AND DISCUSSION ,00
,10
,20
,30
,40
,50
,60
,70
,80
,90
,00
,10
,20
,30
,40
,50
,60
,70
Obj%
Alt%
CL ha
CL pt
5 km - 9 km
> 10 %
9 km - 13 km
LP li
0 km - 5 km
CL haye
8 % - 10 %
5 km - 10 km
2 km - 4 km
> 13 km
> 12 km
4 % - 8 %
7 km - 12 km
4 km - 10 km
0 % - 4 %
3 km - 7 km
10 km - 15 km
0 km - 3 km
> 15 km
Watershed ba
slope (L: ,1
urban area (
forest (L: ,
roads (L: ,2
railway (L:
geologie (L:
waterway (L:
OVERALL Fig. 14. The produced land suitability map for for economic and industrial
activity regarding the Bordj Bouarerridj area. Prepared by the researchers on
the QGIS program. The final map gross weight was calculated by raster
calculation using (6): SL 1ROw ∗ROr6 7 1RWw ∗RWr6 7 1Gw ∗Gr6 7
1WWw ∗WWr6 7 1WAw ∗WAr6 7 1Sw ∗Sr6 7 1Fw ∗Fr6 7
1Uw ∗Ur6 (6) where SL is the suitability of land use for economic and
industrial activity, w is the weight of each criterion, r is the
classification of each criterion, RO stands for road, RW for
railway, G for geology, WW for waterways, WA for water, S
for slope, F for forest, and U for urban areas. Fig. 12. Performance sensitivity for nodes. The proportion of consistency was less than 10%, which
means that the distribution between the factors was acceptable. Through hierarchical analysis the proportion of land-use
suitability for economic and industrial activity in the city of
Bordj Bouarerridj was: TABLE II. AHP KEY CRITERIA RATING
criteria
weight
rating
Road
0.2265
2
Railway
0.1531
3
Geology
0.237
8
Hydrographical network
0.571
6
Watershed basins
0.0726
5
Slope
0.1015
4
Forest
0.0318
7
Urban area
0.3338
1 • 13.38% of the total area with a total surface area of
521.793km2 achieved high suitability. • 67.54% of the total area with a total surface area of
504.794km2 achieved moderate suitability. • 12.94% of the total area with a total surface area of
2634.758km2 achieved low suitability. www.etasr.com Vol. 11, No. 5, 2021, 7738-7744 7743 Engineering, Technology & Applied Science Research [5]
T. Everest, A. Sungur, and H. IV.
CONCLUSIONS In this study, we utilized Delphi methodology and AHP and
GIS technologies and a strategy was developed to assess the
land use for economic activities and thus be able to manage the
land consumption of metropolitan areas. The case study of this
research was the state of Bordj Bouarerridj. A group of experts
defined a set of criteria that helped determine the suitability of
regions for economic activity. The AHP hierarchical analysis
method was used to extract land-appropriate measures through
the Expert Choice program. The consistency ratio was 0.03,
which is less than 0.1, and therefore is considered favorable. [10] M. R. Abbaszadeh, M. Moradi, and S. T. Mehrabankhou, "Application
of analytic hierarchy process in analyzing and ranking of non-financial
measures that affect investor decisions," International Journal of
Business Management and Administration, vol. 2, no. 2, pp. 31-42, Feb. 2013. [11] Y. Wind and T. L. Saaty, "Marketing Applications of the Analytic
Hierarchy Process," Management Science, vol. 26, no. 7, pp. 641–658,
Jul. 1980, https://doi.org/10.1287/mnsc.26.7.641. [12] P. Fernandez, S. Mourato, and M. Moreira, "Social vulnerability
assessment of flood risk using GIS-based multicriteria decision analysis. A case study of Vila Nova de Gaia (Portugal)," Geomatics, Natural
Hazards and Risk, vol. 7, no. 4, pp. 1367–1389, Jul. 2016,
https://doi.org/10.1080/19475705.2015.1052021. After using the AHP method, spatial analysis of the study
area was carried out, with the creation of a geographical
database of the criteria specified by the experts in GIS
environment by creating a model using all accounted data to
extract an appropriate map of land use for economic activity. [13] T. L. Saaty, Decision Making for Leaders: The Analytic Hierarchy
Process for Decisions in a Complex World, 3rd ed. Pittsburgh, PA,
USA: RWS Publications, 2013. [14] P. Jankowski, "Integrating geographical information systems and
multiple criteria decision-making methods," International Journal of
Geographical Information Systems, vol. 9, no. 3, pp. 251–273, May
1995, https://doi.org/10.1080/02693799508902036. We can conclude by saying that the combination of the
Delphi method and the hierarchical analysis using Expert
Choice software in GIS environment is a practical scientific
method that gives accurate results in determining the land use
suitability for economic activity, according to the defined
criteria. However, these criteria remain variable factors because
they depend on the specificities of each region. The produced
map may be adapted to identify regions for future housing
development, evaluate the worth of a property, and assist
decision-makers in achieving balanced urban development. [15] P. • 6.14% of the total area with a total surface area of
239.685km2 was not suitable. • 6.14% of the total area with a total surface area of
239.685km2 was not suitable. The results are summarized in Table III. In the final map
(Figure 14) of the city of Bordj Bouarerridj' suitability of land
usage for industrial activities, we note that the areas that
achieved high suitability according to the AHP are the areas
that are away from the urban tissues and near the road. [6]
E. Memarbashi, H. Azadi, A. A. Barati, F. Mohajeri, S. V. Passel, and F. Witlox, "Land-Use Suitability in Northeast Iran: Application of AHP-
GIS Hybrid Model," ISPRS International Journal of Geo-Information,
vol. 6, no. 12, Dec. 2017, Art. no. 396, https://doi.org/10.3390/
ijgi6120396. TABLE III. PERCENTAGE OF AHP STANDARD. Classes
Area (km2)
%
High suitability
521.793
13.38 %
Moderate suitability
2634.758
67.54 %
Low suitability
504.794
12.94 %
No suitability
239.685
6.14 %
Total
3901.023
100 % [7]
M. M. Aburas, S. H. O. Abdullah, M. F. Ramli, and Z. H. Asha’ari,
"Land Suitability Analysis of Urban Growth in Seremban Malaysia,
Using GIS Based Analytical Hierarchy Process," Procedia Engineering,
vol. 198,
pp. 1128–1136,
Jan. 2017,
https://doi.org/10.1016/
j.proeng.2017.07.155. [8]
A. Rikalovic, I. Cosic, and D. Lazarevic, "GIS Based Multi-criteria
Analysis for Industrial Site Selection," Procedia Engineering, vol. 69,
pp. 1054–1063, Jan. 2014, https://doi.org/10.1016/j.proeng.2014.03.090. [9]
S. A. Delbari, S. I. Ng, Y. A. Aziz, and J. A. Ho, "An investigation of
key competitiveness indicators and drivers of full-service airlines using
Delphi and AHP techniques," Journal of Air Transport Management,
vol. 52, pp. 23–34, Apr. 2016, https://doi.org/10.1016/j.jairtraman. 2015.12.004. IV.
CONCLUSIONS Galanis, "The Delphi Method," Archives of Hellenic Medicine, vol. 35, no. 4, pp. 564–570, Jul. 2018. [16] H. A. Linstone and M. Turoff, The Delphi Method: Techniques and
Applications, 1st ed. Reading, MA, USA: Addison Wesley Publishing
Company, 1975. [17] S. Dehimi and M. Hadjab, "Evaluating the quality of life in urban area
by using the Delphi method. A case study of M’sila city/Algeria,"
Romanian Journal of Geography, vol. 2, no. 63, pp. 193–202, 2019. [18] H. I. Mohammed, Z. Majid, Y. B. Yamusa, M. F. M. Ariff, K. M. Idris,
and N. Darwin, "Sanitary Landfill Siting Using GIS and AHP: A Case
Study in Johor Bahru, Malaysia," Engineering, Technology & Applied
Science Research, vol. 9, no. 3, pp. 4100–4104, Jun. 2019,
https://doi.org/10.48084/etasr.2633. III.
RESULTS AND DISCUSSION Özcan, "Determination of agricultural land
suitability with a
multiple-criteria
decision-making
method
in
Northwestern Turkey," International Journal of Environmental Science
and Technology, vol. 18, no. 5, pp. 1073–1088, May 2021,
https://doi.org/10.1007/s13762-020-02869-9. • 6.14% of the total area with a total surface area of
239.685km2 was not suitable. REFERENCES [1]
Mohamed Gherbi, "Instruments of Urban Planning in Algerian City:
Reality and Challenges," Journal of Civil Engineering and Architecture,
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7359/2015.07.007. [19] E. Triantaphyllou and S. Mann, "Using the analytic hierarchy process for
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L. Jie, Y. Jing, Y. Wang, and Y. Shu-xia, "Environmental Impact
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T. D. T., "Using GIS and AHP technique for land-use suitability
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Earth and Allied Sciences (GIS-IDEAS) 2006, Nov. 2006. [21] A. Emrouznejad and M. Marra, "The state of the art development of
AHP (1979–2017): a literature review with a social network analysis,"
International Journal of Production Research, vol. 55, no. 22, pp. 6653–
6675, Nov. 2017, https://doi.org/10.1080/00207543.2017.1334976. [4]
R. Liu, K. Zhang, Z. Zhang, and A. G. L. Borthwick, "Land-use
suitability analysis for urban development in Beijing," Journal of
Environmental Management, vol. 145, pp. 170–179, Dec. 2014,
https://doi.org/10.1016/j.jenvman.2014.06.020. Benaissa & Khalfallah: Industrial Activity Land Suitability Assessment Using Delphi and AHP … www.etasr.com Vol. 11, No. 5, 2021, 7738-7744 Engineering, Technology & Applied Science Research 7744 [22] B. D. Nguyen, D. T. Minh, A. Ahmad, and Q. L. Nguyen, "The Role Of
Relative Slope Length In Flood Hazard Mapping Using Ahp And Gis
(Case Study: Lam River Basin, Vietnam)," Geography, Environment,
Sustainability, vol. 13, no. 2, pp. 115–123, Jun. 2020, https://doi.org/
10.24057/2071-9388-2020-48. [23] F. Elmahmoudi, O. E. K. Abra, A. Raihani, O. Serrar, and L. Bahatti,
"Elaboration of a Wind Energy Potential Map in Morocco using GIS and
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Science Research, vol. 10, no. 4, pp. 6068–6075, Aug. 2020,
https://doi.org/10.48084/etasr.3692. [24] M. Nilashi, K. Bagherifard, O. Ibrahim, N. Janahmadi, and M. Barisami,
"An Application Expert System for Evaluating Effective Factors on
Trust in B2C WebsitesTrust, Security, ANFIS, Fuzzy Logic, Rule Based
Systems, Electronic Commerce," Engineering, vol. 3, no. 11, pp. 1063–
1071, Nov. 2011, https://doi.org/10.4236/eng.2011.311132. [25] A. Ishizaka and A. Benaissa & Khalfallah: Industrial Activity Land Suitability Assessment Using Delphi and AHP … REFERENCES Labib, "Analytic Hierarchy Process and Expert
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220, Dec. 2009, https://doi.org/10.1057/ori.2009.10. [26] D. Yudi and H. H. Azwir, "Reducing Defects Number of Ampoule by
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Geographical Information System (GIS)," Engineering, Technology &
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https://doi.org/10.48084/etasr.1329. [28] E. O. Oyatoye, B. B. Amole, and S. O. Adebiyi, "Patients’ perception of
quality service delivery of public hospitals in Nigeria using analytical
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model for faba bean cultivation using geographic information system
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Ecological Indicators, vol. 63, pp. 37–47, Apr. 2016, https://doi.org/
10.1016/j.ecolind.2015.11.021. [30] R. Saouli, N. Benhassine, and A. Oularbi, "A Spatio-temporal
retrospective of the uban sprawl of Annaba (Algeria)," Journal of
Fundamental and Applied Sciences, vol. 12, no. 2, pp. 825–844, Nov. 2020. [31] A. Ayache, M. Addoun, B. Hellal, and N. Ayad, "Diachronic analysis of
soil occupancy using remote detection tools in tlemcen province
southern (western Algeria)," Journal of Fundamental and Applied
Sciences, vol. 13, no. 1, pp. 75–88, 2021, https://doi.org/10.4314/jfas. v13i1.5. [32] H. Kazemi, S. Sadeghi, and H. Akinci, "Developing a land evaluation
model for faba bean cultivation using geographic information system
and multi-criteria analysis (A case study: Gonbad-Kavous region, Iran),"
Ecological Indicators, vol. 63, pp. 37–47, Apr. 2016, https://doi.org/
10.1016/j.ecolind.2015.11.021. [33] P. Jankowski, "Integrating geographical information systems and
multiple criteria decision-making methods," International Journal of
Geographical Information Systems, vol. 9, no. 3, pp. 251–273, May
1995, https://doi.org/10.1080/02693799508902036. [34] A. R. Al-shabeeb, "The Use of AHP within GIS in Selecting Potential
Sites for Water Harvesting Sites in the Azraq Basin—Jordan," Journal
of Geographic Information System, vol. 8, no. 1, pp. 73–88, Feb. 2016,
https://doi.org/10.4236/jgis.2016.81008. www.etasr.com
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https://zenodo.org/record/6547435/files/Pobreza_Laboral_y_Tecnoanomia_F_Scotognella.pdf, https://zenodo.org/record/6525505/files/Pobreza_Laboral_y_Tecnoanomia_F_Scotognella.pdf, https://zenodo.org/record/7272250/files/Pobreza_Laboral_y_Tecnoanomia_F_Scotognella.pdf
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Pobreza Laboral y Tecnoanomia
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Zenodo (CERN European Organization for Nuclear Research)
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cc-by
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Pobreza Laboral y Tecnoanomia
Francesco Scotognella
Dipartimento di Fisica, Politecnico di Milano, piazza Leonardo da Vinci 32, 20133 Milano (Italy)
e-mail: francesco.scotognella@polimi.it
Abstract
La pobreza laboral puede discutirse en el marco del trabajo de Hannah Arendt, actividad humana
centrada en las necesidades biológicas. María Pía Chirinos subraya la importancia del trabajo en
términos de conjunto de culturas y transiciones, mientras que Nancy Fraser relaciona dicho trabajo
relacionado con los cuidados con la "reproducción social", en la base de cualquier organización
humana. Además, la pobreza laboral puede relacionarse con la tecnoanomia, en la que un rápido
avance tecnológico provoca el colapso de las normas sociales. La tecnoanomia debe ser controlada
y limitada con el trabajo sinérgico de legisladores y científicos.
Abstract
Working poverty can be discussed within the framework of the Hannah Arendt’s labour, human
activity focussed on biological necessities. Maria Pia Chirinos underlines the importance of labour
in terms of ensemble of cultures and transitions, while Nancy Fraser relates such care-related labour
to “social reproduction”, at the basis of any human organization. Moreover, working poverty can be
related to technoanomia, in which a fast technological advancement results in a collapse of social
norms. Technoanomia should controlled and limited by the synergistic work of legislators and
scientists.
Pobreza Laboral: El fenómeno de la pobreza laboral es significativo y afecta a un porcentaje
importante de la población en muchos países.
Los trabajadores pobres proceden de un entorno familiar considerado pobre, que trabaja o busca
trabajo [1]. Lohmann y Marx, utilizando el informe de 2014 de la Organización Internacional del
Trabajo (OIT) [2], escriben que más de 800 millones de personas vivían en 2013 con un máximo de
2 dólares al día [3].
Esta pobreza puede discutirse retomando la teoría de Hannah Arendt sobre la distinción entre labor,
trabajo y acción (labour, work, action) [4], la teoría de María Pía Chirinos sobre la antropología del
trabajo [5–8], y la teoría de Nancy Fraser acerca de la atención en el artículo Contradictions of
Capital and Care [9].
Labor, trabajo y acción en Hannah Arendt: La labor se centra en las tareas relacionadas con las
necesidades biológicas. El trabajo está dirigido a la producción de bienes duraderos que pasan a
formar parte del mundo. La acción es la actividad mediante la cual los seres humanos interactúan
entre sí. En este marco, la pobreza laboral puede vincularse a la labor según Hannah Arendt.
Antropología del trabajo en Chirinos: María Pia Chirinos destaca la importancia de este tipo de
trabajos, inextricablemente ligados a un tesoro de culturas y tradiciones.
Capital y cuidados en Fraser: Haciendo hincapié en el vínculo entre la labor poco cualificada y los
cuidados, Nancy Fraser señala que sin estos trabajos de reproducción social no habría cultura, ni
economía, ni organización política.
El animal racional (Lat.: animal rationale; Gr.: zoon logon echon, ζῷον λόγοϛ ἔχων) es, esencialmente
y en su plenitud, también activo en el trabajo. Por esta razón, lo que Hannah Arendt define como
trabajo no debería ser categorizado como labor poco cualificada, al igual que la labor de un
trabajador pobre no debería ser relegado a la jaula conceptual del trabajo poco cualificado.
Un nuevo parámetro para evaluar la labor de los cuidados, también desde el punto de vista
económico, es su importancia en la sociedad [10].
Tecnoanomia: La pobreza laboral también puede estar vinculada a nuevos tipos de labor, como los
organizados y gestionados en plataformas de trabajo digitales. El hecho de que las plataformas
laborales digitales (digital labour platforms) puedan eludir la regulación y disminuir la calidad del
empleo podría ser un nuevo problema en el mercado laboral. En casos como el de las plataformas
laborales digitales, se puede hablar de tecnoanomia, ya que el rápido avance tecnológico lleva a la
ruptura de las normas sociales (en este caso las relacionadas con el trabajo).
En las plataformas digitales, la distinción entre empleos de baja, media y alta cualificación no es la
única distinción importante. El nivel de control del trabajo, hasta la parcelación de microtareas a
menudo alienantes, es transversal al nivel de competencias y se ha convertido en un nuevo
parámetro clave para evaluar la calidad del trabajo [11].
El término tecnoanomia se basa en el conocido concepto de anomia introducido por Emile Durkheim
en 1893 [12]. Frank W. Elwell describe la anomia como una "desintegración" social debida a la
división del trabajo y al rápido cambio social [13]. La tecnoanomia hace hincapié en los rápidos
cambios debidos a las nuevas tecnologías, que son demasiado rápidos para ser seguidos por el
conjunto de normas sociales.
Dos estrategias podrían ser apropiadas para frenar el problema de la pobreza laboral:
- Limitar la tecnoanomia. El legislador aboga por un derecho laboral positivo que sigue la "ley
natural" del trabajo: el trabajador entiende lo que ocurre en el mundo del trabajo y evita las lagunas
legislativas que limitan la defensa de los trabajadores.
El derecho natural puede considerarse como un "orden jurídico virtual", la base para concebir y
redactar las leyes positivas, como escribe Laura Zavatta, citando a Jacques Maritain [14].
- La comunidad científica apoya al legislador con sus conocimientos técnicos de vanguardia. La
comunidad científica debe apoyar a los demás trabajadores proponiendo sinergias adecuadas entre
el progreso tecnológico y el trabajo.
Bibliografía
[1] R. Peña-Casas, M. Latta, Working poor in the European Union, Office for Official Publications of
the European Communities, 2004.
[2] Global Employment Trends 2014: The risk of a jobless recovery, 2014.
http://www.ilo.org/global/research/global-reports/global-employmenttrends/2014/WCMS_233953/lang--en/index.htm (accessed January 5, 2022).
[3] H. Lohmann, I. Marx, Introduction, in: Handbook on In-Work Poverty, Edward Elgar Publishing,
2018: pp. 1–4. https://doi.org/10.4337/9781784715632.00005.
[4] H. Arendt, The human condition, 2nd ed, University of Chicago Press, Chicago, 1998.
[5] M.P. Chirinos, Un’antropologia del lavoro: il “domestico” con categoria, Universita della Santa
Croce, Roma, 2005.
[6] M.P. Chirinos, Antropologia della dipendenza: il lavoro e la costituzione dell’essere umano,
Acta Philosofica. 16 (2007) 195–212.
[7] M.P. Chirinos, Il Lavoro come Categoria Antropologica, Iustum Aequum Salutare. IV. 2008/4
(2008) 7–20.
[8] M.P. Chirinos, Humanity in Work: Challenging the “Product Paradigm,” Imago Hominis. 16
(2009) 53–63. https://www.imabe.org/imagohominis/imago-hominis-1/2009-work-lifebalance/humanity-in-work-challenging-the-product-paradigm.
[9] N. Fraser, Contradictions of Capital and Care, New Left Rev. (2016) 99–117.
[10] F. Scotognella, Working Poverty in the Framework of the Work Anthropology: A Literature
Review and Considerations on Care-related Labour, ESJ. 18 (2022) 1.
https://doi.org/10.19044/esj.2022.v18n10p1.
[11] E. Fernández-Macías, Automation, digitisation and platforms: implications for work and
employment., Eurofound, Publications Office, LU, 2018.
https://data.europa.eu/doi/10.2806/090974 (accessed April 25, 2022).
[12] E. Durkheim, The division of labor in society, Free Press, New York, 2018.
[13] F.W. Elwell, Emile Durkheim on Anomie, (2003).
https://faculty.rsu.edu/users/f/felwell/www/Theorists/Essays/Durkheim1.htm (accessed April
27, 2022).
[14] L. Zavatta, La concezione dei diritti dell’uomo di Maritain, (2014).
https://www.openstarts.units.it/handle/10077/10206 (accessed April 25, 2022).
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https://www.scielo.br/j/rcf/a/KSgjq95F3YVtJMsj7dTV8Cj/?lang=es&format=pdf, https://www.revistas.usp.br/rcf/article/download/34097/36829, http://old.scielo.br/pdf/rcf/v14n32/v14n32a07.pdf, https://www.redalyc.org/pdf/2571/257119544007.pdf
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El logro del value for money en la gestión pública: consideraciones en torno a los indicadores de eficiencia, eficacia y economía
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Revista Contabilidade & Finanças
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EL LOGRO DEL VALUE FOR MONEY EN LA GESTIÓN PÚBLICA:
CONSIDERACIONES EN TORNO A LOS INDICADORES DE EFICIENCIA, EFICACIA Y ECONOMIA
SEÇÃO INTERNACIONAL
EL LOGRO DEL VALUE FOR MONEY EN LA GESTIÓN
PÚBLICA: CONSIDERACIONES EN TORNO A LOS
INDICADORES DE EFICIENCIA, EFICACIA Y ECONOMÍA
Cristina Aibar Guzmán
Profesora de la Facultad de Ciencias Económicas y Empresariales de la
Universidad de Santiago de Compostela - España
E-mail: efcris@usc.es
RESUMO
ABSTRACT
Entre los instrumentos y técnicas de gestión privada que han sido objeto de atención prioritaria por
parte de la literatura sobre el tema, al considerar que
su implantación en el sector público podría favorecer
el cambio hacia la Nueva Gestión Pública se
encuentran los indicadores de gestión o medidas de
rendimiento de diversa índole que permitan evaluar
las distintas actividades y políticas llevadas a cabo
por una entidad a lo largo del tiempo. Sin embargo,
dentro de dicho conjunto de indicadores el foco de
atención prioritaria de los investigadores y organismos internacionales interesados en el tema así como
el trabajo de los gestores públicos se han centrado
en el desarrollo de los indicadores de eficiencia,
eficacia y economía, cuyo uso se ha universalizado
como elementos básicos del value for money
estrechamente vinculados a los distintos tipos de
responsabilidad pública. En esta línea, el objetivo del
presente trabajo es profundizar en las características
de los indicadores diseñados para medir las tres E’s
de la actuación de las entidades públicas (eficiencia,
eficacia y economía) así como la problemática
asociada a su diseño y definición.
Among the private management instruments
and techniques that have been the subject of great
attention by the literature about this issue,
considering that their implantation in the public
sector could favor the change towards New Public
Management, we find various kinds of management
indices or efficiency measures that allow for the
evaluation of the different activities and policies
realized by an entity over time. Nevertheless,
among this set of indices, the main focus of
attention of researchers and international organs
interested in the issue, as well as the work of public
managers have focused on the development of
efficiency, efficacy and economic indices, whose
use has become widespread as basic elements of
the value for money, closely linked up with the
different kinds of public responsability. In
accordance with this line, this study aims to look
into the characteristics of the indices designed for
measur ing the three E’s of public entities’
performance (efficiency, efficacy and economy) as
well as the problematics associated with their
design and definition.
Palabras-claves: New public management, indicadores de gestión, eficiencia, eficacia, economía.
Keywords: New public management, management
indicators, efficiency, efficacy, economy.
Recebido em 15.01.2003 • Aceito em 04.06.2003
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1. INTRODUCCIÓN
La Segunda Guerra Mundial supuso el inicio del
incremento de la importancia del sector público y la
expansión de su ámbito de actuación a todas las áreas de la actividad económica y social (básicamente,
debido a la necesidad de reconstrucción que
presentaban los países tras el conflicto bélico). Desde entonces, el peso del sector público en la economía
se ha ido incrementando a lo largo del tiempo1 , tal y
como se refleja en los distintos índices que expresan
el peso de la componente pública en la economía
(presión fiscal, relación entre el gasto público y el PIB,
volumen de empleo del sector público,...), hasta el
punto de que en la actualidad aproximadamente el
50% del PIB de los países de la Unión Europea se
canaliza a través del sector público el cual, además,
da empleo en torno al 20% de la población activa de
dichos países.
Este aumento del tamaño, importancia y actividad
experimentado por el sector público ha estado
acompañado por un notable crecimiento del gasto
público, en un entorno caracterizado por una fuerte y
prolongada recesión económica, lo cual, en la mayoría
de los países, ha llevado aparejado un incremento
del déficit público2 , que pone en evidencia el hecho
de que las entidades públicas han estado viviendo
por encima de sus medios, sin que dicho incremento
se haya visto correspondido por un mejor rendimiento
y una mayor eficiencia de las entidades públicas (dado
que, en muchos casos, la calidad y también a veces
la cantidad de los servicios públicos proporcionados
no satisface las exigencias y necesidades de los
destinatarios de los mismos), lo cual, a su vez, ha
dado lugar a un incremento de las demandas de
responsabilidad pública por parte de los ciudadanos.
En esta línea, uno de los requisitos básicos que
desde la década de los ochenta se ha venido exigiendo
a la actuación pública es el desarrollo de una gestión
eficaz, eficiente y responsable orientada al logro de
un mayor value for money que refleje el compromiso
real de los responsables públicos de proporcionar
“bienestar” a los ciudadanos en contrapartida a los
impuestos que recaudan, de forma que, como señala
De Val Pardo (1999) es posible asimilar dicho término
al logro de los valores de eficiencia, eficacia y
economía en la actuación del sector público.
En consecuencia, en la mayoría de los países
desarrollados así como en muchos países en vías de
desarrollo, se han puesto en marcha distintas iniciativas orientadas a la mejora del funcionamiento de las
entidades públicas, las cuales dieron lugar a la
aparición del modelo denominado Nueva Gestión Pública (New Public Management), caracterizado por la
aplicación en el sector público de los principios de
gestión privada y la introducción de criterios de mercado (Hood, 1995; Hopwood, 1994; McKevitt y Lawton,
1994). En este sentido, entre los instrumentos y técnicas de gestión privada que han sido objeto de
atención prioritaria por parte de la literatura sobre el
tema, al considerar que su implantación en el sector
público podría favorecer el cambio hacia la Nueva
Gestión Pública, se encuentran los indicadores de
gestión o medidas de rendimiento.
Así, en las últimas décadas se ha puesto de
manifiesto el interés en el estudio de la problemática
relacionada con el desarrollo e implantación de indicadores de gestión en las entidades públicas a través de la aparición de una importante corriente de
investigación en la que se han discutido diversos aspectos o facetas relacionados con los mismos: antecedentes históricos de los indicadores de gestión,
principales dificultades para su desarrollo, utilidad de
los indicadores de gestión de cara a favorecer la
introducción de las reformas organizativas y de
funcionamiento asociadas al modelo de Nueva
Gestión Pública, experiencias de distintos tipos de
entidades públicas en el desarrollo e implantación de
indicadores de gestión de diversa índole, patrones en
el uso de los indicadores y variables que afectan a
su utilización por parte de las entidades públicas
(Bouckaert, 1992; Buschor, 1994; Carter, 1994; Day y
Klein, 1994; De Lancer Julnes y Holzer, 2001;
Kravchuk y Schack, 1996; López Hernández et al,
2001; Mora Corral y Vivas Urieta, 2001; Simon,
1994,…).
No obstante, hemos de señalar que la atención
prioritaria de los investigadores y organismos
1
En este sentido, Minogue (1997) señala que entre 1980 y 1990 prácticamente se ha doblado el gasto per cápita del gobierno en los países de la OCDE.
2
A este respecto, Montoro (2000, p. 84) señala que en los años setenta “se produce una crisis fiscal del estado y aparecen déficit públicos muy importantes” que comienzan
a considerarse como perturbaciones del equilibrio económico y motivan que “las políticas económicas, en vez de preocuparse tanto por los efectos de las finanzas sobre
la actividad económica, empiecen a preocuparse por el propio equilibrio de los presupuestos públicos”.
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internacionales interesados en el tema, así como el
trabajo de los gestores públicos, se han centrado en el
desarrollo de los indicadores de eficiencia, eficacia y
economía, cuyo uso se ha universalizado como elementos básicos del value for money estrechamente vinculados a los distintos tipos de responsabilidad pública
(De Val Pardo, 1999; Glynn et al, 1995; Minogue, 1997;
Montesinos Julve y Vela Bargues, 1994).
Sin embargo, como señala Perrin (1991, p. 120),
los términos implicados (eficiencia, eficacia y
economía) suelen tener «definiciones, significados y
mediciones imprecisas» de forma que «significan lo
que el académico, o el político, desean que signifiquen
en un momento y contexto particular». En esta línea,
el objetivo del presente artículo es profundizar en las
características de los indicadores diseñados para
medir las tres E’s de la actuación de las entidades
públicas (eficiencia, eficacia y economía) así como
la problemática asociada a su diseño y definición.
2. LOS INDICADORES DE EFICIENCIA
2.1. El concepto de eficiencia y su
aplicación a la gestión pública
La eficiencia se define como la relación entre los
inputs (bienes y servicios) consumidos y los outputs
y resultados obtenidos en el proceso de creación de
valor de la entidad, de forma tal que, como señalan
AECA (1997), Bac (1994) y Simon (1994), el criterio
de eficiencia tiene su base en la gestión empresarial
y supone el logro de un equilibrio positivo en la relación
output-input, es decir, manteniendo los estándares de
calidad, lo cual implica la racionalidad de los consumos (eliminando los desperdicios y consumos
innecesarios).
En opinión de Mora Corral y Vivas Urieta (2001, p.
17) la utilización del concepto de eficiencia en el sector
Inputs
á
Actividades
á
Outputs
directo
á
público debe ir más allá de la simple traslación de las
ideas aplicadas en el ámbito privado ya que es
necesario considerar el valor efectivo que las entidades públicas aportan a los ciudadanos. En esta línea
se manifiesta también Simon (1994) al señalar que la
aplicación del criterio de eficiencia en las entidades
públicas no debe abordarse desde un punto de vista
parcial sino general, lo que exige considerar no sólo
los outputs y resultados sino también los efectos que
tienen las actividades desarrolladas por dichas entidades, los cuales, en determinados casos, pueden
no ser observables en el corto plazo3 .
Surge así la necesidad de una clara definición de
cuáles son los output, resultados y efectos o impactos de las actuaciones llevadas a cabo por una determinada entidad, lo que plantea un primer problema a
la hora de operacionalizar los indicadores de eficiencia.
Puesto que los servicios suministrados por las entidades públicas cubren diversos aspectos y se dirigen
a un público muy amplio y heterogéneo, no existe un
denominador común capaz de expresar en términos
concretos el valor a realizar por dichos servicios. Esto
lleva a autores como Carter (1994), Simon (1994) y
Stewart y Ranson (1994), entre otros, a afirmar normalmente que el rendimiento de las organizaciones
públicas no puede definirse mediante outputs únicos,
dado que una actividad puede tener muchos resultados que pueden ser igualmente deseables y tiene, como
consecuencia, el que los términos output, resultados
y efectos o impactos sean utilizados de forma
intercambiable por los gobiernos y autoridades públicas (Glynn et al, 1995, pp. 217-218).
En este sentido, Carter (1994) distingue entre los
outputs de la actuación de una entidad pública (los
servicios que la misma proporciona a los ciudadanos) y
los resultados de dicha actuación (representados por el
impacto que los servicios suministrados tienen sobre
los receptores), tal y como se representa en la figura 1.
Resultado
intermedio
á
Resultado
final
á
Resultado
Fuente: Blanco Dopico et al (2001)
Figura 1 – Proceso de generación de resultados en las entidades públicas
3
En este sentido, para Metcalfe y Richards (1984, p. 188) la eficiencia en el contexto del sector público se considera “un valor político y una virtud administrativa”.
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Por otro lado, Glynn et al (1995) consideran que
para un determinado proyecto político o programa
público se denomina: (a) outputs, a los bienes y
servicios de naturaleza normalmente tangible
producidos por el mismo, (b) resultados, a las
consecuencias directas que tiene sobre aquellos a
los que está destinado y (c) impactos, a los efectos
más abstractos y generales que produce (por ejemplo
sobre el nivel de ingresos o de empleo en la economía).
La evaluación de los outputs y resultados, si bien
no exenta de dificultades, es menos problemática que
la evaluación de los impactos generales, ya que en
este caso, en opinión de Ballart (1992), es necesario
determinar el grado en el cual los efectos atribuidos a
un programa se deben realmente a éste y no a otros
factores. Ello exige analizar la relación de causalidad
existente entre la introducción de un programa y los
efectos del mismo, identificando y controlando todos
los factores externos que puedan influir en dicha
relación.
Por su parte, Simon (1994) señala un problema
adicional derivado de la existencia, en ocasiones, de
distintas unidades de medición para inputs y outputs
lo cual dificulta la comparación entre ambos e implica
que la eficiencia deba evaluarse en términos relativos (hablaríamos, por tanto, de eficiencias relativas).
Asimismo, dicho autor destaca la necesidad de ampliar el concepto de eficiencia para incluir a todos los
factores implicados en el proceso de creación de valor de la entidad que no son susceptibles de medición
en unidades monetarias, lo que permitirá, como señala
AECA (1997), que no sea indispensable para la
determinación de si una actuación es eficiente el que
ésta sea evaluada en términos monetarios.
Es necesario señalar que no existe unanimidad
entre los distintos autores que han analizado este
concepto en cuanto a la definición y contenido que se
atribuyen a los diferentes tipos de eficiencia (Cansino
Muñoz-Repiso, 2000). Así, para Metcalfe y Richards
(1989) existen cuatro tipos de eficiencia:
- Eficiencia técnica: que expresa la relación entre inputs y outputs en unidades físicas. En
palabras de Albi et al (2000, p. 240) “se dice
que existe eficiencia técnica cuando el sector
público está sobre su frontera de posibilidades
de producción”, es decir, está empleando sus
distintos factores productivos de la mejor forma posible.
-
Eficiencia económica: relaciona el coste de
utilización de los inputs con el valor que
representan los outputs, es decir, implica que
los beneficios generados por la actuación pública son mayores que sus costes. Dentro de
ella es posible distinguir, a su vez, entre:
- Eficiencia asignativa: implica combinar
óptimamente los distintos factores
productivos de tal forma que, manteniendo
los costes incurridos, se obtenga la mayor
cantidad de output posible. Para Albi et al
(2000) la eficiencia asignativa supone que,
además de ser eficiente técnicamente, el
sector público está empleando la
combinación de inputs menos costosa.
- Eficiencia productiva: que supone obtener
la mayor cantidad de output posible al menor coste (es decir, mide la productividad
en el uso de los inputs)
- Eficiencia operativa: que se centra en los
procesos, funciones y objetivos actuales
- Eficiencia adaptativa: valora la flexibilidad para
adaptarse a cambios rápidos que se produzcan
en el entorno, la capacidad para continuar prestando servicios en condiciones de calidad y la
versatilidad para hacer frente a las nuevas demandas de servicios públicos por parte de los
ciudadanos.
Finalmente, López Díaz (1998) diferencia entre
eficiencia en la provisión de los servicios públicos y
eficiencia de producción. La primera relaciona la oferta de servicios públicos con las necesidades sociales
de los ciudadanos, mientras que la segunda se refiere
a la utilización adecuada de los recursos dada la
tecnología existente.
2.2. La evaluación de la eficiencia de la
actuación pública
Dado que el grado de eficiencia alcanzado por una
determinada actuación o en la prestación de servicio
público viene dado por la relación existente entre los
recursos consumidos y los producidos en el
desempeño de dicha actuación, los indicadores de
eficiencia tratan de evaluar si dicha relación entre
inputs y outputs es o no adecuada, es decir, miden el
nivel de aprovechamiento de los recursos utilizados
en el desarrollo de los distintos procesos llevados a
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cabo por las entidades públicas mediante su
comparación con el volumen de outputs o prestaciones
obtenidos (AECA, 2002).
En este sentido, se considera que una actuación
es eficiente cuando se obtiene el máximo valor posible
manteniendo constante la cantidad de recursos consumidos o bien cuando se obtiene un determinado
valor con un menor consumo de recursos (AECA,
1997; Mora Corral y Vivas Urieta, 2001).
A la hora de definir los indicadores de eficiencia,
AECA (2002, p. 21) propone dos grandes áreas
respecto a las cuales evaluar la eficiencia de un determinado servicio público:
• La eficiencia del personal: tanto en términos
reales, mediante la comparación del volumen
de output o prestaciones obtenidas y el número de personas empleadas para su logro, como
en términos económicos, comparando el conjunto de outputs producidos con el coste
inherente al personal empleado para su
obtención.
• La eficiencia global del servicio: supone calcular el coste unitario de cada output mediante la
comparación del total de prestaciones
obtenidas en un servicio con el total de recursos aplicados en su proceso de desarrollo.
Por otra parte, Montesinos Julve (1993) considera
que puede hablarse de eficiencia media cuando los
indicadores se elaboran tomando como base los
totales de un período y de eficiencia marginal cuando
dichos indicadores se plantean como una relación
incremental.
Finalmente, en opinión de AECA (1997), la
evaluación de los indicadores de eficiencia puede
realizarse de distintas formas: (a) mediante su
comparación con unos estándares predeterminados
establecidos en condiciones de desempeño óptimas,
(b) utilizando series cronológicas que permitan analizar
el progreso alcanzado a lo largo del tiempo y (c) realizando un análisis cross-section que permita, previa
homogeneización del contenido de los indicadores,
comparar distintas entidades en un momento concreto. Asimismo, se destaca la utilidad del análisis
envolvente de datos (data envolvement analysis) para,
mediante técnicas de programación lineal, calcular
medidas de eficiencia que permitan relacionar el nivel
real de desempeño y el óptimo para cada servicio o
actividad.
3. LOS INDICADORES DE EFICACIA
3.1. Consideraciones relativas a la necesidad
de eficacia en la actuación pública
Como señalan Polidano et al (1997), la eficiencia
en la gestión de los recursos y actividades es una
condición necesaria pero no suficiente en el contexto
de la Nueva Gestión Pública, dado que se requiere,
además, establecer los propósitos u objetivos a los
que debe tender la actuación de las entidades públicas y evaluar dicha actuación con respecto a los
mismos. Surge así la necesidad de analizar el
concepto de eficacia, estrechamente relacionado con
la eficiencia. En este sentido, Metcalfe y Richards
(1989, p. 63) consideran que la distinción convencional entre política y administración “hace la eficacia
lógicamente previa a la eficiencia, pero, por la misma
razón, hace que la evaluación de la eficacia dependa
de una definición previa de objetivos”.
Ello supone que la distinción entre ambos
conceptos sea difícil puesto que, como afirman
Metcalfe y Richards (1984, pp. 63-64), «el limite entre eficiencia y eficacia es permeable» de forma que,
en opinión de Simon (1994), prácticamente hasta
finales del siglo XIX, los términos eficacia y
eficiencia eran utilizados indistintamente puesto que
se consideraban casi sinónimos. No obstante, en el
Third Report of the House of Commons Select
Committee on the Treasury and Civil service (19811982) ya se plantea una clara distinción entre ambos conceptos al definir la eficiencia como la relación
entre los inputs consumidos y los outputs obtenidos,
dados los objetivos y teniendo en cuenta los medios
disponibles, y la eficacia como el progreso alcanzado
en el logro de los objetivos de la entidad (Metcalfe y
Richards, 1984).
Con carácter general, se define la eficacia como
la relación existente entre los outputs, resultados y
efectos derivados de la actuación llevada a cabo por
una entidad, y los objetivos de la misma de forma tal
que permite medir el grado en que se han alcanzado
dichos objetivos. Ello supone considerar que una determinada actividad no puede ser efectiva, aún cuando
produzca resultados valiosos, si no alcanza los objetivos para los cuales ha sido realizada.
En consecuencia, la evaluación de la eficacia
de la actuación de una organización requiere que
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previamente se haya realizado un proceso de
planificación en el que se establezcan los objetivos que se pretenden lograr 4 . En este sentido,
Kanter y Summers (1994), consideran que, dado
que los objetivos considerados claves para una
entidad pueden variar a lo largo del tiempo con las
distintas etapas de su ciclo de vida, las medidas o
indicadores de eficacia variarán también en
consonancia con el cambio experimentado por los
objetivos. Por otro lado, AECA (1997) amplia esta
definición al considerar también la comparación
entre los resultados obtenidos y un ideal u óptimo,
configurándose así el término eficacia como una
medida de la mejora continua de la organización.
Partiendo de la idea de que, como señala Blanco
Dopico (1991), es difícil definir la eficacia en base a
una única dimensión, Lovell (1995) considera que dicho
término está abierto a descomposición. En esta línea,
Glynn et al (1995) distinguen varios tipos de eficacia
a partir de la diferenciación entre:
- eficacia substantiva: grado en que los resulta-
dos de las actividades desarrolladas se
corresponden con los objetivos establecidos
- eficacia evaluativa: capacidad para medir y
mejorar la eficacia substantiva.
Dentro de la primera es posible distinguir entre
eficacia directiva (la cual se expresa mediante la
eficacia de outputs, es decir, si se obtienen o no los
outputs preestablecidos) 5 y eficacia política (que
mide el grado en que los resultados e impactos de
una actividad son consistentes con los proyectados)6 .
Esta última se divide, a su vez, en eficacia de resultados (que relaciona los resultados obtenidos con
aquellos deseados por los distintos stakeholders7 ) y
eficacia de impactos, la cual, debido a la naturaleza
intangible de los impactos derivados de la actuación
pública, es más difícil de medir. Por su parte, la
eficacia evaluativa depende de las capacidades técnicas y organizativas de la entidad para realizar la
evaluación del grado de eficacia alcanzado. La figura 2 resume esta tipología.
Eficacia
Þ
Þ
Evaluativa
Sustantiva
Þ
Þ
Þ
Þ
Política
Para la evaluación
de la eficacia
De
outputs
De
resultados
Þ
Þ
Directiva
Þ
Capacidad
organizacional
Þ
Capacidad
técnica
De
impactos
Fuente: Glynn et al (1995, 220)
Figura 2 – Tipos de eficacia
4
A este respecto, Ballart (1992, p. 26) destaca la necesidad de establecer objetivos específicos ya que “los objetivos retóricos [...] no sirven como base a partir de la cual
enjuiciar el éxito”.
5
Dichos outputs pueden medirse en términos cuantitativos o cualitativos.
6
Esta clasificación tiene que ver con la distinción planteada por AECA (1997) entre objetivos de hechos y objetivos de resultados.
7
En esta línea, Kanter y Summers (1994 consideran que un problema común a la hora de definir la eficacia es la tendencia a favorecer, implícita o explícitamente, los objetivos
de determinados stakeholders sobre los de otros por lo que argumentan sobre la necesidad de desarrollar una definición que contemple los intereses de los múltiples
stakeholders de la entidad.
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Por otro lado, un tipo particular de eficacia es la
eficacia presupuestaria, la cual está vinculada a los
presupuestos por programas.
3.2. La evaluación de la eficacia de las
entidades públicas
Los indicadores de eficacia tratan de evaluar si
los resultados alcanzados se ajustan a los inicialmente
previstos, es decir, miden el grado de cumplimiento
de los objetivos. En consecuencia, como hemos
señalado anteriormente, la evaluación de la eficacia
de la actuación de una organización requiere que se
haya realizado previamente un proceso de
planificación por medio del cual se establezcan los
objetivos que se pretenden lograr.
En este sentido, el hecho de que las entidades
públicas con carácter general carecen de objetivos
claramente definidos supone una primera dificultad
a la hora de desarrollar los indicadores de eficacia
en este tipo de entidades. Sin embargo, en opinión
de Simon (1994), los problemas asociados a la
definición de objetivos en las entidades públicas
no constituyen la única fuente de dificultades para
la evaluación de la eficacia en el sector público ya
que es necesario, además, establecer el grado en
el cual cada objetivo debería alcanzarse, es decir,
determinar “unos niveles de ejecución que se
traduzcan en criterios de rendimiento” (Paramés
Montenegro, 1988, p. 140), lo cual, como señalan
McKevitt y Lawton (1994), está condicionado no sólo
por consideraciones de carácter político y
económico, sino también por las connotaciones históricas y culturales que tradicionalmente han estado vinculadas a la actuación pública y al papel del
sector público.
El objetivo de los indicadores de eficacia es señalar
las posibles desviaciones existentes entre los outputs
obtenidos y los esperados y constituir la base para la
identificación y análisis de las causas de dichas
desviaciones. En este sentido, AECA (2002) señala
que dichas desviaciones deben determinarse tanto
desde una perspectiva global o general de cada
servicio, como con referencia a los ámbitos económicofinanciero y operativo asociados al mismo, comparando, respectivamente, los ingresos o gastos reales
con los presupuestados y el volumen de output realizado con el previsto.
Finalmente, como señala AECA (1997) la valoración
de este tipo de indicadores requiere la utilización de
técnicas basadas en los outputs tales como las
auditorías operativas o de actuación.
4. LOS INDICADORES DE ECONOMÍA
4.1. El atributo de economía en la actuación
pública
El atributo de economía se centra en los inputs
(financieros, humanos y materiales) de la organización
y a las condiciones en que se lleva a cabo su
adquisición por parte de una determinada entidad. En
concreto, hace referencia a la búsqueda de la relación
precio-calidad-cantidad más favorable para la
adquisición de los recursos por parte de un organismo público..
En consecuencia, como señalan Montesinos Julve
y Vela Bargués (1994), su logro requiere el
conocimiento no sólo de las necesidades de recursos
de la entidad, sino de las características del mercado
en el que dichos recursos pueden ser adquiridos.
4.2. Los indicadores de economía
Dado que la evaluación de la economía tiene como
objetivo medir el grado en que las condiciones de
adquisición de los inputs han sido ventajosas para la
entidad, AECA (1997) plantea la necesidad de analizar
cuatro elementos vinculados a dichas condiciones de
adquisición (coste, cantidad adecuada, calidad
preestablecida y tiempo adecuado), cuya comparación
con sus valores ideales expresa, a juicio de Navarro
Galera (1997), la economía como grado de acierto en
la adquisición.
Sin embargo, en opinión de Brusca Alijarde y
Cóndor López (1999, p. 70), dentro de los indicadores
de economía, los más utilizados “son aquellos que
hacen referencia a los costes, estableciendo las
comparaciones oportunas con los costes previstos”.
En este sentido, AECA (1997) sugiere la posibilidad
de aplicar las siguientes técnicas para la
comprobación del coste de los inputs:
• Análisis descendente de los costes: tiene
como objetivo detectar y descomponer los
costes irregulares a fin de detectar las áreas
en las que se presentan ineficiencias.
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•
•
•
Cristina Aibar Guzmán
Árbol de costes: supone la aplicación de la técnica anterior a fin de identificar los costes irregulares derivados de decisiones de carácter
político.
Perfil de actividad: se trata de un sistema de
análisis descendente en el que se consideran
únicamente las áreas de actuación susceptibles
de mejora distinguiendo, además, entre
actividades fundamentales u obligatorias y
actividades discrecionales.
Comparaciones internas: supone la comparación
de costes y otras variables que caracterizan
un servicio entre distintas unidades dentro de
una misma entidad8 .
ECONOMIA
Compra de inputs
as menor coste
5. RELACIONES ENTRE LOS
CONCEPTOS DE EFICIENCIA, EFICACIA
Y ECONOMÍA EN LA GESTIÓN PÚBLICA
Como hemos señalado anteriormente, los conceptos
de eficiencia, eficacia, economía y value for money se
encuentran estrechamente interrelacionados. En este
sentido, las figuras 3 y 4 reflejan las interrelaciones
existentes entre dichos conceptos. La primera de ellas
(figura 3) los presenta en relación con el escalonamiento
básico (recursos, resultados, objetivos) para el logro
del value for money, mientras que la segunda (figura 4)
refleja las distintas comparaciones que plantea las
definiciones de los mismos.
EFICIENCIA
Relación óptima
entre inputs y
outputs
=
Input físico
Coste input
é
é
Vale lo
que cuesta
EFICACIA
Impacto de los
outputs sobre los
objetivos
*
Output físico
Input físico
*
Valor output
Output físico
Fuente: elaboración propia en base a López Casasnovas (1995, pág. 61)
Figura 3 – Eficiencia, eficacia y economía en la Gestión Pública
INPUTS
PREVISTOS
Þ
Þ
OUTPUTS
INTERMEDIOS
Þ
Þ Þ
Þ
Economia
OUTPUTS
REALES
Þ
Þ
Eficiencia
Þ Þ
INPUTS
REALES
Þ
OUTPUTS
PREVISTOS
Eficacia
Fuente: AECA (1997, p. 26)
Figura 4 – Interrelación entre Economía, Eficacia y Eficiencia
8
Su aplicación suele verse dificultada por el carácter heterogéneo de la actividad desarrollada por los distintos departamentos de una entidad que reduce la posibilidad
de hacer comparaciones.
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EL LOGRO DEL VALUE FOR MONEY EN LA GESTIÓN PÚBLICA:
CONSIDERACIONES EN TORNO A LOS INDICADORES DE EFICIENCIA, EFICACIA Y ECONOMIA
Finalmente, la figura 5 refleja las interrelaciones
existentes entre dichos conceptos relacionándolo con
el proceso de creación de valor. En ella se observa
que el logro del value for money debe contemplarse
desde una perspectiva de equilibrio ya que, por
ejemplo, la búsqueda de mejores niveles de economía
y eficiencia, a través de la reducción de la cantidad o
calidad de los recursos utilizados, puede poner en
peligro la consecución de los objetivos de la entidad,
reduciendo así la eficacia de la misma.
Value for Money
Economia
Objetivos
Þ
Outputs
Þ
Inputs
Eficacia
Þ
Coste
Eficiencia
Mercado
Proceso de
transformación
Población
objetivo
Fuente: elaboración propia a partir de Montesinos Julve y Vela Bargues (1994, p. 405) y AECA (1997, p. 27)
Figura 5 – Economía, Eficacia y Eficiencia desde la perspectiva del proceso de creación de valor
COMENTARIOS FINALES
A lo largo de las páginas precedentes hemos
venido argumentando distintas cuestiones relativas a
los conceptos de eficiencia, eficacia y economía en
la gestión pública y al desarrollo por parte de las entidades públicas de indicadores que permitan su
evaluación. La elección de estos tres conceptos ha
estado basada en el hecho de que los mismos son
los que se encuentran más directamente relacionados con el concepto de value for money en la actuación
pública y, por tanto, los que han sido objeto de un
mayor interés por parte de las entidades que se han
planteado el desarrollo e implantación de indicadores
de gestión.
Sin embargo, hemos de señalar que los indicadores de eficiencia, eficacia y economía no constituyen
los únicos indicadores de que necesitan disponer los
gestores públicos a fin de llevar a cabo una actuación
más competitiva sino que, por el contrario, a lo largo
del tiempo dicho conjunto de indicadores se ha venido
ampliando cada vez más a fin de incluir indicadores
que permitan incorporar en el ámbito de decisión de
los gestores públicos, criterios de calidad así como
valores políticos y sociales que hagan compatible el
logro simultáneo de una mayor racionalidad en el uso
de los recursos disponibles con el cumplimiento de
las funciones económicas y sociales que justifican la
existencia y razón de ser del sector público.
Así, a lo largo del tiempo se han venido añadiendo
nuevos elementos a las tres E’s tradicionales que
definen el concepto general de value for money
(eficiencia, eficacia y economía) de tal forma que
actualmente se habla hasta de nueve E’s entre las
que se incluyen, además de las ya mencionadas, los
indicadores de efectividad, equidad, excelencia, entorno, evolución sostenible y ecología.
No obstante, hemos de señalar que no se trata de
un conjunto inconexo de medidas de rendimiento sino
que, por el contrario, dichos indicadores se encuentran
fuertemente interrelacionados, siendo posible en
muchos casos establecer relaciones causa-efecto
entre las actuaciones llevadas a cabo para mejorar
una faceta determinada y los resultados que se
aprecian en las restantes dimensiones del desempeño
de una entidad pública.
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Cristina Aibar Guzmán
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https://openalex.org/W2944031191
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http://ejournal.umm.ac.id/index.php/jmb/article/download/5291/5160
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Indonesian
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EVALUASI KELAYAKAN BISNIS PADA RUMAH PEMONDOKAN (KOST) MAHASISWA DI SEKITAR UNIVERSITAS MUHAMMADIYAH MALANG
|
Manajemen Bisnis
| 2,018
|
cc-by-sa
| 5,069
|
EVALUASI KELAYAKAN BISNIS PADA RUMAH PEMONDOKAN
(KOST) MAHASISWA DI SEKITAR UNIVERSITAS MUHAMMADIYAH
MALANG Hellen Mayora Violetha
Department of Management FEB UMM
E-mail: faceless_void21@yahoo.com ABSTRACT The purpose of the study is to evaluate the feasibility of students boarding house
around University of Muhammadiyah Malang. The analytical tool are Net Present
Value, Payback Period, Average Rate of Return, Internal Rate of Return,
Profitability Index.The results of the analysis of boarding house owned by Mr. Rofiq show that the Net Present Value is 226.968.193,1 rupiahwhich is more than
null (eligible). Value Payback Period is six years one month and seven days which
is less than 20 years (feasible). Internal Rate of Return is 17,2063 % which is
higher than COC, it is declared eligible.The value of Average Rate of Return is 43
% which is more than 15 % (feasible). The value of Profitability Index is 1,44
which is more than one (feasible).The results of the analysis of boarding house
owned by Mrs. Atnah show that Net Present Value is 22.370.869,3 rupiah which
is more than null, it is declared eligible. Value Payback Period is twelve years
two months and twentythree days is less than 20 years (feasible). Internal Rate of
Return is 17,8111 % which is higher than COC, it is declared eligible. The value
of Average Rate of Return is 38 % which is more than 17 % (feasible). The value
of Profitability Index is 1.058 which is more than one (feasible). In other words,
the investments wasconducted by both the owner of boarding house was
proceed.The results of the analysis of boarding house owned by Mr. Sofi show
that Net Present Value is minus 170.035.625,2 rupiah which is not more than null
(unfit). Value Payback Period is 32 years 2 months 12 days is more than 20 years
(not feasible). The results of the value of the Internal Rate of Return is 3,7435 %
is less than COC which is declared unfit. The value of Average Rate of Return is
2% which is not expected (less than 15%) declared unfit. The value Profitability
Index is 0.32 which is less than one, it is declared unfit. Keywords:Net Present Value, Internal Rate of Return, Payback Period, Average
Rate of Return, Profitability Index. PENDAHULUAN baik negeri maupun swasta, selain
ituKota Malang dipenuhi berbagai
sekolah,
lembaga
pendidikan
nonformal atautempat-tempat kursus,
serta
sejumlah
pondok Kota Malang telah dikenal
sebagai Kota Pendidikan, maka dari
itu perkembangan pendidikan di Kota
tersebut sangat pesat. Hal ini terlihat
dari banyaknya jumlah universitas 49 50 pesantren.Pendidikandi Kota Malang
memiliki
fasilitas
penunjang
yang
cukup
memadai
seperti
tempat
pemondokan,
toko
buku,
super
market, plaza, pusat pelayananan
kesehatan masyarakat serta fasilitas
penunjang lainnya yang tak kalah
penting adalah adanya angkutan
umum (transpotasi) yang tersedia ke
penjuru kota (memiliki 25 jalur),
yang
menghubungkan
3
(tiga)
terminal yang ada di Kota Malang,
yaitu
terminal
Arjosari
(arah
Surabaya), terminal Gadang (arah
Blitar), terminal Landungsari (arah
Jombang/Kediri). besar maupun perusahaan dalam
skala kecil. Hal
ini
dapat
memberi
pengaruh
positif
dari
sisi
pertumbuhan
ekonomi. Namun
ketatnya
persaingan
ekonomi
membuat
pengusaha
harus
mengetahui
bagaimana
caranya
membuat sebuah usaha agar dapat
bertahan dan berkembang di era
globalisasi
ini. Pengusaha
perlu
mengupayakan peningkatan efisiensi
dan efektivitas pengelolaan usaha
agar mampu mencapai hasil yang
optimal dari sumber daya yang
tersedia. Hal ini berkaitan dengan
keputusan investasi yang tepat guna,
karena keputusan investasi yang tepat
akan memberi keuntungan finansial
bagi pemilik, dan keuntungan bagi
ekonomi makro. Sebaliknya jika
keputusan
investasi
salah
akan
berdampak macetnya sebuah usaha. besar maupun perusahaan dalam
skala kecil. Hal
ini
dapat
memberi
pengaruh
positif
dari
sisi
pertumbuhan
ekonomi. Namun
ketatnya
persaingan
ekonomi
membuat
pengusaha
harus
mengetahui
bagaimana
caranya
membuat sebuah usaha agar dapat
bertahan dan berkembang di era
globalisasi
ini. Pengusaha
perlu
mengupayakan peningkatan efisiensi
dan efektivitas pengelolaan usaha
agar mampu mencapai hasil yang
optimal dari sumber daya yang
tersedia. Hal ini berkaitan dengan
keputusan investasi yang tepat guna,
karena keputusan investasi yang tepat
akan memberi keuntungan finansial
bagi pemilik, dan keuntungan bagi
ekonomi makro. Sebaliknya jika
keputusan
investasi
salah
akan
berdampak macetnya sebuah usaha. (www.malangkota.go.id) Dewasa
ini
menyebabkan
munculnya sebuah peluang usaha
rumah pemondokan. Kota Malang
merupakan
potensi
daerah
yang
memiliki nilai jual dan daya saing
baik di tingkat regional maupun
nasional. Dalam era globalisasi dunia
pendidikan
menghadapi
berbagai
tantangan
dalam
menghadapi
perubahan
yaitu
dengan
adanya
tuntutan
masyarakat
memperoleh
fasilitas pendidikan yang baik dan
berkualitas. p
y
Mendirikan
suatu
usaha
diperlukan strategi yang tepat agar
usaha dapat terus bertahan dan
berkembang. Dalam
mempertahankan
sebuah
usaha,
pemilik perlu membuat perencanaan
dan membuat keputusan investasi
yang tepat. Banyak kendala yang
dihadapi oleh pengusaha kecil dan
menegah dalam menjalankan dan
mempertahankan usahanya. Maka
dari itu diperlukan analisis studi
kelayakan
bisnis
yang
dapat
memudahkan
pemilik
dalam
membuat perencanaan dan membuat
keputusan investasi, selain itu dapat
menilai sebuah usaha apakah usaha
tersebut layak atau tidak. Mendirikan
suatu
usaha
diperlukan strategi yang tepat agar
usaha dapat terus bertahan dan
berkembang. Dalam Persaingan dunia usaha di era
globalisasi
ini
telah
mengalami
kemajuan yang sangat pesat. Hal ini
ditandai dengan berdirinya usaha-
usaha
baru
mulai
dari
usaha
makanan, pakaian, percetakan, kost-
kostan, dan sebagainya. Banyaknya
peluang
usaha
dan
keinginan
manusia
untuk
mandiri
dalam
mendapatkan
penghasilan
dan
didukung dengan banyaknya jumlah
penduduk
menyebabkan
banyak
orang mendirikan sebuah unit usaha
baru, baik itu perusahaan dalam skala Studi
kelayakan
bisnis
merupakan
penelitian
terhadap
rencana bisnis yang tidak hanya
menganalisis layak atau tidaknya
bisnis di bangun, tetapi juga saat 51 kemudian dijadikan sebuah tempat
kost. dioperasikan
secara
rutin
dalam
rangka pencapaian keuntungan yang
maksimal untuk waktu yang tidak
ditentukan. Manfaat
dari
studi
kelayakan bisnis adalah pemilik
usaha dapat mengetahui sumber
informasi yang berkaitan dengan
usahanya dari berbagai aspek yang
dapat dijadikan sebuah keputusan
investasi. Beberapa
usaha
yang
menyediakan
jasa
kost
seperti
kategori kost yang telah dijelaskan
sebelumnya
berada
di
kawasan
Universitas Muhammadiyah Malang. Rumah pemondokan murni tanpa ada
pemiliknya yang tinggal bersama
adalah milik Bapak Rofiq dengan
fasilitas 22 kamar, kemudian rumah
pemondokan yang tinggal bersama
dengan pemilik adalah kost milik Ibu
Atnah dengan fasilitas 15 kamar, dan
yang terakhir ada seorang dengan
sengaja membeli rumah kemudian
dijadikan sebuah tempat kost adalah
milik Bapak sofi. Malang merupakan salah satu
kota yang menjadi tujuan perantauan
oleh orang-orang dari luar kota
maupun luar pulau. Salah satu
alasannya adalah karena banyaknya
sarana pendidikan tersedia. Hal itu
menyebabkan banyaknya pendatang
di Malang baik untuk menempuh
pendidikan
di
perguruan
tinggi,
maupun untuk bekerja. Maka dari itu,
banyak orang memanfaatkan peluang
tersebut dengan mendirikan usaha
rumah
pemondokan
(kost)
mahasiswa. (www.malangkota.go.id) membeli rumah untuk dijadikan
kost/kontrakan, pendapatannya tetap
di tahun 2011 sampai 2012 namun
naik pada 2013, hal inirelative kecil
dibandingan dengan
rumah
pemondokan
lainnyayang sama dengan kriteria
rumah pemondokan milik Bapak
Sofi. (www.malangkota.go.id) Usaha
semacam
ini
sangat
berpotensi
karena
minat
konsumen
yang
terus-menerus
mencari dan membutuhkan rumah
pemondokan (kost). p
Rumah pemondokan (kost)
mahasiswa di sekitar Universitas
Muhammadiyah malang ini memiliki
kelebihan dan kekurangan masing-
masing. Dari ketiga usaha tersebut,
bila ditinjau dari sisi pendapatan rata-
rata mengalami peningkatan setiap
tahunnya
dan
setiap
rumah
pemondokan (kost) tarifnya berbeda-
beda disesuaikan dengan fasilitas
yang
disediakan. Adapun
data
mengenai pendapatan usaha rumah
pemondokan
(kost)
di
sekitar
Universitas Muhammadiyah Malang
(UMM) dapat disajikan pada Tabel 1. Tabel 1 menunjukkan data
pendapatan
usaha
rumah
pemondokan (kost) mahasiswa di
sekitar Universitas Muhammadiyah
Malang menggunakan data 3 tahun
terakhir. Untuk kriteria murni kost,
pendapatan setiap tahun dari tahun
2011
sampai
2013
mengalami
kenaikan,
untuk
kriteria
rumah
tangga yang memanfaatkan kelebihan
ruangnya untuk kost pendapatan
selalu naik dari tahun 2011 sampai
2013 dan yang terakhir seorang yang Rumah pemondokan (kost)
mahasiswa di sekitar Universitas
Muhammadiyah malang ini memiliki
kelebihan dan kekurangan masing-
masing. Dari ketiga usaha tersebut,
bila ditinjau dari sisi pendapatan rata-
rata mengalami peningkatan setiap
tahunnya
dan
setiap
rumah
pemondokan (kost) tarifnya berbeda-
beda disesuaikan dengan fasilitas
yang
disediakan. Adapun
data
mengenai pendapatan usaha rumah
pemondokan
(kost)
di
sekitar
Universitas Muhammadiyah Malang
(UMM) dapat disajikan pada Tabel 1. Rumah
pemondokan
merupakan jasa penginapan bagi
sebagian orang yang membutuhkan
lokasi tempat tinggal sementara agar
mudah
menjangkau
lokasi
yang
dijadikan
tempat
menempuh
pendidikan ataupun tempat bekerja. Rumah pemondokan terbagi menjadi
beberapa
macam,
yaitu
rumah
pemondokan yang murni tanpa ada
pemiliknya yang tinggal bersama,
rumah pemondokan yang tinggal
bersama dengan pemilik rumah,
karena ada rumah tangga yang
memiliki
kelebihan
ruang
dan
memiliki potensi untuk dibuat usaha
kost, dan berikutnya ada seorang
dengan
sengaja
membeli
rumah (
)
p
j
p
Tabel 1 menunjukkan data
pendapatan
usaha
rumah
pemondokan (kost) mahasiswa di
sekitar Universitas Muhammadiyah
Malang menggunakan data 3 tahun
terakhir. Untuk kriteria murni kost,
pendapatan setiap tahun dari tahun
2011
sampai
2013
mengalami
kenaikan,
untuk
kriteria
rumah
tangga yang memanfaatkan kelebihan
ruangnya untuk kost pendapatan
selalu naik dari tahun 2011 sampai
2013 dan yang terakhir seorang yang 52 membeli rumah untuk dijadikan
kost/kontrakan, pendapatannya tetap
di tahun 2011 sampai 2012 namun
naik pada 2013, hal inirelative kecil
dibandingan
dengan
rumah
pemondokan
lainnyayang sama dengan kriteria
rumah pemondokan milik Bapak
Sofi. dilanjutkan
atau
tidak
sehingga
tujuan dari penelitian ini adalah
untuk mengetahui dan mengevaluasi
kelayakan usaha rumah pemondokan
(kost)
mahasiswa
di
sekitar
Universitas Muhammadiyah Malang. TINJAUAN PUSTAKA Penelitian
yang
dilakukan
oleh Wahyu Sutrisno yang diadakan
pada tahun 2013 yaitu evaluasi
kelayakan bisnis pada usaha jasa king
laundry. Penelitian ini bertujuan
untuk menganalis layak atau tidak
layak usaha jasa King Laundry. Penelitian ini menggunakan metode
penilaian investasi yaitu PP (Payback
Period), NPV (Net Present Value),
IRR (Internal Rate of Return), ARR
(Average rate of return), dan PI
(Profitability Indeks). Variabel yang
digunakan dalam peneltian ini adalah
aspek studi kelayakan bisnis yang
terdiri
dari
aspek
pasar
dan
pemasaran,
aspek
teknis
dan
teknologi, aspek manajemen dan
organisasi, aspek ekonomi dan social,
dan aspek keuangan. Tabel 1. Pendapatan usaha rumah
pemondokan tahun 2011-2013
(dalam Rupiah)
Sumbe : Rumah Pemondokan (kost) mahasiswa sekitar
UMM
Nama
2011 2012 2013
Keterangan
Kost
Bapak
Rofiq
(Murni
Kost)
117.960.000
128.520.000
139.080.000
Jumlah
kamar
sebanyak 22
Kost Bu
Atnah
( Rumah
Tangga)
65.700.000
69.300.000
72.000.000
Jumlah
kamar
sebanyak 15
Rumah
Bapak
Sofi
yang di
beli
untuk
kost
9.000.000
9.000.000
10.000.000
Jumlah
kamar 2 ,
kamar
mandi,
dapur, luas
tanah 136
m2, dan luas
bangunan 54
m2 Tabel 1. Pendapatan usaha rumah
pemondokan tahun 2011-2013 Dari
ketiga
rumah
pemondokan tersebut, pendapatan
yang diperoleh dari kost milik Bapak
Rofiq
menunjukkan
adanya
peningkatan, dan pendapatan milik
Ibu Atnah mengalami peningkatan
namun kenaikannya tidak sebanyak
kost Bapak Rofiq, dan rumah milik
Bapak Sofi, harga yang ditawarkan
terlalu kecil sehingga pendapatan
yang diperoleh dari rumah tersebut
sangat
minim
meskipun
ada
kenaikan. Berdasarkan
hasil
analisis
capital
budgeting
menunjukkan
bahwa Nilai Payback Period (PP) <
umur investasi 5 tahun yaitu sebesar
3 tahun 5 bulan 7 hari. Nilai Net
Present
Value
(NPV)
sebesar
Rp1.194.615,53, nilai Internal Rate
of Return (IRR) sebesar 18,678% >
COC, Nilai Average Rate of Return
(ARR) sebesar 62,78% > tingkat
keuntungan yang diharapkan dan PI
sebesar 1,03 > 1. Dari hasil penelitian
yang
dilakukan
tersebut
dapat
disimpulkan bahwa usaha jasa King
Laundry layak untuk dilanjutkan. Berdasarkan
data
yang
diperoleh, menunjukkan usaha rumah
pemondokan cenderung meningkat
dan selalu diminati oleh pasar. TINJAUAN PUSTAKA Hal
ini perlu ditindak lanjuti apakah
ketiga
usaha
tersebut
layak Investasi menurut Haming
dan
Basalamah
(2003:3)
secara
umum diartikan sebagai keputusan 53 Sucipto(2010:4)
dalam
bukunya
ruang lingkup studi kelayakan bisnis
menyatakan terdapat tiga manfaat
yang ditimbulkan dari adanya studi
kelayakan bisnis, yaitu : 1) Manfaat
financial,2)Manfaat
ekonomi
nasional, 3) Manfaat sosial mengeluarkan
dana
pada
saat
sekarang untuk membeli aktiva riil
(tanah,
rumah,
mobil,
dan
sebagainya) atau aktiva keuangan
(saham,obligasi, reksadana, wesel
dan sebagainya) dengan tujuan untuk
mendapatkan penghasilan yang lebih
besar di masa yang akan datang. Aspek pasar dan pemasaran
merupakan
aspek
yang
harus
diperhatikan dalam memulai sebuah
usaha, karena sekecil apapun sebuah
usaha harus memiliki pasar yang
jelas dan memiliki strategi serta
kebijaksanaan
pengelolaan
pemasaran yang tepat guna. Aspek
pemasaran berisi
rencana-rencana
yang
akan
dilaksanakan
dalam
pemasaran produk, serta beberapa
data
yang
mendukung
rencana
tersebut. Studi
pasar
akan
memberikan
gambaran
mengenai
intensitas
persaingan,
informasi
tentang kebutuhan dan keinginan
konsumen,
pendapatan
rata-rata
calon
konsumen,
ketersediaan
saluran distribusi, dan kondisi sarana
angkutan. Studi kelayakan bisnis dalam
arti
sempit
adalah
merupakan
penelitian terhadap rencana bisnis
yang tidak hanya menganalisis layak
atau tidaknya suatu bisnis dibangun,
tetapi juga pada saat bisnis tersebut
beroperasi
secara
rutin
dengan
berhasil
untuk
memperoleh
keuntungan yang maksimal secara
ekonomis. Studi kelayakan bisnis dalam
arti luas adalah penelitian yang
mendalam tentang dapat tidaknya
atau layak tidaknya rencana bisnis
dilakukan
dengan
berhasil
dan
menguntungkan
(tidak
hanya
keuntungan
ekonomis/finansial),
akan
tetapi
cenderung
melihat
kemanfaatan yang lebih luas (makro)
bagi daerah atau lokasi dimana bisnis
tersebut dilaksanakan. Aspek Teknis dan Teknologi
merupakan aspek yang berkaitan
dengan kapasitas proyek, lokasi, tata
letak alat produksi, bentuk bangunan
(bertingkat atau tidak), kajian atas
bahan dan sumbernya, desain produk,
dan analisis biaya produksi. Kasmir dan Jakfar (2003)
dalam
bukunya
studi
kelayakan
bisnis menyatakan studi kelayakan
bisnis adalah suatu kegiatan yang
mempelajari
secara
mendalam
tentang suatu kegiatan atau usaha
atau bisnis yang akan dijalankan,
dalam rangka menentukan layak atau
tidak usaha tersebut dijalankan. Aspek
manajemen
dan
organisasi merupakan aspek yang
cukup
penting
dianalisis
untuk
kelayakan suatu usaha. Tujuan studi
kelayakan aspek manajemen dan
organisasi adalah untuk mengetahui
apakah dalam pembangunan dan
implementasi
bisnis
dapat
direncanakan,
dilaksanakan,
dan
dikendalikan sehingga bisnis rencana
bisnis layak atau tidak layak. TINJAUAN PUSTAKA Tujuan
pentingnya
melakukan studi kelayakan bisnis
menurut
Sucipto
(2010:5):
Menghindari
risiko
kerugian,
memudahkan
perencanaan,
memudahkan pelaksanaan pekerjaan,
memudahkan
pengawasan,
memudahkan pengendalian.Menurut Tujuan
pentingnya
melakukan studi kelayakan bisnis
menurut
Sucipto
(2010:5):
Menghindari
risiko
kerugian,
memudahkan
perencanaan,
memudahkan pelaksanaan pekerjaan,
memudahkan
pengawasan,
memudahkan pengendalian.Menurut 54 menyamkan nilai sekarang investasi
dengan niali sekarang penerimaan-
penerimaan kas bersih. Aspek Ekonomi dan Sosial
merupakan aspek studi kelayakan
bisnis
yang
bertujuan
untuk
mengetahui seberapa jauh lingkungan
luar
memberikan
peluang
dan
sekaligus ancaman bagi rencana
bisnis. Selain
itu
juga
untuk
mengetahui apa saja yang dapat
disumbangkan oleh proyek bisnis
bagi lingkungan luar, jika bisnis
sudah direalisasi. Metode Payback Period (PP)
merupakan teknik penilaian terhadap
jangka waktu (periode) pengembalian
investasi suatu usaha dengan cara
mengukur
seberapa
cepat
suatu
investasi kembali, dengan demikian
metode
ini
mengukur
rapidity
kembalinya dana investasi bukan
mengukur profitability. Aspek keuangan merupakan
aspek yang digunakan untuk menilai
keuangan
perusahaan
secara
menyeluruh dan merupakan salah
satu aspek yang sangat penting untuk
dinilai
kelayakannya. Tujuan
penilaian aspek keuangan adalah
untuk
mengetahui
prakiraan
pendanaan dan aliran kas proyek
bisnis, sehingga dapat diketahui
layak atau tidaknya suatu rencana
bisnis
yang
dimaksud. Aspek
keuangan menilai sumber dana, biaya
investasi, estimasi aliran kas (Cash
Flow), biaya modal (Cost of Capital),
Kriteria Penilaian Investasi. Metode Average Rate of
Return (ARR) adalah metode yang
mengukur seberapa besar tingkat
keuntungan rata-rata yang diperoleh
dari suatu investasi dengan cara
membandingkan laba setelah pajak
(EAT)
terhadap
rata-rata
investasinya. Metode Profitability
Index
(PI)adalah
metode
yang
menghitung
perbandingan
antara
nilai sekarang penerimaan kas bersih
di masa yang akan datang (selama
umur
investasi)
dengan
nilai
sekarang
investasi
(initial
investment). Metode Net Present Value
(NPV)
adalah
metode
yang
menghitung
selisih
antara
nilai
sekarang investasi (capital outlays)
dengan nilai sekarang penerimaan-
penerimaan kas bersih (present value
of proceed) baik dari operastional
cash flow maupun dari terminal cash
flow pada masa yang akan datang
(selama umur investasi). METODE PENELITIAN Penelitian ini menggunakan
jenis penelitian studi kasus yakni
jenis penelitian yang relevan dari
hasil penelitian pada obyek penelitian
yang
mencoba
mengetahui
dan
memecahkan
permasalahan
yang
dihadapi
perusahaan.Data
yang
digunakan
dalam
penelitian
ini
adalah
data
primer
dan
data
sekunder. Data primer adalah data
yang diperoleh atau dikumpulkan
langsung dari sumber asli (tanpa
perantara). Dalam hal ini data primer
tersebut berupa hasil wawancara
yang dilakukan oleh peneliti kepada
pemilik usaha Rumah Pemondokan
(kost) mahasiswa. Metode
Internal
Rate
of
Return (IRR) adalah tingkat bunga
yang menjadikan NPV sama dengan
nol, karena present value dari cash
flow pada tingkat bunga tersebut
sama dengan internal investasinya. Metode Internal Rate of Return
(IRR)
adalah
metode
yang
menghitung
tingkat
bunga
yang 55 pada aspek pasar dan pemasaran
adalah
aspek pasar
memberikan
informasi permintaan pasar (Market
Demand) atas hasil usaha dalam
penelitian
ini
berupa
volume
penjualan rumah pemondokan (kost)
mahasiswa, tarif yang dikenakan,
segmentasi dan target pasar, dan
pesaing usaha. sedangkan aspek
pemasaran memberikan informasi
tentang strategi dan siasat pemasaran
atau promosi yang dilakukan oleh
pemilik kost. Data sekunder adalah data
yang diperoleh dalam bentuk yang
sudah jadi, sudah dikumpulkan dan
diolah oleh pihak lain. Data sekunder
dalam penelitian ini yaitu data
laporan keuangan usaha, sumber
modal yang digunakan dan data
pendapatan pada rumah pemondokan
(kost) mahasiswa. Dalam mengumpulkan data
yang diperlukan untuk penelitian,
maka Teknik pengumpulan data yang
dilakukan dalam penelitian ini adalah
dengan
melakukan
observasi,
wawancara,
dan
dokumentasi. Observasi
yaitu
menggunakan
pengamatan
secara
langsung
di
lapangan. Hal ini dilakukan dalam
rangka
melakukan
pengamatan
secara
langsung
kegiatan
usaha
rumah
pemondokan
(kost)
mahasiswa. Analisis aspek teknis dan
teknologi
memberikan
informasi
deksripsi
produk,
fasilitas
yang
tersedia, biaya operasi, dan tata letak
(layout) kost mahasiswa. Aspek
organisasi
dan
manajemen
Memberikan informasi perencanan
(planning),
pengorganisaian
(organizing),
pelaksanaan
(actuating),
dan
pengawasan
(controlling). tugas dan tanggun
jawab personil, dan gaji insentif. Wawancara/interview, dalam
penelitian ini teknik pengumpulan
data
di
lakukan
dengan
cara
melakukan
tanya
jawab
dengan
pemilik rumah pemondokan (kost). Hal ini dilakukan dalam rangka
mengambil data mengenai sejarah
berdirinya
rumah
pemondokan
(kost), gambaran umum usaha rumah
pemondokan
(kost)
mahasiswa. Dokumentasi dalam penelitian ini
proses pengumpulan data dilakukan
dengan cara mempelajari dokumen
pendukung serta mencatat secara
langsung data yang tersedia pada
obyek penelitian. Hal ini dilakukan
dalam
rangka
memperoleh
data
berupa: Laporan keuangan, data
jumlah kamar, data tarif rumah
pemondokan, data fasilitas rumah
pemondokan. METODE PENELITIAN Analisis aspek ekonomi dan
sosial mengungkap pengaruh dan
manfaat yang didapat dari berdirinya
usaha rumah pemondokan (kost)
mahasiswa terhadap perkembangan
daerah dan penduduk usaha rumah
pemondokan (kost) yang terkait, dan
biaya sosial yang telah dikeluarkan. Aspek Keuangan Menentukan
pembiayaan proyek, berapa besarnya
kebutuhan dana dan sumbernya,
biaya dana baik untuk keperluan
investasi maupun untuk keperluan
modal kerja proyeksi arus kas bersih,
biaya penyusutan, bunga pinjaman,
dan terminal cash flow. Melakukan
penilaian investasi dengan kriteria
penilaian investasi yang terdiri dari
metode Net Present Value (NPV),
metode Internal Rate of Raturn
(IRR), metode Payback Periode, Data yang telah terkumpul
dapat
dianalisis
dengan
menggunakan teknik Analisis yaitu 56 banyak konsumen. Tidak ada strategi
pemasaran secara khusus, hanya
memberi informasi dengan memberi
banner di rumah tersebut bahwa
rumah terebut disewakan. Hal itu
cukup berpengaruh, karena banyak
konsumen yang menghubungi no
telepon yang tertera pada banner,
disamping itu pemilik menitipkan
pada petugas keamanan agar ikut
mempromosikan rumah Bapak Sofi
kepada
konsumen
dan
hasilnya
rumah tersebut sejak di kontrakan
selalu ada konsumen yang berminat. metode Average Rate of Return
(ARR), metode Profitability Index
(PI). metode Average Rate of Return
(ARR), metode Profitability Index
(PI). HASIL
PENELITIAN
DAN
PEMBAHASAN Berdasarkan aspek pasar dan
pemasaran, rumah pemondokan milik
Bapak Rofiq dinyatakan layak karena
produk yang ditawarkan telah sesuai
dengan harga yang ditawarkan, fasilitas memadai dengan harga yang
terjangkau. Strategi yang dilakukan
dalam memasarkan usaha rumah
pemondokan
seperti
promosi
penjualan
dengan
menggunakan
brosur,
juga
menyebar
dengan
sendirinya dari mulut ke mulut
memberi
pengaruh
terhadap
permintaan
usaha
rumah
pemondakan milik Bapak Rofiq
selebihnya hanya memasang papan
sebagai informasi terima kost. Berdasarkan Aspek Teknis
dan Teknologi, Rumah pemondokan
milik Bapak Sofi dinyatakan layak
karena
rumah
pemondokan
ini
menyediakan
fasilitas
lengkap
meliputi kamar 4mx3m sebanyak 19
kamar, dan 3 kamar lainnya 5mx4m
setiap kamar terdapat tempat tidur,
lemari pakaian, meja, kursi dengan
fasilitas pendukung seperti ruang
tamu,
ruang
santai,
dapur
dan
beberapa kamar mandi, dan lahan
parkir, lokasi rumah pemondokan
sangat strategis dekat dengan jalan
raya dan kampus UMM. Sama halnya dengan rumah
pemondokan
milik
Ibu
Atnah,
dengan memasang harga yang lebih
murah namun fasilitas disesuaikan
dengan
harga,
cukup
menarik
perhatian
karena
terkesan
lebih
murah dengan sistem pembayaran
bulanan. Promosi penjualan dengan
menyebarkan
brosur
semenarik
mungkin memberi pengaruh positif,
selebihnya informasi tersebar dari
mulut ke mulut dan memasang papan
sebagai informasi terima kost, maka
dilihat
dari
aspek
pasar
dan
pemasaran usaha rumah pemondokan
milik Ibu Atnah dinyatakan layak. Rumah pemondokan milik
Ibu
Atnah
berdasarkan
aspek
teknologi dinyatakan layak karena
menyediakan fasilitas kamar 4mx4m
sebanyak 15 kamar, setiap kamar
mendapat tempat tidur, meja dan
lemari dengan harga yang sesuai. Fasilitas pendukung lainnya adalah
lahan parkir yang luas, dapur, ruang
tamu, ruang santai, dan beberapa
kamar
mandi. Lokasi
rumah
pemondokan tidak dekat dengan
jalan raya tetapi masih mudah
mengaskes apa pun karena banyak
penjual makanan, percetakan, sarana
ibadah, sarana kesehatan dan lain-
lain. Rumah pemondokan milik
Bapak Sofi disewakan dengan harga
yang murah setiap tahunnya fasilitas
memadai dan berada di kawasan
perumahan yang asri, dengan kondisi
bangunan yang baru dan harga yang
terjangkau mampu menarik perhatian 57 Rumah pemondokan milik
Bapak Sofi dari aspek teknis dan
teknologi dinyatakan layak karena
lokasi rumah yang berada di kawasan
perumahan
New
bukit
villa
sengkaling memberi kesan asri dan
memilik pemandangan yang indah. Bangunan rumah yang nyaman dan
baru dengan luas bangunan 54 m2,
luas tanah 136 m2, jumlah kamar 2,
kamar mandi 1, ruang tamu, dapur,
halaman depan dan belakang yang
luas dengan fasilitas 2 tempat tidur,
meja, dan kursi. Rumah ini dekat
dengan
pos
keamanan
sehingga
mudah mengawasi keamanan dari
rumah ini. HASIL
PENELITIAN
DAN
PEMBAHASAN Bila dilihat dari Hasil analisis Net
Present Value (NPV) positif atau > 0
maka
dinyatakan
layak. Nilai
Payback Period (PP) kurang dari
umur ekonomis maka PP dinyatakan
layak. Tabel 3. Kesimpulan Hasil Kriteria
Kelayakan Investasi Rumah
Pemondokan Milik Ibu Atnah
Kriteria
Investasi
Hasil
Keterangan
NPV
-
170.035.625,2
Tidak layak karena
NPV bernilai negatif
-170.035.625,2< 0
IRR
4,4571%
Tidak layak karena
IRR 4,4571% < 15%
PP
32,17
Tidak layak karena
PP 32 tahun, 2 bulan,
12 hari lebih dari
umur ekonomis yaitu
20 tahun
ARR
2%
Tidak layak karena
ARR 2< r = 15%
PI
0,32
Tidak layak karena
PI 0,32 < 1
Sumber: Data keuangan rumah pemondokan
milik Ibu Atnah
Hasil nilai Internal Rate of
Return (IRR) lebih besar > COC
maka dinyatakan layak, nilai Average
Rate of Return (ARR) lebuh besar
dari
tingkat
keuntungan
yang
diharapkan maka dinyatakan layak. dan nilaiProfitability Index (PI) ≥ 1
maka usaha ini dinyakan layak. Pengaruh lainnya adanya perubahan
lingkungan
dengan
bermunculan
usaha-usaha sejenis maupun tidak
sejenis,
dan
adanya
perubahan
lingkungan secara fisik yang lebih
baik. rumah pemondokan milik Bapak
Rofiq
dari
aspek
keuangan
dinyatakan layak. Tabel 3menunjukkan hasil
analisis usaha rumah pemondokan
milik Ibu Atnah menunjukan bahwa
biaya
investasi
yang
telah
dikeluarkan dari modal sendiri dan
pinjaman
bank,
bisa
menjadi
menguntungkan dari tahun ke tahun. Bila dilihat dari Hasil analisis Net
Present Value (NPV) positif atau > 0
maka
dinyatakan
layak. Nilai
Payback Period (PP) kurang dari
umur ekonomis maka PP dinyatakan
layak. Ketiga rumah pemondokan
tersebut
memiliki
biaya
khusus
yangdipergunakan untuk biaya sosial
sebagai aksi sosial yang dilakukan
oleh pemilik usaha. Tabel 2. Kesimpulan Hasil Kriteria Kelayakan Rumah Pemondokan Kelayakan Rumah Pemondokan y
Milik Bapak Rofiq
Kriteria
Investasi
Hasil
Keterangan
NPV
226.968.193,1
Layak karena NPV
bernilai positif
226.968.193,1 > 0
IRR
17,2063 %
Layak karena IRR
17,2063 % > 15%
PP
6,104
Layak karena PP yaitu 6
tahun, 1 bulan, 7 hari <
umur ekonomis yaitu 20
tahun
ARR
43%
Layak karena ARR 43%
> r = 15%
PI
1,44
Layak karena PI 1,44 ≥ 1
Sumber: Data keuangan rumah pemondokan
milik Bapak Rofiq Milik Bapak Rofiq Tabel 3. HASIL
PENELITIAN
DAN
PEMBAHASAN sehingga
memudahkan
dalam
melakukan pengawasan, dan dibantu
dengan 1 orang tenaga kerja sebagai
pembantu untuk mengawasi dan
membersihkan rumah pemondokan. Pemberian gaji yang disesuaikan
dengan
jenis
pekerjaan
beserta
tambahan tunjangan hari raya dan
fasilitas tempat tinggal diharapkan
dapat memotivasi kerja bagi pekerja. p
j
g p
j
Rumah pemondokan milik
Bapak Sofi dilihat dari aspek
manajemen dan organisasi layak
karena meskipun pemilik tinggal di
luar kota hanya bisa memantau pada
saat hari libur, dan memantau via
telepon atau sms kepada konsumen,
usaha ini tetap berjalan. Tidak ada
tenaga
kerja
yang
dipercayakan
untuk mengawasi rumah milik Bapak
Sofi karena memang tidak diperlukan
tenaga kerja dalam usaha ini. Selama
ini pemilik menggunakan sistem
kepercayaan
dengan
melakukan
pendekatan kepada konsumen agar
terjalin komunikasi yang baik dan
hubungan yang baik. Berdasarkan
Aspek
manajemen dan organisasi, rumah
pemondokan milik Bapak Rofiq
dinyatakan layak karena dilihat dari
Planning, Organizing, Actuating, dan
Controling pemilik menjalankannya
dengan tepat, dengan jumlah tenaga
kerja 2 orang cukup melancarkan
aktivitas usaha rumah pemondokan. Jumlah Karyawan akan ditambah
apabila pemilik melakukan ekspansi. Dengan
melakukan
pengawasan
sesering mungkin, respon yang cepat,
dan terbuka terhadap kritik dan saran
dari
konsumen
membuat
rumah
pemondokan ini terus mengalami
kemajuan. Pemberian
gaji
yang
disesuaikan dengan jenis pekerjaan
beserta tambahan tunjangan hari raya
diharapkan dapat memotivasi kerja
bagi pekerja. Berdasarkan Aspek ekonomi
dan sosial, rumah pemondokan milik
Bapak
Rofiq
dinyatakan
layak,
karena banyak dampak positif yang
ditimbulkan dari berdirinya rumah
pemondokan milik Bapak Rofiq, baik
keuntungan bagi pemilik maupun
banyak
pihak
luar
baik
secara
finansial maupun secara fisik. Begitu
pula dengan rumah pemondokan
milik Ibu Atnah, berdirinya usaha ini
memberi dampak positif bagi pemilik
maupun pihak-pihak lain. Rumah pemondokan milik
Ibu
Atnah
dilihat
dari
aspek
manajemen dan organisasi dinyatkan
layak, karena pemilik melakukan
perencanaan
hingga
mengambil
keputusan berdasarkan pengamatan
yang dilakukan sehari-hari, karena
tinggal satu atap dengan konsumen, Rumah pemondokan milik
Bapak sofi juga memberi dampak
positif terutama bagi pemilik secara
finansial dan dengan adanya yang
menempati rumah tersebut, akan ada
yang memelihara rumah tersebut. 58 n
n
k
n
h
n
s
l
n
n
l
n
n
h
i
. t
0
k
r
. n
a
e
t
rumah pemondokan milik Bapak
Rofiq
dari
aspek
keuangan
dinyatakan layak. Tabel 3menunjukkan hasil
analisis usaha rumah pemondokan
milik Ibu Atnah menunjukan bahwa
biaya
investasi
yang
telah
dikeluarkan dari modal sendiri dan
pinjaman
bank,
bisa
menjadi
menguntungkan dari tahun ke tahun. HASIL
PENELITIAN
DAN
PEMBAHASAN Kesimpulan Hasil Kriteria
Kelayakan Investasi Rumah
Pemondokan Milik Ibu Atnah
Kriteria
Investasi
Hasil
Keterangan
NPV
-
170.035.625,2
Tidak layak karena
NPV bernilai negatif
-170.035.625,2< 0
IRR
4,4571%
Tidak layak karena
IRR 4,4571% < 15%
PP
32,17
Tidak layak karena
PP 32 tahun, 2 bulan,
12 hari lebih dari
umur ekonomis yaitu
20 tahun
ARR
2%
Tidak layak karena
ARR 2< r = 15%
PI
0,32
Tidak layak karena
PI 0,32 < 1
Sumber: Data keuangan rumah pemondokan
milik Ibu Atnah Tabel 3. Kesimpulan Hasil Kriteria Tabel 3. Kesimpulan Hasil Kriteria
Kelayakan Investasi Rumah
Pemondokan Milik Ibu Atnah Berdasarkan Tabel 2 hasil
analisis usaha rumah pemondokan
milik
Bapak
Rofiq
menunjukan
bahwa biaya investasi yang telah
dikeluarkan
bisa
menjadi
menguntungkan dari tahun ke tahun. Bila dilihat dari Hasil analisis Net
Present Value (NPV) positif atau >0
maka dinyatakan layak. Payback
Period
(PP) diperoleh < umur
ekonomis maka dinyatakan layak. Hasil nilai Internal Rate of Return
(IRR) lebih besar > COC maka
dinyatakan layak, nilai Average Rate
of
Return
(ARR)
>
tingkat
keuntungan yang diharapkan yaitu
maka
dinyatakan
layak. dan
nilaiProfitability Index (PI) ≥ 1 maka
usaha ini dinyakan layak. Hasil
analisis menyatakan bahwa usaha Sumber: Data keuangan rumah pemondokan
milik Ibu Atnah Hasil nilai Internal Rate of
Return (IRR) lebih besar > COC
maka dinyatakan layak, nilai Average
Rate of Return (ARR) lebuh besar
dari
tingkat
keuntungan
yang
diharapkan maka dinyatakan layak. dan nilaiProfitability Index (PI) ≥ 1
maka usaha ini dinyakan layak. Berdasarkan
hasil
analisis
menyatakan bahwa usaha rumah
pemondokan milik Ibu Atnah dilihat
dari aspek keuangan dinyatakan
layak. 59 Tabel 4menunjukkan hasil
analisis usaha rumah pemondokan
milik Bapak Sofi menunjukan bahwa
modal yang telah dikeluarkan untuk
usaha kurang menguntungkan. Dari
sisi pertumbuhan selalu meningkat
namun penerimaan yang diterima
kurang
bila
dikurangi
biaya
pengeluaran di awal-awal usaha. Bila
dilihat dari Nilai Net Present Value
(NPV) Hasil analisis Net Present
Value (NPV) negatif atau < 0 maka
dinyatakan
tidak
layak. Nilai
Payback Period (PP) diperoleh lebih
dari umur ekonomis maka dinyatakan
tidak layak. Berdasarkan hasil dari aspek
keuangan, khusunya untuk rumah
pemondokan milik Bapak Sofi, perlu
ditindak lanjuti mengapa hasil dari
aspek keuangan tidak layak. Hal ini
dikarenakan untuk tipe rumah yang
dimiliki
Bapak
Sofi
seharusnya
memasang tarif lebih tinggi agar
mendapat
keuntungan
di
awal
pendirian usaha. Dalam menentukan
harga perlu dipertimbangkan baik-
baik
dan
disesuiakan
dengan
spesifikasi rumah milik Bapak Sofi,
agar
tidak
merugi. HASIL
PENELITIAN
DAN
PEMBAHASAN Karena
pendapatan yang diperoleh relative
minim,
sebaiknya
tidak
mengeluarkan
biaya
yang
tidak
diperlukan terlebih dahulu karena
pendapatan
lebih
kecil
dari
pengeluaran. Tabel 4. Kesimpulan hasil
kriteriakelayakan Investasi
RumahPemondokan Milik Bapak
Sofi
Kriteria
Investasi
Hasil
Keterangan
NPV
22.370.869,3
Layak karena NPV
bernilai positif Rp
22.370.869,3 > 0
IRR
18
Layak karena IRR 18%
> 17%
PP
12,23
Layak karena PP 12
tahun, 2 bulan, 23 hari
kurang dari umur
ekonomis yaitu 20 tahun
ARR
38%
Layak karena ARR 38%
> r = 17%
PI
1,058
Layak karena PI 1,058 ≥
1
Sumber:
Data
keuangan
rumah
pemondokan milik Bapak Sofi. Tabel 4. Kesimpulan hasil
kriteriakelayakan Investasi
RumahPemondokan Milik Bapak SIMPULAN Berdasarkan
hasil
analisis
aspek pasar dan pemasaran, aspek
teknis
dan
teknologis
(produksi/operasi) aspek manajemen
dan organisasi, aspek ekonomi dan
sosial, aspek keuangan menunjukan
bahwa usaha rumah pemondokan
milik Bapak Rofiq dan milik Ibu
Atnah layak untuk dilanjutkan, untuk
rumah pemondokan milik Bapak Sofi
berdasarkan hasil analisis aspek pasar
dan pemasaran, aspek teknis dan
teknologis (produksi/operasi), aspek
manajemen dan organisasi, aspek
ekonomi dan sosial usaha rumah
pemondokan milik Bapak Sofi layak
untuk dilanjutkan, sedangkan dari
aspek keuangan menunjukan bahwa
usaha rumah pemondokan milik
Bapak
Sofi
tidak
layak
untuk
dilanjutkan. Sumber:
Data
keuangan
rumah
pemondokan milik Bapak Sofi. Sumber:
Data
keuangan
rumah
pemondokan milik Bapak Sofi. Hasil nilai Internal Rate of
Return
(IRR)
<
COC
maka
dinyatakan tidak layak, nilai Average
Rate of Return (ARR) < tingkat
keuntungan yang diharapkan maka
dinyatakan
tidak
layak. dan
nilaiProfitability Index (PI) < 1 maka
usaha ini dinyatakan tidak layak. Dari hasil analisis menyatakan bahwa
usaha rumah pemondokan milik
Bapak
Sofi
dilihat
dari
aspek
keuangan dinyatakan tidak layak. 60 DAFTAR PUSTAKA DAFTAR PUSTAKA Kasmir, S.E., MM dan Jakfar, S.E., MM
2003. Studi Kelayakan Bisnis. Prenada Media, Jakarta Timur. Abdul Halim. 2005. Analisis Investasi. Salemba Empat, Jakarta. Murdifin Haming, S.E., M.Si dan Salim
Basalamah, S.E., M.Si. 2003. Studi
Kelayakan Investasi Proyek dan
Bisnis. Penerbit PPM, Jakarta. Agus
Sucipto,
M.M,
2010. Studi
Kelayakan
Bisnis.UIN-Maliki
Press, Malang. Santoso, Singgih dan Tjiptono, Fandy,
2001. Riset Pemasaran: Konsep
dan Aplikasi dengan SPSS. PT Elex
Media Komputindo, Jakarta. Didit Herlianto dan Triani pujiastusi, 2009. Studi Kelayakan Bisnis. Graha
Ilmu, Yogyakarta Wiliam,
Gordon
dan
Jeffry,
1995. Investment. Prentice Hall Inc. Gitman, Lawrence J. 2000. Principles of
Managerial
Finance,
edisi
9. Addisom Wesley Publising Co,
New York. Wahyu Sutrisno, 2013.Evaluasi Kelayakan
Bisnis pada Usaha Jasa King
Laundry.Universitas
Muhammadiyah Malang. H. Indriyo Gitosudarmo, M.Com (Hons)
dan Drs.H Basri, M.M. 2002. Manajemen
Keuangan Edisi 4. Penerbit BPFE-
YOGYAKARTA. UU
no. 17
2002
Tentang
Pajak
Penghasilan Pasal 11. http://www.malangkota.go.id/mlg. Perkembangan Pendidikan di kota
Malang. Diunduh pada tanggal 18
November 2013. Husein Umar, 2003. Studi Kelayakan
dalam Bisnis Jasa. PT Gramedia
Pustaka Utama, Jakarta. http://www.PenyusutanAktivaTetapMenur
utPajakTerbaruAkuntansiItuMudah
.com.Penyusutan
aktiva
tetap
berdasarkan pajak. Diunduh pada
tanggal 04 maret 2014. Iman Soeharto, 1990. Manajemen Proyek
industri (persiapan, pelaksanaan,
pengelolaan). Penerbit erlangga,
Jakarta.
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https://openalex.org/W2515398420
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https://europepmc.org/articles/pmc4997700?pdf=render
|
English
| null |
Physical activity and relaxation in the work setting to reduce the need for recovery: what works for whom?
|
BMC public health
| 2,016
|
cc-by
| 12,219
|
Abstract Background: To recover from work stress, a worksite health program aimed at improving physical activity and
relaxation may be valuable. However, not every program is effective for all participants, as would be expected
within a “one size fits all” approach. The effectiveness of how the program is delivered may differ across individuals. The aim of this study was to identify subgroups for whom one intervention may be better suited than another by
using a new method called QUalitative INteraction Trees (QUINT). Methods: Data were used from the “Be Active & Relax” study, in which 329 office workers participated. Two
delivery modes of a worksite health program were given, a social environmental intervention (group motivational
interviewing delivered by team leaders) and a physical environmental intervention (environmental modifications). The main outcome was change in Need for Recovery (NFR) from baseline to 12 month follow-up. The QUINT
method was used to identify subgroups that benefitted more from either type of delivery mode, by incorporating
moderator variables concerning sociodemographic, health, home, and work-related characteristics of the participants. Results: The mean improvement in NFR of younger office workers in the social environmental intervention group was
significantly higher than younger office workers who did not receive the social environmental intervention (10.52;
95 % CI: 4.12, 16.92). Furthermore, the mean improvement in NFR of older office workers in the social environmental
intervention group was significantly lower than older office workers who did not receive the social environmental
intervention ( −10.65; 95 % CI: −19.35, −1.96). The results for the physical environmental intervention indicated that the
mean improvement in NFR of office workers (regardless of age) who worked fewer hours overtime was significantly
higher when they had received the physical environmental intervention than when they had not received this type of
intervention (7.40; 95 % CI: 0.99, 13.81). Finally, for office workers who worked more hours overtime there was no effect
of the physical environmental intervention. Conclusions: The results suggest that a social environmental intervention might be more beneficial for younger
workers, and a physical environmental intervention might be more beneficial for employees with a few hours overtime
to reduce the NFR. * Correspondence: erwintak2@gmail.com
1Netherlands Organisation for Applied Scientific Research TNO, Schipholweg
77, Leiden, The Netherlands
Full list of author information is available at the end of the article Physical activity and relaxation in the work
setting to reduce the need for recovery:
what works for whom? Margriet A. G. Formanoy1, Elise Dusseldorp1,2,3, Jennifer K. Coffeng4, Iven Van Mechelen2, Cecile R. L. Boot4,5,
Ingrid J. M. Hendriksen1,5 and Erwin C. P. M. Tak1* Margriet A. G. Formanoy1, Elise Dusseldorp1,2,3, Jennifer K. Coffeng4, Iven Van Mechelen2, Cecil
Ingrid J. M. Hendriksen1,5 and Erwin C. P. M. Tak1* © 2016 The Author(s). 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. Formanoy et al. BMC Public Health (2016) 16:866
DOI 10.1186/s12889-016-3457-3 Formanoy et al. BMC Public Health (2016) 16:866
DOI 10.1186/s12889-016-3457-3 Background Not taking enough
time to relax increased the NFR and was associated with
exhaustion [14]. Furthermore, within a group of service
employees, enjoyable and restful within workday breaks
improved their need for recovery [15]. The results of the “Be Active & Relax” project showed
that none of the delivery modes were effective in redu-
cing the NFR [17]. Therefore, it was not recommended
to implement the current interventions because the NFR
did not significantly differ as a result of applying either
delivery mode. And although some significant effects
were found on work-related outcomes, these were only
small (e.g., contextual performance, dedication, task per-
formance, and absorption [21]). To identify subgroups for whom one intervention might
be better suited than another, several statistical methods
are available. Based on the characteristics of the partici-
pants measured at baseline (so called moderator variables),
the subgroups can be determined a priori (e.g., based on
previous literature; also called confirmatory analysis) or a
posteriori, that is, induced from the data (also called ex-
ploratory analysis). The classical approaches to identify
subgroups are analysis of variance with paired compari-
sons, and multiple regression analysis with pre-defined
interaction effects. A disadvantage of these methods is that
they can handle only a small number of moderator vari-
ables. For the situation of many potential moderator vari-
ables that might interact with the treatment variable and
no clear a priori hypotheses, several new methods were de-
veloped in the last decade based on recursive partitioning
(see [22] for an enumeration), among which QUalitative
INteraction Trees (QUINT) [23, 24]. These new methods
can handle a large number of potential moderators in the
analysis. Most of these methods optimize treatment-
subgroup interactions in general, while QUINT aims at
only identifying subgroups that differ in direction of the
treatment effect (so called qualitative treatment-subgroup
interactions). Because our interest was in discovering such
subgroups, QUINT was used in this study. However, some studies did not succeed in improving
the need for recovery by a worksite health program [16]
or the effect did not sustain in the long-term [17]. One
possible cause of this phenomenon could be that the
studies focused on the effect(s) of a worksite health pro-
gram for all workers included in the study (i.e., the treat-
ment main effect). However, the question is whether the
assumption that a program is equally effective for all
workers is realistic. Background Therefore, it might be very useful to define subgroups
that may benefit more from a certain worksite health
program. The present study focuses on the identification
of such subgroups using data from the “Be Active &
Relax” study. Work stress and work-related health problems are a
major problem in modern organizations [1]. When
chronically exposed to high levels of psychological job
demands, job variety, little autonomy or support from
others, it may lead to increased stress levels as well as
health problems [1]. Work-related stress can cause
substantial economic costs due to lost productivity and
absenteeism [2]. A 2010 European survey (n = 21,703)
showed that 28 % of the workers experienced work-
related stress [3]. The worksite health program “Be Active & Relax” was
developed to reduce the NFR in office workers [19] via the
increase of daily physical activity and relaxation. The ef-
fectiveness of the intervention was investigated in a trial
using a 2 × 2 factorial design. The design factors were two
different strategies of delivering the “Be Active & Relax”
program, in other words, two delivery modes. One
delivery mode was a “social environmental intervention”,
which consisted of a counseling style that focused on
behavioral change in groups and was derived from
motivational interviewing at the individual level [20]. The
other delivery mode was a “physical environmental inter-
vention”, which consisted of encouraging physical activity
and relaxing behavior by making changes in the physical
environment. An early precursor for work-related health problems is
a higher Need for Recovery (NFR; [4]). NFR is described
as the need to recuperate and unwind from work-
induced effort and represents the short-term workload
effects of a day at work [5]. It can be seen as an inter-
mediate variable between psychosocial work characteris-
tics and work-related health problems [6, 7]. Several health behavior strategies were studied to re-
duce work stress and work-related health problems,
and/or NFR, among which physical activity and relax-
ation. Evidence was found that physical activity is valu-
able in unwinding from work [8, 9]. Sufficient physical
activity, that is, three to five times a week for 15 to
45 min, resulted in lower work stress [10], reduced ab-
senteeism [11], and improved job satisfaction [12]. In
addition, participating in relaxation activities diverted
the mind from work and improved self-esteem-feelings
that are essential for recovery [13]. Trial registration: NTR2553 Keywords: Motivational interviewing, Social environmental intervention, Environmental modifications, Physical activity,
Worksite health promotion, Relaxation program, Need for recovery, Work, QUINT * Correspondence: erwintak2@gmail.com
1Netherlands Organisation for Applied Scientific Research TNO, Schipholweg
77, Leiden, The Netherlands
Full list of author information is available at the end of the article © 2016 The Author(s). 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. Formanoy et al. BMC Public Health (2016) 16:866 Formanoy et al. BMC Public Health (2016) 16:866 Page 2 of 15 Interventions The delivery modes of the physical activity and relax-
ation intervention of the “Be Active & Relax” study were
systematically developed [19] using a modified version of
the intervention mapping protocol [25]. Two types of
delivery modes were chosen: a social environmental
intervention and a physical environmental intervention. g
The research questions of the present study were: Background It may be that a “one size fits all” ap-
proach is not suitable in this case. In other words, the
effectiveness of an intervention may differ across indi-
viduals. For example, Taris et al. [18] suggested that
working overtime on a recurrent character may influ-
ence the results of workplace health interventions in two
different ways. First, as a result of working overtime, less
time is available for recovery. Second, people who work
overtime recurrently are prone to make “unhealthy
choices” concerning lifestyle, such as smoking and con-
suming high levels of alcohol. By incorporating (some
of) these moderator variables into the design of the
study, one can target the right population. Page 3 of 15 Page 3 of 15 Page 3 of 15 Formanoy et al. BMC Public Health (2016) 16:866 Formanoy et al. BMC Public Health (2016) 16:866 The aim of this study was to identify which intervention
in the “Be Active & Relax” study [19] - the social environ-
mental or the physical environmental intervention-best
suited which group of office workers. It was hypothesized
that one or more moderator variables might influence the
effect on the primary outcome measure, which was
change in NFR from baseline to follow-up. Social environmental intervention (SEI) 1) Did the effect of the social environmental 1) Did the effect of the social environmental
intervention differ for subgroups of workers and did
the effect of the physical environmental intervention
differ for subgroups of workers? Four group motivational interviewing sessions were con-
ducted by the team leaders (i.e., after receiving a 2-day
training) with the office workers of their own team. The
main aim of the sessions was to stimulate physical activity
and relaxation by group motivational interviewing ses-
sions (e.g., during session number two, office workers
were asked to fill in a worksheet stating their goals and
subsequent rewards). The social environmental interven-
tion was supported by a web-based social media platform. 2) More specifically, for both interventions, which
subgroup of workers showed a positive effect
(e.g., a better outcome in NFR) and which
subgroup showed a negative effect (e.g., a worse
outcome in NFR)? Study design and study population For this study, data were used from the “Be Active &
Relax” study [19]. From September 2011 to September
2012, 329 office workers participated. They were all
recruited at one business location of a Dutch financial
service provider. All participants signed an informed
consent. This study was a cluster-randomized controlled
trial (with three data collection time points) to reduce
the NFR in office workers using a physical activity and
relaxation program. The study protocol was approved by
the Medical Ethics Committee of the VU University
Medical Center (Amsterdam, The Netherlands). Add-
itional details regarding the study design and methods of
the “Be Active & Relax” study have been published else-
where [19]. Methods Based on previous studies [26–32], the aim was to facili-
tate daily physical activity and relaxation in the work en-
vironment, by changing the coffee corners (by adding a
bar with bar chairs and a giant wall poster visualizing a
relaxing environment), the open office environment
(exercise balls and curtains to reduce background noise),
the meeting rooms (a standing table and a giant wall
poster visualizing a relaxing environment), and the en-
trance hall (a table tennis table, lounge chairs for infor-
mal meetings, and footsteps on the floor to promote
stair walking). The interventions PEI and the SEI are de-
scribed in more detail elsewhere [19]. Study design and study population Outcome measure
h The primary outcome measure of the study was NFR,
which was assessed using the NFR after work scale [5]. The questionnaire for the participants was in Dutch. The
scale is originally a Dutch scale and is validated for the
Dutch population [5]. The scale has good reliability
(Cronbach’s alpha = 0.88) and validity. The validity was
demonstrated by estimating the associations of NFR with
psychosocial risk factors (e.g., emotional load and physical
exertion) [5]. Furthermore, it was shown that the NFR has
satisfactory test-retest reliability (ICCs 0.68 to 0.80) and is
sensitive to detect change in increase in work related fa-
tigue due to the increase in working hours (effect size
0.40) [26]. The NFR after work scale is a sum score of 11
dichotomous items, expressed as a percentage, represent-
ing short-term effects of a day at work. Example questions
of this scale included: “I find it hard to relax at the end of
a working day”, “when I get home from work, I need to be
left in peace for a while”. A hundred percent NFR corre-
sponds to a sum score of 11, and represents a very high
need for recovery. Our outcome for the analyses is change A 2 by 2 full factorial design was applied, resulting
in four experimental conditions (see Table 1). Condi-
tions with the social environmental intervention are
referred to as SEI+ and conditions without as SEI−. The same applies to the conditions with the physical
environmental intervention (PEI+) or without (PEI−). Table 1 Two by two factorial design, the social environmental
intervention (SEI) and the physical environmental intervention
(PEI), including the number of participants per condition
PEI
Total
PEI+
PEI−
SEI
SEI+
63
94
157
SEI−
76
96
172
Total
139
190
329 Table 1 Two by two factorial design, the social environmental
intervention (SEI) and the physical environmental intervention
(PEI), including the number of participants per condition Formanoy et al. BMC Public Health (2016) 16:866 Page 4 of 15 Page 4 of 15 Page 4 of 15 in NFR from baseline to follow-up (at 12 months). The
follow-up score at 12 months was taken to ascertain that
the time period was long enough to be able to measure ef-
fect of SEI and / or PEI. The follow-up score was sub-
tracted from the baseline score. Moderator variables The “Be Active & Relax” study contained many potential
moderator variables. From these variables, we selected
the most relevant ones. These were chosen based on the
expertise of the researchers, supplemented with modera-
tors that were identified by a literature search. The
search was performed in several databases (i.e., PubMed,
PsycInfo and Picarta) using the following entries: work-
site health promotion, intervention, exercise, workplace,
relaxation, and need for recovery. This resulted in vari-
ables concerning socio-demographic variables (e.g. age
[33], sex, BMI), health and home related variables (e.g. physical activity in free time [34], mental health variables
[35], detachment at home) and work related variables
(e.g. vitality, team commitment, organizational commit-
ment). For an overview, see Appendix 1. Their univariate
effects on NFR were analyzed [16]. The algorithm of QUINT starts with all persons in one
node (the root node). Then it searches for a moderator
that optimizes the qualitative treatment-subgroup inter-
action. The search is performed among all possible mod-
erators, all possible split points on each moderator and
all possible assignments of the leaves to the subgroups. The best combination of moderator, split point, and as-
signment is used to split the total group into two nodes
(the “current” leaves). For the next splits, the procedure
is repeated in each of the current leaves, and now the
combination of moderator, split point, current leaf node,
and assignment of the new leaves to the partition classes
is chosen that optimizes the qualitative treatment-
subgroup interaction. Outcome measure
h A positive score means an
improvement and represents an absolute reduction in
NFR from baseline to 12 months. The minimal relevant
difference on the NFR after work scale was set at 12 [26]. subgroups of persons. This implies that the treatment ef-
fect may be positive for one subgroup, and negative for
another subgroup. As such, these types of interactions are
essential for optimal treatment assignment. In a QUINT analysis, three types of subgroups are dis-
tinguished that are involved in (a) qualitative treatment-
subgroup interaction(s). We will elucidate these sub-
groups for one of our treatment factors, that is, the
social environmental intervention with categories SEI+
(n = 157) and SEI−(n = 172). A QUINT analysis parti-
tions the total group of office workers in the following
types of subgroups (i.e., partition classes): 1) a group of
persons for whom SEI+ has on average a better outcome
than SEI−; 2) a group for whom SEI−has a better out-
come than SEI+, and 3) a group for whom it does not
make any difference. The partitioning criterion of the
QUINT algorithm takes into account the difference in
treatment outcome and the number of participants in
subgroups 1 and 2. The difference in treatment outcome
can be specified as a treatment effect size (i.e., Cohen’s
d) or as a crude difference in means; the corresponding
partitioning criterion is called “effect size criterion” or
“difference in means criterion”, respectively. Statistical analyses
Introduction of QUINT The best pruned tree is the smallest tree
that satisfies the one-standard-error rule, that is, it has a
bias-corrected criterion value higher or equal to the
maximum bias-corrected criterion value minus its stand-
ard error. The pruning procedure is described in detail
in Appendix 2 of an earlier paper [23]. In an extensive
simulation study, it was shown that the inferential errors
(Type I and Type II errors) of QUINT were small if the
total sample size was higher or equal to 400, dmin
equaled 0.30, and the true treatment effect size was large
(i.e., |d| ≥1). If the sample size was lower or equal to
300, the recovery of tree complexity was acceptable only
for smaller trees (i.e., trees with one or two splits) [23]. intervention. In both analyses, improvement in NFR was
used as the outcome variable and all moderator variables
were used as splitting candidates. The number of office
workers used in this analysis was 304 (25 of the 329 of-
fice workers were deleted due to missing values on one
or more moderators). In the final QUINT analyses, the
variable “general health” was deleted from the set of
moderator variables, because eight office workers had a
missing value only on this variable and this variable did
not appear to be important (i.e., it was not selected as
splitting variable). By deleting “general health”, the
number of office workers in the final analysis increased
to 312. As was mentioned before, two partitioning criteria can
be used in a QUINT analysis, either the effect size criter-
ion or the difference in means criterion. Our outcome
measure can be treated as ordinal (i.e., ratings), as well
as numeric (i.e., percentages); therefore, in a first series
of QUINT analyses, the effect size criterion was used,
and in a second series, the difference in means criterion
was used. In this way, it could also be checked whether
the solution was stable (i.e., the same) or not. In both
series of analyses, a value of 25 was used as minimal
sample size per intervention in a leaf, dmin was set at
0.299, and default values of the weights of the partition-
ing criterion were used (see supplementary materials in
Appendix 1). A large number of bootstrap samples (i.e.,
B = 1000) was used and the one-standard-error pruning
rule. Statistical analyses
Introduction of QUINT Using a new technique like QUINT has some advan-
tages. Compared to the more classical approach of ana-
lysis of variance (where the “one size fits all” paradigm is
predominate), one advantage of the QUINT method is
its ability to identify subgroups that differ in direction of
the intervention effect. The results of QUINT give prac-
tical indications on how to optimally assign workers to
an intervention. Other advantages are that QUINT can
handle a large number of moderator variables (in this
study, 25 in total) and can easily identify higher order
intervention-subgroup interactions, both of which are
not possible when using the classical post hoc analysis. Furthermore, the bias-corrected pruning procedure of
the method guarantees that no spurious interaction ef-
fects will be found. The splitting process stops if the value of the partition-
ing criterion cannot be increased anymore. Furthermore,
several stopping criteria are taken into account during
the splitting process, among which a qualitative inter-
action condition (i.e., after the first split, the absolute
value of the standardized mean difference in treatment
outcome in each of the two leaves should exceed a crit-
ical minimum value (dmin; the default value is 0.30; this
can be seen as a check of whether a qualitative inter-
action is present in the data), and a minimal sample size
per treatment (i.e., in each leaf, a minimum number of
participants is in each treatment group for reliable esti-
mation of the treatment means; by default this number
is 10 % of the treatment group sample size). After fitting
the full tree (with the total number of leaves as L), the
tree is pruned back using a bootstrap-based bias-
correction procedure [22]. This procedure results in QUINT is based on a binary recursive partitioning algo-
rithm, which is an algorithm that recursively splits the
data in two groups and thus resulting in a binary tree. The
partitioning criterion of QUINT maximizes qualitative
treatment-subgroup interactions. In general, treatment-
subgroup interactions imply that the effect of a treatment
variable on an outcome variable depends on the levels of
(an) other variable(s). These levels define subgroups of
persons. In case of qualitative treatment-subgroup inter-
actions, the treatment effect differs in direction for specific Formanoy et al. BMC Public Health (2016) 16:866 Page 5 of 15 Page 5 of 15 bias-corrected criterion values for trees with number of
leaves is 1 to L. Application of QUINT to the data of the “Be Active & Relax”
study The QUINT analyses were performed using the R-package
“quint” [24, 36] in the R version 3.0.2 (R Core Team,
2013). Before starting these analyses, two pre-conditions
were tested. The first condition of QUINT is that each
person is randomly assigned to an intervention group;
however, in the “Be Active & Relax” study, the social en-
vironmental intervention was randomized at department
(i.e., different service departments of a financial service
provider) level, and the physical environmental interven-
tion was stratified on department level, i.e., one stratum
with environmental modifications and the other stratum
without environmental modifications. To check if the nesting of office workers within depart-
ments could be disregarded, the intra cluster correlation
coefficient (ICC) was computed, using improvement in
NFR as outcome variable, and the departments as clusters. The ICC was 0.02, indicating that the amount of variance
attributed to the departments was very low, and could be
neglected. The second condition of QUINT is that the
data include only 2 intervention groups, whereas the “Be
Active & Relax” study concerned a 2 by 2 design, implying
four intervention groups. A re-analysis of the data of the
“Be Active & Relax” study using a full factorial analysis of
variance (i.e., including main effects of the social en-
vironmental intervention and the physical environmental
intervention, and the interaction effect between the inter-
ventions) with improvement in NFR as outcome variable,
revealed that the interaction effect was not significant (nor
the two main effects). This result showed that the effect of
the social environmental intervention on NFR did not de-
pend on the physical environmental intervention. This
allowed us to treat the two factors (i.e., the social environ-
mental and the physical environmental intervention) sep-
arately (e.g., analogous to the approach of Raveaud [37]). Statistical analyses
Introduction of QUINT Furthermore, independent t-tests were performed
in each leaf of the pruned tree to test the difference in
means of the two groups. It should be noted that the sig-
nificance level of these t-tests is somewhat inflated, due
to the data-induced subgroups. Therefore, also the bias-
corrected effect sizes in the leaves were estimated using
the validation procedure for small data sets (recom-
mended by Dusseldorp & Van Mechelen [23]). Main effects of the delivery modes The office workers in the social environmental interven-
tion group showed a mean improvement in NFR of 3.82,
and those who did not receive the social environmental
intervention showed
a mean improvement
of 1.17
(Table 2); the difference in means was 2.65 (SD = 23.63;
effect size d = 0.11), and the main effect of the social en-
vironmental intervention was not significant (p >0.05;
indicated by an independent t-test). The office workers
in
the
physical
environmental
intervention
group
showed a mean improvement in NFR of 4.59, and those
who did not receive the physical environmental inter-
vention showed a mean increase of 0.85 (Table 2); the
difference in means was 3.75 (SD = 23.60; effect size
d = 0.16), and the main effect of the physical environmen-
tal intervention was not significant (p > 0.05; indicated by To answer the two research questions, two QUINT
analyses were performed; one for the social environmen-
tal intervention and one for the physical environmental Formanoy et al. BMC Public Health (2016) 16:866 Page 6 of 15 Table 2 Descriptive statistics for all variables involved in re-analyses of data from the “Be Active & Relax” study. The potential moder-
ators were all measured at baseline (i.e., before receiving a physical activity and relaxation program). The statistics are given for both
delivery modes: the social intervention and the physical environmental intervention (N = 312) Table 2 Descriptive statistics for all variables involved in re-analyses of data from the “Be Active & Relax” study. The potential moder-
ators were all measured at baseline (i.e., before receiving a physical activity and relaxation program). The statistics are given for both
delivery modes: the social intervention and the physical environmental intervention (N = 312) delivery modes: the social intervention and the physical environmental intervention (N = 312)
Delivery mode: social environmental
intervention
Delivery mode: physical environmental
intervention
Yes n = 149
No n = 163
Yes n = 132
No n = 180
Variable
Range
Mean
(SD)
Mean
(SD)
Mean
(SD)
Mean
(SD)
Outcome
Improv. in need for recovery
−100.0
81.82
3.82
(25.32)
1.17
(21.98)
4.59
(23.38)
0.85
(23.75)
Potential moderators
Need for recovery at baseline
0.00
100.00
30.42
(28.90)
30.36
(28.88)
32.18
(30.28)
29.08
(27.75)
Age (in years)
19.00
63.00
42.46
(10.05)
41.77
(9.89)
41.63
(10.39)
42.45
(9.63)
Sex (male vs. Main effects of the delivery modes female)
0
1
0.62
(0.48)
0.63
(0.48)
0.62
(0.49)
0.63
(0.48)
Level of education
1.00
3.00
2.29
(0.86)
2.41
(0.77)
2.46
(0.76)
2.27
(0.84)
Cohabiting (yes vs. no)
0
1
0.75
(0.43)
0.77
(0.42)
0.8
(0.40)
0.74
(0.44)
Mother country (Neth. vs. other)
0
1
0.93
(0.26)
0.91
(0.29)
0.93
(0.25)
0.91
(0.29)
Body Mass Index
17.10
39.19
25.18
(4.35)
24.87
(3.74)
24.61
(3.56)
25.31
(4.34)
Mental Health
2.00
6.00
4.5
(0.72)
4.51
(0.73)
4.42
(0.69)
4.57
(0.74)
Detachment at home
1.00
7.00
4.76
(1.33)
4.9
(1.35)
4.8
(1.39)
4.86
(1.31)
Relaxation at home
2.00
7.00
5.16
(1.02)
5.25
(1.11)
5.05
(1.07)
5.33
(1.05)
Physical activity (in MET-min.)
375
29610
7527
(4234)
7521
(3937)
7066
(4018)
7860
(4095)
Vitality
2.00
7.00
5.00
(0.96)
5.06
(1.00)
4.92
(0.97)
5.11
(0.98)
Team commitment
1.00
5.00
4.07
(0.65)
4.14
(0.68)
3.99
(0.64)
4.19
(0.67)
Organizational commitment
2.57
5.00
4.00
(0.47)
4.08
(0.44)
3.97
(0.44)
4.09
(0.46)
Supervisor support
1.00
4.00
2.87
(0.51
2.89
(0.48)
2.86
(0.53)
2.89
(0.47)
Colleague support
2.00
4.00
3.09
(0.38)
3.09
(0.37)
3.05
(0.37)
3.12
(0.37)
Job demands
1.50
4.00
2.82
(0.49)
2.71
(0.40)
2.78
(0.46)
2.75
(0.44)
Decision authority
1.00
4.00
2.98
(0.53)
2.99
(0.54)
2.98
(0.56)
2.99
(0.52)
Job insecurity
1.00
3.00
1.55
(0.39)
1.65
(0.48)
1.58
(0.42)
1.62
(0.45)
Skill discretion
1.83
4.00
3.03
(0.37)
3.09
(0.37)
3.1
(0.39)
3.03
(0.35)
Working overtime (in hrs. p. wk.)
0.00
40.00
2.85
(6.05)
3.19
(7.78)
2.74
(6.70)
3.25
(7.22)
Detachment at work
1.00
7.00
3.48
(1.39)
3.54
(1.34)
3.46
(1.30)
3.54
(1.41)
Relaxation at work
1.00
7.00
3.53
(1.25)
3.69
(1.31)
3.45
(1.19)
3.74
(1.33)
Walking during lunch
1
5
2.78
(1.45)
2.94
(1.47)
2.86
(1.39)
2.87
(1.52)
Active during lunch
1
4
1.92
(1.04)
1.91
(1.04)
1.83
(0.98)
1.97
(1.08) around 300), the smaller pruned trees were preferred to
the larger ones to take a conservative approach. There-
fore, the results of the first series of analysis are de-
scribed below and presented in Figs. 1 and 2, those from
the second series are presented in Appendix 2. an independent t-test). The mean values (or percentages)
on all included moderator variables for the groups with
and without each delivery mode are shown in Table 2. Results of the QUINT analyses The first series of QUINT analyses resulted in the same
full trees as the second series of analyses. However, the
pruning results were different: the pruned trees from the
effect size criterion were smaller than those from the
difference in mean criterion. Because the sample size of
this study was relatively small for a QUINT analysis (i.e., The result for the social environmental intervention
was a pruned tree with two leaves. The split of the tree
(Fig. 1) involved the variable “Age” and a split point of
46.5 years. It should be noted that in the leaves of Fig. 1,
the effect sizes d are expressed as the standardized
mean difference between the group with the social Formanoy et al. BMC Public Health (2016) 16:866 Page 7 of 15 Fig. 1 Pruned tree for social environmental intervention with moderator variable “Age” with two leaves (File ‘Fig. 1 pruned.png’). Legend: Pruned
tree involving the variable Age and a split point of 46.5 years; the effect sizes d are expressed as the standardized mean difference between the
group with the social environmental intervention (SEI+) and the group without (SEI−); For the leaf assigned to P1 (i.e., the left green leaf) the
effect size d is positive, while for the leaf assigned to P2, the effect size d is negative Fig. 1 Pruned tree for social environmental intervention with moderator variable “Age” with two leaves (File ‘Fig. 1 pruned.png’). Legend: Pruned
tree involving the variable Age and a split point of 46.5 years; the effect sizes d are expressed as the standardized mean difference between the
group with the social environmental intervention (SEI+) and the group without (SEI−); For the leaf assigned to P1 (i.e., the left green leaf) the
effect size d is positive, while for the leaf assigned to P2, the effect size d is negative means = 10.52; 95 % CI: 4.12, 16.92); bias-corrected effect
size d = 0.32, Table 3). Furthermore, the results showed that older office
workers (> 46.5 years) who received the social environ-
mental intervention had a worse outcome than older of-
fice workers who did not receive the social environmental
intervention (Leaf 2 in Fig. 1; difference in means = -10.65;
95 % CI: −19.35, −1.96); bias-corrected effect size d = -0.22,
Table 3). environmental
intervention
(SEI+)
and
the
group
without
(SEI−). Results of the QUINT analyses As
a
consequence,
for
the
leaf
assigned to P1 (i.e., the left green leaf), the effect size
d is positive, while for the leaf assigned to P2, the ef-
fect size d is negative. The results indicated that youn-
ger office workers (≤46.5 years) who received the social
environmental intervention had a better outcome in NFR
than younger office workers who did not receive the social
environmental intervention (Leaf 1 in Fig. 1; difference in means = 10.52; 95 % CI: 4.12, 16.92); bias-corrected effect
size d = 0.32, Table 3). Furthermore, the results showed that older office
workers (> 46.5 years) who received the social environ-
mental intervention had a worse outcome than older of-
fice workers who did not receive the social environmental
intervention (Leaf 2 in Fig. 1; difference in means = -10.65;
95 % CI: −19.35, −1.96); bias-corrected effect size d = -0.22,
Table 3). Fig. 2 Pruned tree for physical environmental intervention with moderator variable “Working overtime” with two leaves (File ‘Fig. 2 pruned.png’). Legend: Pruned tree involving the variable Working overtime and a split point at 2.25 h indicating that office workers who worked fewer hours
overtime (≤2.25) had a better outcome with the physical environmental intervention than without the physical environmental intervention
(Leaf 1) and office workers who worked more hours overtime (> 2.25) had a worse outcome with the physical environmental intervention than
without (Leaf 2) Fig. 2 Pruned tree for physical environmental intervention with moderator variable “Working overtime” with two leaves (File ‘Fig. 2 pruned.png’). Legend: Pruned tree involving the variable Working overtime and a split point at 2.25 h indicating that office workers who worked fewer hours
overtime (≤2.25) had a better outcome with the physical environmental intervention than without the physical environmental intervention
(Leaf 1) and office workers who worked more hours overtime (> 2.25) had a worse outcome with the physical environmental intervention than
without (Leaf 2) Formanoy et al. BMC Public Health (2016) 16:866 Page 8 of 15 Table 3 Descriptive statistics in the leaves of the quint results for the social environmental intervention (SEI; Fig. 1) and the physical
environmental intervention (PEI; Fig. 2). Discussion This study examined which subgroups benefit more
from which delivery mode of a physical activity and re-
laxation program during work on NFR. The results from
the QUINT analysis suggest that a social environmental
intervention might be more beneficial for younger
workers, and a physical environmental intervention
might be more beneficial for employees with a only few
hours overtime. Furthermore, Hughes et al. [39] found an individual-
ized program to be more effective than a program where
older university workers could choose health modules
on their own, and received generic health e-mail tips. Participants from the individualized intervention re-
ported a borderline significant reduction in percentage
energy from fat at 6 months (p = .063) and a significant
reduction at 12 months (p < .05) and the group reported
significantly more minutes of moderate physical activity
than did controls at 6 and 12 months (p < .05). At last, a
significant decline in waist circumference at 6 months
was achieved by the intervention group that was main-
tained at 12 months (p < .05). Stress and smoking did
not change significantly. These results underline the
claims for individualizing the approach for older office
workers. Age Several studies found age to be an important factor
in the effectiveness of worksite health programs [38,
39]. The review by Rongen et al. [38] demonstrated
that the effectiveness of workplace health promotion
programs was larger in younger populations (mean
age of <40, with a difference in effect size of -0.17;
95 % CI -0.23, −0.17). Specifically, 18 studies were
compared that had studied an intervention aimed at a
healthy lifestyle (such as physical activity, healthy nu-
trition, weight loss, or smoking cessation). The deliv-
ery mode varied from a tailored fitness program to
counseling sessions focusing on physical activity and
nutrition. The authors of this review advised to target
specific
age
populations
in
worksite
interventions. The study also found workplace health promotion
programs
to
be
more
effective
in
white
collar
workers. Results of the QUINT analyses The mean values and standard deviations on improvement in Need for Recovery (NFR) are
displayed (i.e., a higher score reflects a larger reduction in NFR from baseline to 12 month follow-up), and the treatment outcome
diff differences
n
Mean
SD
n
Mean
SD
Difference in means (95 % CI)
Bias-corrected effect size d
Fig. 1
SEI+
SEI−
Leaf 1
90
8.29
22.27
107
−2.23
23.20
10.52 (4.12, 16.92)*
0.32
Leaf 2
59
−3.00
28.22
56
7.66
17.89
−10.65 (−19.35, -1.96)*
−0.22
Fig. 2
PEI+
PEI−
Leaf 1
103
6.15
23.90
128
−1.25
25.39
7.40 (0.99, 13.81)*
0.13
Leaf 2
29
−0.94
20.90
52
6.01
18.35
−6.95 (−16.26, 2.36)
−0.08
Note. CI confidence interval; *p < .05, estimated by independent t-test latter difference concerned an increase of NFR in a
non-experimental design and was determined over a
longer period (2 years). The result for the physical environmental interven-
tion was also a pruned tree with two leaves. The split
of the tree involved the variable “Working overtime”
and a split point at 2.25 h (Fig. 2). The results indi-
cated that office workers who worked fewer hours
overtime (≤2.25) had a better outcome with the
physical environmental intervention than without the
physical environmental intervention (Leaf 1 in Fig. 2;
difference in means = 7.40; 95 % CI: 0.99, 13.81); bias-
corrected effect size d = 0.13). Furthermore, the re-
sults showed that office workers who worked more
hours overtime (> 2.25) had a worse outcome with
the physical environmental intervention than without,
but this effect was not significant (Leaf 2 in Fig. 2;
difference in means = −6.95; 95 % CI: −16.26, 2.36);
bias-corrected effect size d = −0.08, Table 3). Comparison with other studies
Observed change in NFR Although the “Be Active & Relax” study demonstrated
no significant effect on the main outcome NFR [16],
differential treatment efficacy was found in our study. The changes in NFR found from baseline to 12-
month follow-up in the intervention subgroups (i.e.,
8.29 for the social environmental and 6.15 for the
physical environmental intervention) were somewhat
lower than the change found by De Croon et al. [26],
that is, a difference in NFR of 12.6. However, the Page 9 of 15 Formanoy et al. BMC Public Health (2016) 16:866 Page 9 of 15 The social environmental intervention had a negative
effect on older workers. It could be that younger em-
ployees had a more positive attitude toward the social
environmental intervention than older employees. fruits and vegetables in full-time workers. Working
overtime simply reduces the amount of time and en-
ergy available to be physically active [18]. It could be that the participants in this study who
worked more hours overtime did not make as much
use of the environmental modifications as the partici-
pants who did not work overtime because of time
constraints. As a result, working overtime may have
interfered with fully benefitting from the environmen-
tal modifications. Office workers who work overtime
on a regular basis probably need a different interven-
tion to change their lifestyle and make them more
physically active. It may be advisable for organizations
to pay attention to the work process when working
overtime is structural. Office workers who work over-
time structurally may be a vulnerable group. Not only
do they have stress as a result of working extra hours,
but they may also be less susceptible for interventions
aimed at physical activity and relaxation. Furthermore, there is some evidence that being a
good role model as a team leader in terms of healthy
behaviors may be important for those offering the
social environmental intervention to support physical
activity and relaxation [16]. In addition, other charac-
teristics of the team leaders (i.e., age, gender, ethni-
city, or level of education) might have influenced the
effectiveness of the social environmental intervention. Finally, the older participants in our study (> 46.5 years
old) may have been healthier than the office workers
who have left the job (healthy worker effect). The
baseline NFR score of the older participants was
relatively low (i.e., 28.0) and did not differ signifi-
cantly from the younger ones. Limitations of this study y
There are some limitations of this study. First, the
compliance of the sample. Not all participants in the
“Be Active & Relax” study fully participated in the so-
cial and in the physical environmental interventions
[42]. The reach (% of participants that attended at
least one sessions or used at least one element) for
the social and physical environmental intervention
ranged from 45 to 76 % [42]. A barrier for not at-
tending the social environmental intervention sessions
were, for example, having a holiday. An important
barrier for the participants for not using the physic-
ally activating and relaxing elements in the physical
environmental intervention was office workers indicat-
ing that they did not have enough time. Although the
level of reach is comparable to other worksite health
programs (mostly below 50 % [43]); this could have
underestimated the results in the QUINT analysis. Comparison with other studies
Observed change in NFR This finding is in con-
trast with the studies of Kiss et al. [40] and Mohren
et al. [41], who found that older workers had a
higher NFR than younger workers. Kiss et al. [40]
found that the mean recovery score (40.9) was sig-
nificantly higher in the group of the older public
sector workers as compared to the mean score (33.6)
of younger workers (p < 0.05). Mohren et al. [41]
studied day workers and discovered that the highest
levels of NFR were observed in the age group of 46–
55 years. The relative risk for developing elevated
NFR was highest in the age groups 36–45 years (RR
1.30; 1.07–1.58) and 46–55 years (RR 1.25; 1.03–
1.52) in men and 46–55 years (RR 1.36; 1.04–1.77)
in women when compared to the reference group. Looking at the evaluation of the “Be Active &
Relax” study [42], it is remarkable that the element in
the environmental modifications that was most phys-
ically activating (e.g. table tennis table) was not used
much. It was shown that the percentage that used the
table tennis table at least once on a scale from 0
(never) to 5 (always) was on average 17 % of the 35
participants that were evaluated. Implications for research and practice The implications of the results of our study are three-
fold. Firstly, the results suggest that a “one size fits
all” approach does not hold for the worksite health
program “Be Active and Relax”. For other worksite
health programs, this commonly used approach may
also not work. Instead, we recommend the developers
of such programs to carefully consider possibilities to
tailor the program to specific subgroups. Secondly,
the study shows that an advanced exploratory ana-
lyses method, like QUINT, is able to indicate for
which subgroups of workers a worksite health pro-
gram is beneficial and for which subgroups it is not. And thirdly, the results suggest that group motiv-
ational interviewing by team leaders is not an appro-
priate strategy for older workers to reduce their need
for recovery. In addition, changing the work environ-
ment to facilitate daily physical activity and relaxation
at the worksite is not appropriate for workers who
work more hours overtime. More research is needed
to investigate which type of health program would
work for these specific subgroups of office workers. Based on the results, we advise to take age and work-
ing overtime into account when developing a health
program for office workers, and also when designing
health intervention evaluation studies. A second limitation of the study was that the exist-
ence of possible subgroups that benefit more from
the combination of the social and physical environ-
mental intervention was not explored due to sample
size restrictions. Although the statistical analyses did
not show the need for doing this (due to a non-
significant interaction effect between the social envir-
onmental intervention and the physical environmental
intervention), it cannot be ruled out that subgroups
exist that might benefit especially from the combined
intervention. The generalizability of the interventions to other
work environments is questionable, because the inter-
ventions were specifically tailored to the target popu-
lation by using intervention mapping. Furthermore,
our study population consisted of 60 % males and
57 % was highly educated, which does not represent
the general Dutch working population. This was due
to the fact that the financial service provider’s work-
force involves mainly highly educated, male and white
collar employees. Finally, the “Be Active & Relax” study was not a full
RCT. The social environmental intervention group
was randomized, but the physical environmental inter-
vention was not (for details, see [19]). Working overtime
k Working overtime means that a person is putting in
more hours than agreed upon in his or her contract. Although the threshold for working overtime to bene-
fit from environmental modifications in this study is
rather low (on average 2.25 h per week), underlying
mechanisms may explain why office workers who
work overtime do not benefit fully from the delivery
mode environmental modifications. Research
indicates
two
possible
mechanisms
for
overtime work causing an increase in stress levels
[18]. The first is that high levels of overtime may lead
to lack of recovery because of the shorter periods of
rest between working days. This can affect recovery
time and increase the exposure time to work stress,
in turn resulting in adverse health and well-being. The second mechanism affects health indirectly and
concerns behavioral decisions and habits (i.e., lifestyle
behaviors) of workers. Taris et al. found overtime to
lead to lower levels of physical activity and intake of Although no significant effect was found on the
main outcome measure of NFR in the study by Cof-
feng et al. [16], secondary outcome measures did dif-
fer
significantly. In
the
combined
environmental
intervention group (n = 92), exhaustion and vigorous
physical activities decreased significantly, and small
breaks at work and active commuting increased sig-
nificantly compared to the control group. The social Page 10 of 15 Page 10 of 15 Formanoy et al. BMC Public Health (2016) 16:866 environmental intervention group (n = 118) showed a
significant reduction in exhaustion, and sedentary be-
havior at work, and a significant increase in small
breaks at work and leisure activities compared to the
control group. In the physical environmental interven-
tion group (n = 96), stair climbing at work and active
commuting significantly increased, and sedentary be-
havior at work decreased significantly compared to
the control group. research, larger sample sizes are recommended to
confirm our hypotheses. The software of QUINT al-
lows the user to choose between several parameters,
for example, the minimum sample size per interven-
tion condition in a leaf and the total number of
bootstrap samples used for the pruning procedure. In
the present study, we found that results were more
stable using a minimum intervention sample size of
25 (instead of the default value of 10 %), and a total
number of bootstrap samples of 1000 (instead of the
default value of 25). Implications for research and practice Therefore, cau-
tiousness is needed in the interpretation of the results
of the physical environmental intervention; the benefi-
cial effect for office workers with a few hours over-
time might not have been caused by the intervention,
due to the non-random assignment. Working overtime
k However, generalizability of recommending the social
environmental intervention for younger office workers
and using the physical environmental intervention for
office workers who do not work overtime should be
interpreted cautiously. Conclusions This study was conducted to explore whether subgroups
may benefit more from a social or a physical environ-
mental intervention to reduce the NFR by using the
QUINT method. Specifically, the results suggest possible roles for
age and working overtime: younger workers benefitted
(in terms of a reduced need for recovery) from a so-
cial environmental intervention and employees who
worked fewer hours overtime benefitted from a phys-
ical environmental intervention. It is recommended to
incorporate age and working overtime as stratification
variables in future research into the need for recovery
among office workers to confirm these results, and to
tailor the interventions to specific groups. Methodological issues In the present study, the QUINT analyses were per-
formed using the effect size criterion and the differ-
ence in means criterion. Both analyses led to the
same full trees, but different pruned trees. Due to
the relatively low sample size (N = 312) for a QUINT
analysis, we took a conservative approach and pre-
ferred the smaller trees (for detecting complex inter-
actions, N ≥400 is recommended [23]). In future Page 11 of 15 Formanoy et al. BMC Public Health (2016) 16:866 Table 4 The potential moderators were all measured at baseline (i.e., before receiving a physical activity and relaxation program)
and are divided into background variables, health and home related variables and work related variables
# it
α or r
Example of an item with answer
categories and score range
Questionnaire
Studies indicating a possible
modifying effect
Background
Age
1
N/A
“What is your date of birth?”
N/A
Steenstra (2009) [33]; Demou
(2012) [48], Bauman (2012) [49]
Sex
1
N/A
“What is your gender?”; 2
categories “man” or “woman”
N/A
Demou (2012) [48], Bauman
(2012) [49]
Level of education
1
N/A
“What is the highest level of
education you have completed?;
8-point scale from 1”no education”
to 8 “university degree”
N/A
Cohabiting
1
N/A
“What is your current marital
status?”; 6 categories (such as
married, living with another, etc)
N/A
Mother country
1
N/A
“In what country were you
born?”; 2 categories: the
Netherlands or other
N/A
Health/home
BMI
2
N/A
“What is your weight?” en “How
tall are you?”
N/A
Colkesen (2011) [34]
General health
1
N/A
“In general, how would you say
your health is?”; 5-point scale
from 1 “excellent” to 5 “poor”
Rand-36; Van der Zee (1993) [44]
Demou (2012) [48], Colkesen
(2011) [34], Bauman (2012) [49]
Mental health
9
α = 0.90
“Did you feel full of pep?”; 6-
point scale from 1 “none of the
time” to 6 “all of the time”
Rand-36; Van der Zee (1993) [44]
Demou (2012) [48]; Dishman
(2010) [35]
Detachment at home
4
α = 0.94
“After work, I don’t think about
work at all”; 7-point scale from 1
“never” to 7 “almost always”
The recovery experience
questionnaire; Sonnentag (2007)
[14] and De Bloom (2011) [45]
Physical activity
7
r = 0,97
“Do you walk/cycle to work? On
how many days per week? What
is the average time it takes? Dutch version of the Job
Content Questionnaire Karasek
(1998) [47] Methodological issues Rate
the intensity: light, moderate,
vigorous”
Short Questionnaire to Assess
Health Enhancing Physical
Activity (SQUASH); Wendel-Vos
(2003) [46]
Colkesen (2011) [34]
Relaxation at home
4
α = 0.92
“After work, I do relaxing things”;
7-point scale from 1 “never” to 7
“almost always”
The recovery experience
questionnaire; Sonnentag (2007)
[14] and De Bloom (2011) [45]
Work related
Vitality
5
α = 0.83
“At my work I feel bursting with
energy”; 6-point scale from 1
“never” to 6 “always”
Part of Utrecht Work
engagement Scale (UWES);
Demerouti (2001)
Strijk (2012) [17]
Team commitment
3
α = 0.82
“What I feel for this team – I am
proud”; 5-point scale from 1 “to-
tally agree” to 5 “totally disagree”
Organizational
commitment
8
α = 0.79
“I find it important that my job
go’s well”; 5-point scale from 1
“strongly agree” to 5 “strongly
disagree”
Meyer (2002) [50]; Lohela (2009)
[51]
Supervisor support
4
α = 0.78
“My supervisor is concerned
about the welfare of those under
him”; 4-point scale from 1
“strongly agree” to 4 “strongly
disagree”
Dutch version of the Job
Content Questionnaire Karasek
(1998) [47]
Choi (2011) [52]
Appendix 1
Relevant moderator variables measured in the “Be Active & Relax” Project Appendix 1
Relevant moderator variables measured in the “Be Active & Relax” Project The recovery experience
questionnaire; Sonnentag (2007)
[14] and De Bloom (2011) [45]
Part of Utrecht Work
engagement Scale (UWES);
Demerouti (2001)
Strijk (2012) [17] Meyer (2002) [50]; Lohela (2009)
[51]
Choi (2011) [52] Dutch version of the Job
Content Questionnaire Karasek
(1998) [47] Formanoy et al. Methodological issues BMC Public Health (2016) 16:866 Page 12 of 15 Table 4 The potential moderators were all measured at baseline (i.e., before receiving a physical activity and relaxation program)
and are divided into background variables, health and home related variables and work related variables (Continued)
Colleague support
4
α = 0.76
“People I work with are
competent in doing their jobs”;
4-point scale from 1 “strongly
agree” to 4 “strongly disagree”
Dutch version of the Job
Content Questionnaire Karasek
(1998) [47]
Choi (2011) [52]
Job demands
5
α =0.70
“My job requires working very
fast”; 4-point scale from 1
“strongly agree” to 4 “strongly
disagree ”
Dutch version of the Job
Content Questionnaire Karasek
(1998) [47]
Lohela (2009) [51], Van Laethem
(2013) [53]
Decision authority
3
α =0.68
“My job allows me to make a lot
of decisions on my own”; 4-point
scale from 1 “ strongly agree” to
4 “strongly disagree ”
Dutch version of the Job
Content Questionnaire Karasek
(1998) [47]
Mullan (1985) [54]
Skill discretion
6
α =0.67
“My job requires that I learn new
things”; 4-point scale from 1
“strongly agree” to 4 “strongly
disagree ”
Dutch version of the Job
Content Questionnaire Karasek
(1998) [47]
(WEBA)
Job insecurity
3
α =0.68
“During the past year were you
in a situation where you faced
job loss or layoff?”; 3-point scale
from 1 “no” to “actually layed off”
Dutch version of the Job
Content Questionnaire Karasek
(1998) [47]
Karlsson (2010) [55], Lohela
(2009) [51]
Working overtime
1
N/A
“On average, how many hours of
overtime do you put in per
week?”
Taris (2011) [18]
Detachment at work
4
α = 0.93
“During a break at work I forget
about my work”; 7-point scale
from 1 “never” to 7 “almost
always”
Adapted question (at work
was added) of “The recovery
experience questionnaire”;
Sonnentag (2007) [14] and
De Bloom (2011) [45]
Relaxation at work
4
α = 0.88
“During a break at work I use the
time to relax”; 7-point scale from
1 “never” to 7 “almost always”
Adapted question (at work
was added) of “The recovery
experience questionnaire”;
Sonnentag (2007) [14] and
De Bloom (2011) [45]
Notes: # it number of items, α Cronbach’s α, r Pearson’s correlation coefficient, N/A not applicable Dutch version of the Job
Content Questionnaire Karasek
(1998) [47]
Karlsson (2010) [55], Lohela
(2009) [51]
Taris (2011) [18] Acknowledgements
W
ld lik t
th g
We would like to thank all teamleaders and employees who participated in
the “Be Active & Relax study. The results showed that the social intervention was
especially effective for the younger employees with a high
Organization commitment (Leaf 2 in Fig. 3; bias-corrected
effects were d = 0.38 and difference in means = 10.40). Furthermore the social environmental intervention was Appendix 2 counter-effective for older persons working less than one
hour of overtime (Leaf 3 in Fig. 3; d = -0.97; bias-corrected
effects were d = −0.67; difference in means = −16.29). Results of the second series of analyses Results of the second series of analyses The full tree that resulted from the QUINT analysis
for the social environmental intervention and for the
physical environmental intervention using the Differ-
ence in means criterion was equal to the full trees
using the Effect size criterion (the results from the
latter were described in the Results section). However,
the pruning procedure resulted in larger trees for
both the social environmental intervention and the
physical environmental intervention, that is, trees with
four leaves (see Figs. 3 and 4). For the social en-
vironmental intervention, additional splitting variables
were Organization commitment and Working over-
time (Fig. 3). For the physical environmental intervention, additional
splitting variables were Team commitment and Physical
activity (Fig. 4). The results showed that the physical en-
vironmental intervention was especially effective for the
employees working less hours overtime with a high
Team commitment and a lower level of Physical activity
(Leaf 2; bias-corrected effects were d = 0.42 and differ-
ence in means = 11.40). Funding
Thi
j Funding
This project was funded by Fonds Nuts Ohra. The contribution of Iven van
Mechelen was funded by the Research Fund KU Leuven (Award Number:
GOA/15/003) and the Belgian Interuniversity Attraction Poles programme
(Award Number: IAP/P7/06). g
This project was funded by Fonds Nuts Ohra. The contribution of Iven van
Mechelen was funded by the Research Fund KU Leuven (Award Number:
GOA/15/003) and the Belgian Interuniversity Attraction Poles programme
(Award Number: IAP/P7/06). Page 13 of 15 Formanoy et al. BMC Public Health (2016) 16:866 Fig. 4 QUINT solution for the delivery mode “physical environmental intervention”, using the Difference in means criterion ig. 4 QUINT solution for the delivery mode “physical environmental intervention”, using the Difference in means criterion Fig. 3 QUINT solution for the delivery mode “social environmental intervention”, using the Difference in means criterion ig. 3 QUINT solution for the delivery mode “social environmental intervention”, using the Difference in means criterion Page 14 of 15 Page 14 of 15 Formanoy et al. BMC Public Health (2016) 16:866 Authors’ contributions MAGF and ED wrote the initial manuscript. ED and IVM performed the data
analysis, with input from MAGF and ECPMT. ECPMT had a role in supervision. JKC, CRLB en IJMH were responsible for the management of the trial and
data collection. All authors contributed to the further writing and approved
the final version of the manuscript. 13. Hahn VC, Binnewies C, Sonnentag S, Mozja EJ. Learning how to recover
from job stress: effects of a recovery training program on recovery,
recovery-related self-efficacy, and well-being. J Occup Health Psych. 2011;16(2):202–16. 14. Sonnentag S, Fritz C. The recovery experience questionnaire: development
and validation of a measure for assessing recuperation and unwinding from
work. J Occup Health Psych. 2007;12:204–21. 14. Sonnentag S, Fritz C. The recovery experience questionnaire: development
and validation of a measure for assessing recuperation and unwinding from
work. J Occup Health Psych. 2007;12:204–21. Ethics approval and consent to participate For this study, data were used from the “Be Active & Relax” study [19]. From
September 2011 to September 2012, 329 office workers participated. They
were all recruited at one business location of a Dutch financial service
provider. All participants signed an informed consent. This study was a
cluster-randomized controlled trial (with three data collection time points) to
reduce the NFR in office workers using a physical activity and relaxation
program. The study protocol was approved by the Medical Ethics Committee
of the VU University Medical Center (Amsterdam, The Netherlands). Additional
details regarding the study design and methods of the “Be Active & Relax” study
have been published elsewhere [19]. 17. Strijk JE, Proper KI, Van der Beek AJ, Van Mechelen W. A worksite vitality
intervention to improve older workers’ lifestyle and vitality-related
outcomes: results of a randomised controlled trial. J Epidemiol Community
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health: a longitudinal study among full-time employees. Int J Behav Med. 2011;18(4):352–60. 19. Coffeng JK, Hendriksen IJM, Duijts SF, Proper KI, van Mechelen W, Boot CRL. The development of the be active & relax ‘Vitality in Practice’ (VIP) project
and design of an RCT to reduce the need for recovery in office employees. BMC Public Health. 2012;12(1):592–617. 19. Coffeng JK, Hendriksen IJM, Duijts SF, Proper KI, van Mechelen W, Boot CRL. The development of the be active & relax ‘Vitality in Practice’ (VIP) project
and design of an RCT to reduce the need for recovery in office employees. BMC Public Health. 2012;12(1):592–617. Consent for publication
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Not applicable. Not applicable. 16. Coffeng JK, Boot CR, Duits SF, Twisk JW, Mechelen W, Hendriksen IJ. Effectiveness of a worksite social & physical environment intervention on
need for recovery, physical activity and relaxation; results of a randomized
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recovery. Eur J Work Organ Psychol. 2006;15:218–40. The Data Protection officer of VU University Medical Center has ethical
restrictions to making our data publicly accessible, because the data can lead to
personally identifiable health information. The data includes the name of the
company, age, education, being overweight, and other personal characteristics. The readers may contact Cécile Boot (crl.boot@vumc.nl) to request the data. The data will be available upon request to all interested researchers. 10. Norris R, Carroll D, Cochrane R. The effects of aerobic and anaerobic training on
fitness, blood pressure, and psychological stress and well-being. J Psychosom
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1 1Netherlands Organisation for Applied Scientific Research TNO, Schipholweg
77, Leiden, The Netherlands. 2Department of Psychology, Catholic University
of Leuven, Tiensestraat 102, Leuven, Belgium. 3Methodology and Statistics,
Institute of Psychology, Leiden University, Wassenaarseweg 52, Leiden, The
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TEXTBOOK ANALYSIS ON COLLEGE ACADEMIC WRITING
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TEXTBOOK ANALYSIS ON
COLLEGE ACADEMIC WRITING
Handoyo Puji Widodo
Politeknik Negeri Jember, Jember
Abstract: When no specific materials are available particularly on EFL writing
courses, the selection and use of a textbook are of great priority. For this reason,
this article analyzes a textbook on college academic writing in an EFL contextIndonesia. In this analysis, I employed the in-depth method using the three
phases of the textbook analysis, concerning the three main features of the textbook: (1) goal and organization, (2) contents-inputs, models, and exercises, and
(3) the suitability of the textbook viewed from aims, beliefs about writing, the
roles of the teacher, the role of the students, and the roles of the textbook as a
whole. The extent to which the selected textbook reflected the recent views of
the teaching and learning of writing skill was also investigated. The results show
that the author echoed his clear goal and organization. The contents of the textbook regarding the inputs, models, exercises, and writing assignments reflected
the features of academic writing required for college students.
Key words: academic writing, process writing, text functions
The status of writing in language teaching has accelerated tremendously since 30
years ago. In the teaching of writing, the sequence of activities typically involves:
(1) familiarization: learners study grammar and vocabulary, usually through a text;
(2) controlled writing: learners imitate given patterns, often from substitution tables; (3) guided writing: learners manipulate model texts; and (4) free writing:
learners employ the patterns they have developed to write a letter, a paragraph, an
essay, and the like (Richards, 2002). Writing in a second or foreign language is regarded as one of the most difficult skills for a learner to master, particularly in free
academic writing. The difficulty is due to the need to generate and organize ideas
using an appropriate choice of vocabulary, sentence, and paragraph organization
and to turn such ideas into a readable text (Richards and Renandya, 2002).
109
110 TEFLIN Journal, Volume 18, Number 2, August 2007
For this reason, there should be appropriate materials, which cater for the students needs. Teaching materials are regarded as a key factor in most language
programs (Richards, 2001). For teachers, textbooks or course books either required
or supplementary provide content and teaching-learning activities, which shape
much of what happens in the classroom (Celce-Murcia, 2001). Furthermore,
Wright (cited in Lee, 2003: 165) says that teaching materials (e.g. textbooks) assist
to define the goals of the syllabus and the roles of teachers and learners within the
instructional process.
In EFL writing courses where no specific teaching materials for academic
writing exist, the selection and use of a textbook or course book are crucial issues.
Teachers, in this respect, are required to be able to make informed judgments about
the textbooks and other related teaching materials (Richards, 2001; Lee, 2003). In
other words, the selection and use of the textbook both required and supplementary
is based on its analysis or evaluation.Language materials design and development
take into account such factors as: teachers, learners, language use and function,
tasks, designs, content, and adoption so that the teaching-learning activities are
successfully conducted. In this line, this paper attempts to analyze a textbook on
college academic writing in the context of EFL in Indonesia.
METHODS FOR ANALYZING OR EVALUATING TEXTBOOKS
In general, textbook analysis or evaluation could be carried out through three
main methods: impressionistic, checklist, or in-depth methods (McGrath, 2002).
First, the impressionistic method refers to achieving a general impression of the
materials in the textbook. This analysis, as Stervick (cited in McGrath, 2002: 2526) suggests, includes: 1) three qualities: strengths pay off for the learners, lightness learnability, and transparency clarity of organization and presentation; 2)
three dimensions: linguistic, social, and topical; and 3) four essential components
of lessons: opportunities for language use, sample of language, exploration of vocabulary, and exploration of phonological, orthographic or grammatical form. Second, the checklist method comprises three items, i.e. comparison, identification, or
verification. This method is easy to do since teachers only check the list of the
items while analyzing or evaluating a certain textbook. Last, the in-depth method
encompasses such items as the aims and content of the materials, learners needs,
teachers needs, and material functions as a classroom resource. In other words,
this analysis deals with learner needs and interest, learner approaches to language
learning, and the teaching-learning approach in the teacher s own classroom.
Widodo, Textbook Analysis on College Academic Writing 111
Phases of Textbook Analysis
Of the three methods listed above, I focus on analyzing or evaluating a textbook on academic essay writing, through the three-phase, in-depth method (Lee,
2003). In the first phase, the analysis includes the goal and organization of the textbook. In the second phase, the analysis focuses on contents (e.g. inputs, models,
and exercises) of the textbook. In the last phase, the analysis deals with the suitability of the textbook with the aims, beliefs about writing, the roles of the teacher, the
role of students, and the roles of the textbook as a whole in an EFL context, Indonesia. The textbook analysis is presented in Figure 1 below.
Phase 1
Goal and Organization
Phase 2
Contents
(Inputs, Models, & Exercises)
Phase 3
Suitability
(Goal, Belief about Writing Teaching, & Roles
of Teacher, Students, & Textbook)
Figure 1: Phases of Textbook Analysis
In the following section, the extent to which the selected textbook reflects the
recent views of the teaching and learning of writing skill is discussed in detail.
The textbook chosen is College Writing Skills written by John Langan. This
textbook was published by The McGraw-Hill Companies, Inc in 1996 with the
ISBN of 0-07-036442-7. This textbook has been chosen on the basis of my teaching experience in teaching writing as well as materials design and development for
writing courses at college and university levels.
112 TEFLIN Journal, Volume 18, Number 2, August 2007
FINDINGS
The Extent to which the Selected Textbook Reflects Recent Views of the
Teaching and Learning of Writing Skill
The selected textbook on College Writing Skills reflects a process-based writing approach which focuses on how learners experience a series of activities or
steps in writing. This approach is reflected in Chapter 1 of the textbook and the author s statement in the introduction. The author of the textbook treats writing as a
process comprising four main stages: planning (pre writing), drafting (writing), and
revising and editing. This is related to the process-based approach suggested by
Seow (2002). The four stages of the writing process is presented in Figure 2.
Process Activated
Planning
Drafting
Process Terminated
Editing
Revising
Figure 2. The Writing Process (Seow, 2002: 315)
Some authors (e.g. Dirby, Kirby, and Liner, 2004; Seow, 2002; and Terrible,
1996) also emphasize that process oriented writing, when implemented in the
classroom, also should incorporate three other stages externally imposed on students by the teacher, i.e. responding, evaluating, and post writing (e.g. reading
aloud, displaying, or publishing). Related to the three additional stages, the author
of the textbook incorporates two activities, i.e. responding and evaluating essays
through peer review. Such activities are the key aspects of the process-based approach, and they have a central role in the successful implementation of process
writing, since they provide , students with an opportunity to improve their composition and to write better. More importantly, the students are trained to think critically. Is presented in Figure 3.
Widodo, Textbook Analysis on College Academic Writing 113
Planning
Drafting
Peer/Tutor Review
Revising
Editing
Figure 3: Cycle of Process Writing
Unfortunately, the author of the textbook does not recommend post writing
activities, such as reading aloud, sharing, or publishing, as suggested by Dirby,
Kirby, and Liner (2004); Hyland (2003); Seow (2002); and Terrible (1996).
Hyland (2003) concludes that the process approach to teaching writing emphasizes learners as the independent writers of texts so that a teacher allows her/his
students to develop their abilities to plan, define a rhetorical problem, and propose
and evaluate solutions. Response is crucial in assisting learners to move through
the stages of the writing process; therefore, various means of providing feedback
are used, including teacher-student conferences, peer response, audio taped feedback, and reformulation.
In addition to incorporating the process-based approach, the author sees writing as a functions or the means for achieving the ends (purposes) of writing as
Hyland (2003) points out. In this respect, the author introduces different patterns of
essays, in Chapter 2: Types of Essay Development. In this chapter, the author describes such functional patterns of essays as: description, narration, examples,
process, cause and effect, comparison and contrast, definition, division and classification, and argumentation. The author believes that such patterns are fundamental
to academic writing for college students. They should enable student to develop effective essays through the creation of introductory, body, and concluding paragraphs as well as transitions and develop different types of essays. To sum up, texts
could then be seen as composed of structural entities such as Introduction-BodyConclusion, and particular organizational patterns such as narration, description,
and argumentation are discussed and practiced.
In conclusion, the author views writing as a process since the students need to
go though some activities, and as text functions in developing different types of essays. In short, the author views writing not only as a process, but also as an en-
114 TEFLIN Journal, Volume 18, Number 2, August 2007
deavor to communicate in social context in which he tries to encourage learners to
use language patterns to achieve coherent, purposeful composition.
The Appropriateness of Goals, Organization, and Contents of The Selected
Textbook
The first phase analysis focuses upon the appropriateness of the goal and organization of the textbook, and the second phase analysis concentrates on the appropriateness of the contents of the textbook.
Goal
Regarding the goal, the author states that College Writing Skills will help
students master the traditional five-paragraph essay. It is a very practical book with
a number of special features to aid instructors and students (p. xi). One of the features indicating that the textbook is process-based is that the author mentions
Writing is treated as a process. The second chapter, Important Factors in Writing, discusses prewriting, rewriting, and editing. In addition, many writing assignments are accompanied by Suggestions on How to Proceed that give stepby-step directions in the process of writing an essay. In addition, from the phrase
the traditional five-paragraph essay, we could conclude that the students need to
know three general surface structures of an essay, that is, one introductory paragraph, two body paragraphs, and one concluding paragraph. In this respect, the author also emphasizes that the students should come up with such a format through
a series of activities or a process. Another feature that the author incorporates a text
function-based approach is found in the second chapter of the textbook (pp. 121122 and 124) which discusses the paragraph-pattern essays. To sum up, the goal of
the textbook is clearly stated along with the essential features of the textbook.
Organization
In terms of the organization, the textbook was published in 1996 to aid in the
teaching and learning of academic writing particularly for college students in an international context. The textbook, as the core material, as accompanied by three
supplementary materials, i.e. Instructor s Edition made up of the student text, Instructor s Guide featuring hints to the instructor, a model syllabus, and answers for
all the activities and tests in the text and Instructor s Manual and Test Bank
Widodo, Textbook Analysis on College Academic Writing 115
including material in the Instructor s Guide along with thirty supplementary activities and tests in which such activities and tests offer practice in a wide range of
skills covered in the textbook, from generating and narrowing a thesis to outlining
essays to editing papers for common mistakes in grammar. The textbook offers
color printing only on the headline of each point. The textbook is structured into
four chapters. The first chapter Basic Principles of Essay Writing comprises: (1)
Introduction to the Essay Form, (2) Important Factors in Writing, (3) The First
and Second Steps in Essay Writing, (3) The Third and Fourth Steps in Essay Writing, and (4) Four Bases for Evaluating Essays; each sub-chapter is consistently accompanied with some arguments and clear instructions. The second chapter
Types of Essay Development discusses nine main patterns of essay development
(description, narration, examples, process, cause and effect, comparison and contrast, definition, division and classification, and argumentation) in which each pattern of essay development succinctly comprises: a short input (explanation), a
model essay, classroom activities, and writing assignments. The third chapter
Special Skills provides explanations about taking essay exams, writing a summary,
writing a report, writing a resume and job application letter, using the library, and
writing a research paper. With such skills, I think that the author is committed to
developing students writing skills by providing additional writing skills, which
could support the students learning needs (i.e. taking essay exams and writing a
report) and their future career (e.g. writing a resume and job application letter).
Each description of the skills is consistently accompanied with models and activities. The fourth chapter Handbook of Sentence Skills offers explanations about
grammar, mechanics, punctuation, and word use (vocabulary); each of which is
accompanied with activities and review tests. Additionally, this chapter treats practice, which focuses on editing tests for sentence-skills mistakes consisting of 12 editing tests with clear directions.
In conclusion, the textbook is clearly and consistently organized along with its
activities. The organization of the textbook reflects its goal and requirements of
academic writing for college students.
Contents
In the second phase, I now continue my analysis procedure by carrying out an
in-depth analysis in order to investigate the explicit nature (i.e. contents) of the
chapters in the selected textbook. As previously mentioned, the textbook is divided
into 4 chapters: (1) Basic Principles of Essay Writing, (2) Types of Essay Devel-
116 TEFLIN Journal, Volume 18, Number 2, August 2007
opment, (3) Special Skills, and (4) Handbook of Sentence Skills in which such
chapters provide sufficient classroom activities and writing assignments. Nevertheless, in this sense, only two chapters (Basic Principles of Essay Writing and Types
of Essay Development) are thoroughly analyzed. This analysis meets McDonough
and Shaw s suggestion (cited in Lee, 2003: 174) that:
In order to perform an effective internal inspection of materials, we are required
to examine at least two units of a piece or set of materials. This also enables us to
find out any characteristic relationship between chapters or units in the textbook.
Chapter 1 and Chapter 2 have been chosen since the two are fundamental aspects for writing academic essays, particularly for college students of intermediate
level. Chapter III Special Skills could be treated to equip students with further writing skills, and Chapter IV could be treated as part of references for sentence problems, which exist in students essays.
Arriving at a closer inspection of the sequence of activities included in Chapters 1 and 2, each chapter starts with points which will be discussed. In detail,
Chapter 1 is divided into five sub-chapters, including (1) Introduction to the Essay
Form explaining the importance of supporting a point in writing, the difference
between a paragraph and an essay, and the general structure of an essay; (2) Important Factors in Writing discussing the importance of your attitude about writing,
developing a subject, keeping a journal, prewriting, outlining, revising, editing, and
proofreading; (3) The First and Second Steps in Essay Writing showing how to
start an essay with a thesis and support the thesis with specific evidence/details; (4)
The Third and Fourth Steps in Essay Writing providing explanations about organizing and connecting specific details in the body paragraph(s) of an essay, starting
and ending an essay with effective introductory and concluding paragraphs, and
writing clear, error free sentences; and (5) Four Bases for Evaluating Essays
explaining how to evaluate an essay for unity, support/details, coherence, and sentence skills. I think that the sequence of the sub-chapters in Chapter 1 gives basic
understanding to students how to write an essay consisting three levels: introductory, body, and concluding paragraphs; this is consistent with the author s goal that
help students master the traditional five-paragraph essay (p. xi). It is also
worth noting that the author provides pre writing techniques, such as brainstorming, free writing, diagramming, making a list, preparing a scratch outline, and outlining. Such techniques will enable students to have opportunities for methods of
generating ideas, as Raimes (2002) suggests. Furthermore, the author s explana-
Widodo, Textbook Analysis on College Academic Writing 117
tions about the introductory paragraph consisting of a general statement and thesis; the body paragraphs developing the thesis stated in the introductory paragraph along with the relevant details; and the concluding paragraph are well introduced. Such concepts along with the exercises will assist the students to comprehend and write an essay easily. Interestingly, in sub-chapter 4 Four Bases for
Evaluating an Essay, the author reminds students/learners of other essential aspects
of writing an essay; four of which are unity, coherence, details, and grammar by
supplying the evaluation checklists for an essay. According to Raimes (2002), this
will encourage the students to be involved in editing and proofreading activities. Of
the five sub-chapters, the author provides succinct explanation or input session,
model or example of the input, and exercises. The four forms of activities allow the
students to have input and practice so that they are well prepared for the next chapter, that is, Types of Essay Development, for example. Unfortunately, the author
does not provide assignments for students to reinforce the materials in each subchapter so that they could do some exercises outside the classroom. Regardless of
this shortcoming, in Chapter 1, the author assigns the students to write an essay that
include required points.
Chapter 2 is divided into 10 sub-chapters, including (1) Introduction to Essay
Development discussing definitions of the patterns of essays explained later on,
point of view in writing, writing for specific purpose and audience, peer review,
and essay checklists; (2) Description, (3) Narration, (4) Examples, (5) Process, (6)
Cause and Effect, (7) Comparison and Contrast, (8) Definitions, (9) Division and
Classification, and (10) Argumentation. In terms of point of view in writing, the
author mentions three approaches or points of view; three of which are first person
(e.g. I, me, mine, we, our, and us), commonly used in narrative essays based upon
personal experience; second person (i.e. you), generally found in a process or procedure essay; and third person (e.g. she, he, her, him, they, their, and them), commonly found in most of the academic essays. In this sense, the author tries to remind students of appropriate use of pronouns in writing an academic composition.
It could be concluded therefore, that the author applies the text function-based
approach to selecting the types of essays, which are assigned to students or on
which the students work. It is quite interesting to observe that the author instructs
the students to outline their draft before they develop an essay and use peer review
before their assignments are submitted. The us of peer review, indicates that the author considers writing as a process, which requires prewriting and feedback from
others as important elements in writing a better essay. Furthermore, the author believes that peer review offers some benefits. This idea is compatible with Hyland s
118 TEFLIN Journal, Volume 18, Number 2, August 2007
(2003: 199) statement that such an activity will encourage students to participate
actively, get involved in authentic communicative context, and develop critical
reading skills. To facilitate peer review, the author provides checklists for essay
feedback for the teacher and the learners and essay evaluation for the teacher.
The core materials of the second chapter are the types of essay development.
In this respect, the author provides the input, models, exercises, and writing assignments. In the input, the author provides a short introduction to types of essay
by giving models. Some topics (i.e. basement or garage, exam room, shyness, love,
how to gain or lose weight, how to relax, punishment for certain crimes, two
teachers, two games, team player, optimist, clothing, school subjects, lower attendance, and missing school to go to work) are familiar with the students, so they
could easily draft or develop an essay. To know whether the students understand
the concept, the author provides exercises in terms of essay structures explained in
Chapter 1. Besides, the writing assignments are provided for writing independently
on the basis of one of the topics given. The focus is on having the students develop
a type of essay assigned for outside-classroom activities.
To sump, in Chapters 1 and 2, the author divides the chapters into subchapters in which each sub-chapter consistently comprises: inputs, models, exercises, and writing assignments. The inputs and models are clearly explained along
with some problems. The author emphasizes sequential steps for developing patterns of essays on the basis of the writer s purposes. I think that the author provides
essay topics that are quite familiar with EFL college students particularly in Indonesia.
DISCUSSION OF THE FINDINGS
The Suitability of The Selected Textbook with The EFL College Students in
Indonesia
In this last phase, I would like to make an attempt of drawing together the
various aspects of the textbook as previously noted at Phases 1 and 2, and I would
suggest the underlying goal, beliefs about writing, the roles of the teacher, the role
of students, and the roles of the textbook as a whole, as Richards (2001) emphasizes.
Widodo, Textbook Analysis on College Academic Writing 119
Goal
Drawing from the analyses in the phases 1 and 2, it is obvious that the core
goal of the textbook is the development of the college students essays in English,
concentrating on writing good essays of different types through the integrated approach: process and text function based writing. The exercises included in the textbook are intense. For instance, in Chapter 1, the author consistently provides some
exercises after the input and its model are given, and in Chapter 2, he provides not
only classroom exercises, but writing assignments as well in which some activities
include peer review. In this sense, it could be viewed that the author tries to encourage students to think critically and to work independently without relying too
much on the teacher, and the author believes that writing should be considered as a
process as mentioned in the key features of the textbook, and he assures that student writers ideas could be written on the basis of certain purpose and audience.
From the two chapters, it is apparent that the author is highly committed to the goal
prescribed in the introduction of the textbook. I think the textbook meets the needs
of college students who would like to write an essay at either an intermediate or
post-intermediate level.
Belief about Writing
The author s belief about writing could be presented in the content of the
textbook and may influence the methodology. As previously described in Phases 1
and 2, the methodology is characterized by a basic standardized pattern in which
each concept starts with inputs, models, and exercises. The essay writing element
also shows an underlying belief about rhetorical writing regarding general structure
of a composition (an essay), idea organization, and syntactic patterns. The patterns
of the essays indicate types of essays (e.g. description and argumentation), which
determine the writer s purposes mostly found in academic settings. In other words,
the author clearly sees writing as a medium of communication in different purposes to render a purposeful, communicative composition. He also views writing
as a process through the creation of seven important elements in the essay, such as
surface structure of essays (i.e. introductory, body, and concluding paragraphs) and
idea organization (e.g. unity, coherence, detail, and grammar) in which the students
have to develop a set of strategies for planning, drafting, revising, and editing. The
process approach is used to furnish useful support for the students in achieving
greater control over the cognitive strategies employed in writing. On the other
hand, in Chapter 2 the author provides many writing assignments for developing
120 TEFLIN Journal, Volume 18, Number 2, August 2007
patterns of essays assigned without considering or suggesting time allocation and
opportunity for students to have thorough feedback. Aside from such a drawback,
the exercises designed by the author reflect a process in which the students are required to go through intensive writing work. The author s belief about writing is
compatible with the context of EFL college students in which the process is of
great priority supported with the clarity of goals of writing a certain composition
(e.g. an essay) on the basis of purpose and audience.
The Roles of the Teacher
Since the textbook is accompanied with the three supplementary textbooks,
namely, Instructor s Edition, Instructor s Manual, and Test Bank as previously
mentioned in Phase 1, this indicates that a teacher serves as a facilitator or resource, monitor, motivator, feedback provider, and evaluator. As a facilitator or resource, the teacher provides some inputs that are learnable or comprehensible for
the students; s/he is required to select useful exercises or activities for the students.
As a monitor, the teacher is required to monitor students activities because there
are numerous activities to be done by the students. As a motivator, since the students go through intense activities, the teacher needs to motivate the students to
write better and cooperate with one another and with the teacher. As feedback provider and evaluator, the teacher should provide some comments or suggestions on
the students work, and then he could evaluate the students essays as a result of the
feedback given.
Since the inputs and input models are given prior to the exercises or assignments, the teacher is required to help the students to comprehend the inputs and the
model/examples provided. With the supplementary textbooks given, the teacher
feels more confident in solving some problems in the exercises. In spite of this,
s/he has to be able to wisely and thoroughly arrange the time in the classroom and
evaluation for the students. It seems to me that, for an EFL teacher, this textbook
provides clear models and standard answers, which are internationally standardized, and could motivate the teachers to design and develop writing materials. In
this case, the teacher could further serve as materials designer, developer, or
evaluator.
Widodo, Textbook Analysis on College Academic Writing 121
The Roles of Students
From the organization and content of the textbook particularly in Chapters 1
and 2, focusing on essays, it is suggested that the students begin writing an essay
with a clearly stated thesis; provide logical, detailed support for their thesis; organize the supporting materials effectively; and revise and edit carefully so that the essay is presented in clear, error free sentences. In this sense, it could be obviously
seen that the students serve as competent planners, writers, and feedback providers
of their essays and others essay in a peer review activity. In other words, the author believes that the students could serve not only as planners and writers but also
feedback providers. I think involving students as feedback providers means empowering them in thinking critically. In other words, the author of the textbook
tries to inspire the students to get started writing better.
The Roles of the Textbook
Since all of the concepts, models, and exercises along with the Instructor s
Edition are provided, the textbook serves not only as an informant but also as a
useful guide or reference to facilitate teaching-learning process both in the classroom and outside the classroom. In short, the principal role of the textbook as a
whole is to organize the teaching and learning of English essays for college students. The textbook could serve as a source of learning for students particularly in
an EFL context.
CONCLUSION AND SUGGESTIONS
In conclusion, the analysis in Phase 1 indicates that the author has echoed his
clear goal and organization. The analysis in Phase 2 shows the contents of the textbook in terms of the inputs, models, exercises, and writing assignments. Last, the
analysis in Phase 3 enables proves that the textbook is suitable regarding such issues as: goal, belief about writing, the roles of the teacher, the roles of the students,
and the roles of the textbook as a whole, as Richards (2001) and Raimes (2002)
point out. On the basis of the analyzes above, I recommend that the textbook be
used as one of the learning sources especially in college essay writing courses either core or supplementary in the context of EFL.
122 TEFLIN Journal, Volume 18, Number 2, August 2007
REFERENCES
Celce-Murcia, M. 2001. Teaching English as Second or Foreign Language (3rd
edition). Boston: Heinle & Heinle.
Coffin, C., M.J. Curry, S. Goodman, A. Hewings, T. Lillis, and J. Swann. 2002.
Teaching Academic Writing: A Toolkit for Higher Education. London:
Routledge.
Hyland, K. 2003. Second Language Writing. New York: Cambridge University
Press.
Kirby, D., D.L. Kirby, and T. Liner. 2004. Inside Out: Strategies for Teaching
Writing (3rd edition). Portsmouth: Heinemann.
Lee, Y. 2003. A package for an English paragraph: An evaluation of the coursebook used in two EFL writing courses. English Teaching 58/3: 165 188.
McDonough, J. and C. Shaw. 2000. Materials and Methods in ELT: A Teacher s
Guide. Oxford: Oxford University Press.
McGrath, I. 2002. Materials Evaluation and Design for Language Teaching. Edinburgh: Edinburgh University Press Ltd.
Raimes, A. 2002. Ten steps in planning a writing course and training teachers of
writing. In J. C. Richards and W. A. Renandya (eds.), Methodology in Language Teaching: An Anthology of Current Practice (pp. 306-314). Cambridge: Cambridge University Press.
Richards, J.C. 2001. Curriculum Development in Language Teaching. Cambridge:
Cambridge University Press.
Richards, J. C. 2002. 30 years of TEFL/TESL: A Personal Reflection. RELC Journal 33/2: 1 33.
Richards, J.C. and T. Rodgers. 2001. Approaches and Methods in Language
Learning (2nd edition). New York: Cambridge University Press.
Richards, J.C. and W. A. Renandya. 2002. Methodology in Language Teaching:
An Anthology of Current Practice. Cambridge: Cambridge University Press.
Seow, A. 2002. The writing process and process writing. In J. C. Richards and W.
A. Renandya (eds.), Methodology in Language Teaching: An Anthology of
Current Practice (pp. 315-320). Cambridge: Cambridge University Press.
Terrible, C. 1996. Writing. Oxford: Oxford University Press.
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Universality class of explosive percolation in Barabási-Albert networks
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Scientific reports
| 2,019
|
cc-by
| 10,575
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Universality class of explosive
percolation in Barabási-Albert
networks Received: 28 September 2018
Accepted: 26 March 2019
Published: xx xx xxxx Received: 28 September 2018
Accepted: 26 March 2019
Published: xx xx xxxx MD. Habib E Islam1,2 & M. K. Hassan1 In this work, we study explosive percolation (EP) in Barabási-Albert (BA) network, in which nodes are
born with degree k = m, for both product rule (PR) and sum rule (SR) of the Achlioptas process. For m = 1
we find that the critical point tc = 1 which is the maximum possible value of the relative link density t;
Hence we cannot have access to the other phase like percolation in one dimension. However, for m > 1
we find that tc decreases with increasing m and the critical exponents ν, α, β and γ for m > 1 are found
to be independent not only of the value of m but also of PR and SR. It implies that they all belong to
the same universality class like EP in the Erdös-Rényi network. Besides, the critical exponents obey the
Rushbrooke inequality α + 2β + γ ≥ 2 but always close to equality. PACS numbers: 61.43.Hv, 64.60.Ht, 68.03.Fg, 82.70.Dd. Percolation is still studied in extenso even after more than 60 years of its first formulation1–7. One of the reasons is
that its notion is omnipresent in systems as disparate as spread of forest fire, flow of fluid through porous media,
spread of biological and computer viruses leading to epidemic, formation of public opinion, resilience of systems
etc.8–11. However, the most important reason is that it can describe phase transition and critical phenomena
albeit it requires no quantum and many particle interaction effects into consideration12. To study percolation
we first need to choose a skeleton. Traditionally, physicists like spatially embedded lattices as a skeleton. On the
other hand, prior to 1999 random graph, namely Erdös-Rényi (ER) network, has also been used as a skeleton to
study percolation though mostly by mathematicians13. In recent years use of network as a skeleton has gained
huge interest among scientists in general and physicists in particular owing to two seminal papers of which one
on small-world network and the other on scale-free (SF) network14–16. The idea of scale-free networks proposed
by Barabási and Albert has revolutionized the notion of the graph theory. Universality class of explosive
percolation in Barabási-Albert
networks One of the hallmark features of the SF
network is the presence of hubs which is hold responsible for making the SF network highly resilient to random
removal of links albeit remains vulnerable to targeted removal17,18. The resilience to random attack is in a sense
opposite to the process of percolation. In percolation one studies the conditions leading to the formation of the
giant, a cluster of nodes connected by occupied links that grows linearly with network size N, while in the resil-
ience one checks the condition of the disappearance of the giant. In 2009, Achlioptas et al. proposed a simple variant of the classical random percolation (RP) and applied it in
the ER network19. The skeleton of the ER network consists of N isolated nodes and N(N − 1)/2 number of frozen
links among all the pairs of N nodes. Then at each step two distinct frozen links are picked at random instead
of one which is done in RP. However, ultimately only one of two links, that suppresses the growth of the larger
clusters, is activated and occupied while the other link is discarded for recycle. A specific rule that discourages the
growth of the larger cluster and encourages the smaller ones to grow faster is the now well-known Achlioptas pro-
cess (AP). It has been found that such a simple change in the selection rule proved to cause a dramatic effect in the
final outcome. Indeed, as we keep occupying links following the rules of the AP, we find the emergence of a giant
cluster with a bang. This is in sharp contrast to its classical counterpart and hence it is called “explosive percola-
tion” (EP). Indeed, the corresponding order parameter P, the relative size of the largest cluster smax/N, undergo
such an abrupt transition that it was at first mistaken as a discontinuity and suggested to exhibit the first order or
discontinuous transition. This claim immediately triggered an explosion of scientific activities20–29. To this end, it
is now well settled that the EP transition is actually continuous but with some first order like finite-size effects28–31. yiif
Recently, it has been reported that RP in scale-free weighted planar stochastic lattice (WPSL) belongs to a
universality class which is different from the unique universality class of all the known planar lattices32,33. Earlier, 1University of Dhaka, Department of Physics, Theoretical Physics Group, Dhaka, 1000, Bangladesh. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports BA Model and its Properties p
We begin by discussing the construction process of the BA network. It starts by choosing a seed that may consists
of m0 arbitrarily connected nodes where m0 has to be extremely small compared to the final size N of the network
that we intend to grow. However, it has been found that the observable quantities are independent of the size of
m0 and of the detailed nature of how nodes of the seed are connected. Once a seed is chosen, we add one node
with m links at each time step, where m ≤ m0, and pick m distinct nodes from the existing network such that node
i is picked with probability Π(i) proportional to its own degree ki. It has been shown by BA that the network that
grow following this preferential attchment (PA) rule, Π(i) ∝ ki, exhibits power-law degree distribution
λ
−
~
P k
k
( )
with exponent λ = 3 indeoendent of m. The total number of links in the BA networks of size N is approximately
equal to mN since each node is born with degree m. Each link contributes to degree 2. Thus the total degree is
equal to 2 mN and hence the average degree of the BA network of size N is 2 m and hence for m = 1 it is a tree
network. Network grown in this way can be used as a skeleton provided the snapshots of the network in the same
realization at different times are similar. In ref.35 we have shown that the snapshots of the BA network at different
times are similar and hence we say it exhibits self-similarity. In Fig. (1) we give snapshots of the BA network con-
sisting of 256 nodes grown for m = 1, 2, 5, 10 just to grasp how the value of m keep its signature even in their
visual look.h The question is: Do all the data points follow the power-law decay of P(k) with the same exponent? In Fig. (2a)
we show how the degree distribution varies with m. We can clearly see that the number of data points for small m,
especially the data points for small k, do not fall on the straight line of the log(P(k)) versus log(k) plot. However,
as the m value increases the trend to follow the straight line increases. Universality class of explosive
percolation in Barabási-Albert
networks 2Old Dominion
University, Department of Physics, 4600 Elkhorn Ave Norfolk, VA 23529, USA. Correspondence and requests for
materials should be addressed to M.H.E.I. (email: m2islam@odu.edu) or M.K.H. (email: khassan@du.ac.bd) Scientific Reports | (2019) 9:8585 | https://doi.org/10.1038/s41598-019-44446-2 1 www.nature.com/scientificreports/ Cohen et al. reported that the universality class of RP on scale-free networks is different from the expected uni-
versality class of RP on infinite dimensional ER or Bethe lattice34. Motivated by the role that the scale-free nature
of the skeleton plays in percolation, we study two variants of EP, namely product rule (PR) and sum rule (SR)
under AP, on scale-free BA networks whose nodes are born with degree m. We then address the following ques-
tions: Does the PR-SR universality that holds in the ER network also hold in the BA network? Does EP in the ER
and BA networks belong to the same universality class as expected since ER and BA networks both are infinite
dimensional? If not, does EP in the BA network still obey Rushbrooke inequality? Besides addressing these fun-
damental questions, we find the following results. The critical points depend on m but not the critical expo-
nents. We attempt to explain the significance of order and disorder in percolation using the idea of entropy and
order parameter. We find that, like ferromagnetic transition, percolation too is accompanied by order-disorder
transition.h The rest of the article is organized as follows. In section Sec. II we discuss some of the properties of the BA
network to see how they differ with m. In Sec. III, the explosive percolation model is defined and the idea of
convolution is introduced. Sec. IV contains definitions and properties of entropy and order parameter. In Sec. V we present our results on specific heat and susceptibility. In Sec. VI on the other we discuss about the scaling
properties and the Rusbrooke inequality. The results are discussed and conclusions drawn in Sec. VII. BA Model and its Properties Indeed, we find that already at m = 100 it
appears that almost all the data points, except the finite-size effect, follow the straight line which has slope equal
to 3. Moreover, we observe that the extent of straight line increases with increasing network size revealing that in
the thermodynamic limit it will be a straight line all the way through. The impact of m in the growing network can
be best understood by measuring the inverse harmonic mean (IHM) of the neighbours of each node36. The value
of IHM of the i th node is defined as =
∑=
k
IHM
,
(1)
i
j
k
k
i
1
1
i
j (1) where ki is the number of nearest neighbors of the node i. If we now measure the fraction of the nodes which have
IHM values within a given class, which we call relative frequency, then the resulting histogram plot for different
m value is shown in Fig. (2b). It clearly shows the strong impact of m in the plots. The plots for higher m, such
as m = 70 or 100, seem very different from those for the smaller m such as for m = 1 or m = 2 etc. Thus, the BA
network for different m is significantly different albeit the exponent of the degree distribution is the same. Definition of Explosive Percolation The size of the nodes are 10 consisting of N = 256 nodes are shown to appreciate
nodes are depicted as being proportional to their respect Figure 1. Snapshots of BA networks for m = 1, 2, 5, 10 consisting of N = 256 nodes are shown to appreciate the
impact of m even in the impression. The size of the nodes are depicted as being proportional to their respective
degree. ther hand, to apply the SR, we take the sum Σij = si + sj and Σkl = sk + sl instead of the product and do the same
s for PR. f h
bl
h
l
k
h
b
f h
l
h
f other hand, to apply the SR, we take the sum Σij = si + sj and Σkl = sk + sl instead of the product and do the same
as for PR. One of the problems with percolation on network is the absence of the spanning cluster that spans from one
end to the other end across the system since there is no boundary or end in network. Therefore a definition of
spanning probability for percolation on network is not possible; this in turn makes it a formidable task to find the
critical point and the critical exponent ν of the correlation length accurately. Yet another problem in percolation
in general is that we need to perform many independent realizations. It works fine if the observable quantity, say
X, is obtained directly from the ensemble averaged data. However, sometimes we may need to take derivative of X
to obtain say Y = X′. In such cases, the ensemble average of X does not give smooth enough data for Y regardless
of how big the ensemble size is. The only solution then is to use the idea of convolution as prescribed by Newman
and Ziff in 200040,41. According to their approach, we first need to measure the observable, say Xn, as a function
of the number of occupied links n. Note that we continue the process of occupying links n until n = N links are
occupied or relative density of link t = n/N = 1 is achieved. We now take an ensemble averaged data over many
independent realizations. Definition of Explosive Percolation Once the network of desired size is grown we use that as a skeleton like we earlier used scale-free weighted planar
stochastic lattice and ER network37–39. We assume that all the links of the BA network are temporarily frozen. We first label all the frozen links so that e(i, j) represents a link that establishes a connection between nodes i
and j ensuring that i ≠ j since self connection is forbidden. Earlier, two groups independently studied EP to form
scale-free networks instead of using a scale-free network as a skeleton22,25. In contrast, we use the scale-free BA
network as a skeleton from where we pick a pair of candidate links randomly at each step and follow the rest of the
process exactly like Achlioptas et al.19. Links can be of two types. When occupation of a link connects two isolated
clusters then it results in a larger cluster and it is called inter cluster link. On the other hand, when a link connects
two sites of the same cluster, which does not result in the increase in the cluster size, then the corresponding link
is called intra-cluster link. Initially, every node is a cluster of its own size since all the links are assumed frozen. The process starts by picking two distinct links, say e(i, j) and e(k, l), randomly with uniform probability at each
step. To apply the PR, we then calculate the products, Πij = si × sj and Πkl = sk × sl, of the size of the clusters that the
two nodes on either end of the links e(i, j) and e(k, l) respectively contain. The link with the smaller value of the
products Πij and Πkl is occupied; in case of Πij = Πkl one of the corresponding links is selected randomly. On the Scientific Reports | (2019) 9:8585 | https://doi.org/10.1038/s41598-019-44446-2 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ p
p
/
Figure 1. Snapshots of BA networks for m = 1, 2, 5, 10 consisting of N = 256 nodes are shown to appreciate th
impact of m even in the impression. The size of the nodes are depicted as being proportional to their respectiv
degree. Figure 1. Snapshots of BA networks for m = 1, 2, 5, 10 consi
impact of m even in the impression. Entropy and Order Parameteri py
Entropy H and order parameter P definitely are the two most important quantities of interest in the theory of
phase transition as they are used to define the order of transition. For instance, if they suffer a sudden jump or
discontinuity at the critical point then the transition is first order and else it is called continuous or second order
phase transition. Besides, they are also used as a litmus test of whether the transition is accompanied by symmetry
breaking or not. In the case of symmetry breaking the system undergoes a transition from the disordered state,
characterized by maximally high entropy, to the ordered state, characterized by maximally high order parameter. Note that the highly ordered state is always less symmetric than the highly disordered state. For instance, in ferro-
magnetic to paramagnetic transition we find that P and H undergo such an abrupt or sudden change but without
gap or discontinuity at tc. In the case of percolation, it has been well known that the order parameter changes in
the same fashion as in ferromagnetic transition. However, to know whether percolation transition too is accom-
panied by symmetry breaking or not we have to know how entropy changes at the critical point. Only recently we
proposed a suitable normalized probability and obtained desired behaviour of entropy for the first time by using
it in the definition of Shannon entropy ∑μ
μ
= −
H t
K
( )
log
,
(3)
i
m
i
i ∑μ
μ
= −
H t
K
( )
log
,
i
m
i
i (3) where we choose K = 1 since it merely amounts to a choice of a unit of measure of entropy42. Surely one can use
any normalized probability in Eq. (3) and claim to have measured entropy. Especially, being percolation a prob-
abilistic model, there is no short of normalized probability. Note that originally in the information theory, the
sum in Eq. (3) is taken over individual labelled message. In percolation, we regard each cluster as equivalent to
message hence the sum cannot be over the size of the clusters rather over the individual cluster label. In fact, none
of the existing probabilities which we know in percolation theory can be used to measure entropy although there
have been some attempts43,44. Definition of Explosive Percolation We then use the resulting data in the convolution relation ∑
=
−
=
−
X t
t
t
X
( )
(1
)
,
(2)
n
N
n
N
n
n
1 ∑
=
−
=
−
X t
t
t
X
( )
(1
)
,
n
N
n
N
n
n
1 (2) to obtain X(t) for any value of t. The appropriate weight factor for each n at a given t is ∑
−
=
−
t
t
(1
)
n
N
n
N
n
1
40,41. The
convolution relation takes care of that weight factor and hence helps obtaining a smooth curve for X(t) as a func-
tion of t. Its importance can be best appreciated when one takes the derivative of X(t) and then plot dX(t)/dt as a to obtain X(t) for any value of t. The appropriate weight factor for each n at a given t is ∑
−
=
−
t
t
(1
)
n
N
n
N
n
1
40,41. The
convolution relation takes care of that weight factor and hence helps obtaining a smooth curve for X(t) as a func-
tion of t. Its importance can be best appreciated when one takes the derivative of X(t) and then plot dX(t)/dt as a Scientific Reports | (2019) 9:8585 | https://doi.org/10.1038/s41598-019-44446-2 3 www.nature.com/scientificreports/ Figure 2. (a) Degree distribution of the BA network for different m value. (b) Plots of the relative frequency
distribution of the IHM value as a function of IHM. Figure 2. (a) Degree distribution of the BA network for different m value. (b) Plots of the relative frequency
distribution of the IHM value as a function of IHM. function of t. The resulting plot will be far too smooth compared to the ones without convolution which cannot
be compensated by increasing the ensemble size to obtain ensemble averaged data. function of t. The resulting plot will be far too smooth compared to the ones without convolution which cannot
be compensated by increasing the ensemble size to obtain ensemble averaged data. Entropy and Order Parameteri We assume that there are m distinct, disjoint, and indivisible labelled clusters i = 1, 2, ..., k whose sizes are say
s1, s2, ...., sk respectively after addition of n = tN links. To measure entropy for percolation we use the Shannon
entropy where we choose μi = si/N as the cluster picking probability that a site being picked at random will belong
to the cluster i32,33,37. Note that initially each node is a cluster of its own size s = 1 and hence μi = 1/N for all the
nodes i = 1, 2, ..., N. This is exactly like the state of the isolated ideal gas where all allowed microstates are equally
likely and hence it is expected that entropy is maximum at t = 0. To see what happens at the other extreme, at t = 1,
we first plot histogram of μi versus cluster label i in Fig. (3) for PR with m = 5 only to show what happens to the
probabilities {μ1, μ2, ..., μk} in the vicinity of tc. It is clear from the figure that already near tc = 0.733556, there is a
clear sign of explosive growth to one giant cluster. Thus if we pick one of the nodes at random it is most likely to
belong to the giant cluster and hence the corresponding microsstate is the most likely. The same is true for SR and
for other values of m as well. Thus, it is expected that at t = 1 almost all the nodes will belong to one giant cluster
making μ ≈ 1 and hence entropy at t = 1 should be minimally low. g μ
py
y
Using the cluster picking probability μi in Eq. (3) we have measured entropy and shown in Fig. (4a) for m = 1. We see that for m = 1 the critical point tc = 1. This is like one dimensional percolation problem where we do
not have access to the other phase t > tc since the number of links n for m = 1 cannot exceed N and hence t ≤ 1. Scientific Reports | (2019) 9:8585 | https://doi.org/10.1038/s41598-019-44446-2 4 www.nature.com/scientificreports/ Figure 3. Plots of cluster picking probability μ as a function of cluster label i to show abruptness of its change
as t changes from (a) t = 0.7333 to (b) t = 0.7341. Entropy and Order Parameteri It clearly shows that near tc the giant cluster emerges within an
extremely small change in t. Figure 3. Plots of cluster picking probability μ as a function of cluster label i to show abruptness of its change
as t changes from (a) t = 0.7333 to (b) t = 0.7341. It clearly shows that near tc the giant cluster emerges within an
extremely small change in t. Figure 4. Plots entropy H vs t for m = 1, 3, 5 and m = 10. We find the critical points for m = 1, 3, 5 and m = 10
are 1, 0.7467, 0.73422 and 0.7272 respectively. The entropy plots for all m > 1 share the same shape except the tc
values decreases with increasing m. Figure 4. Plots entropy H vs t for m = 1, 3, 5 and m = 10. We find the critical points for m = 1, 3, 5 and m = 10
are 1, 0.7467, 0.73422 and 0.7272 respectively. The entropy plots for all m > 1 share the same shape except the tc
values decreases with increasing m. However, as we increase the value of m from m = 1 we find that tc < 1 and the tc value decreases with increasing
m. This is expected as we know that the critical point always depends on the mean connectivity which is 2 m for
the BA network. In Fig. (4b) we show entropy versus t curves interpolate nicely between t = 0 and t = 1 for m = 3. The key features of entropy for all other m > 1 is the same except their tc value. We all know entropy measures the
degree of disorder. What is disorder in percolation anyway? Say, initially every cluster is colored with a distinct
color. To do that we need N number different colors. The probability that a node picked at random belongs to, say
green or red or any other color, is 1/N. Seeing a system with number of different colors as many as the number
of clusters will definitely give an impression of a disordered system. Now as we keep adding links, clusters will
merge to form bigger clusters. Entropy and Order Parameteri It means that our entropy
H measures the average degree of ignorance or confusion associated with picking each node and answering
exactly which cluster does it contain. Now as we keep adding links we will have a giant cluster of same color at
t = 1. In this case, we are in the least confused state as we have H(μ) = 0. This is the state we regard as the most
ordered phase. Thus percolation is indeed an order-disorder transition. where disorder is equivalent to degree of
confusion and order is equivalent to certainty.i q
y
In percolation, the order parameter is defined as the relative size of the largest cluster P = smax/N. Note that at
t ≤ tc the largest cluster is miniscule in size which decreases in comparison to N as N increases and hence P → 0
as N → ∞ till t = tc. However, at above tc the size of the largest cluster is proportional to the size of the system N
itself. Thus, in the t > tc regime P is non-zero and for a given value of t its value increases to a constant as N → ∞. Moreover, P increases sharply but without discontinuity near tc and then moves slowly towards unity as t increases
further. This is exactly how magnetization behaves with temperature during the paramagnetic to ferromagnetic
phase and hence regarding P as the order parameter is well justified. Typically, the phase where the order param-
eter is always zero is regarded as the disordered phase provided entropy is significantly high there. On the other
hand, the phase where entropy is minimally low but the order parameter is significantly high is called the ordered
phase. We intend to present both the quantities in the same plot to see whether they compliment each other in
this fashion or not. However, note that the numerical value of the maximum entropy, which is equal to log(N),
is much higher than that of the P, which can at best be equal to unity. Therefore, for better comparison we plot
relative entropy H(t)/H(0) and relative order parameter P(t)/P(1) in an attempt to re-scale their values so that in
either cases their respective maximum values become unity. The plots of re-scaled entropy and order parameter
are shown in Fig. (5a,b) for various m = 1 and m = 5 respectively for product rules only. Entropy and Order Parameteri Consider that when two clusters merge the resulting cluster take the color of the
larger cluster and in the case of merging two equal sized clusters the resulting cluster picks one of the two colors
at random. As we continue adding links we will see a transition across tc to a giant cluster of one single color that
dominates the entire system. At this point, the system will look ordered in the sense that any site we pick at ran-
dom is almost certain that it will belong to the giant cluster. Scientific Reports | (2019) 9:8585 | https://doi.org/10.1038/s41598-019-44446-2 5 www.nature.com/scientificreports/ Figure 5. (a) We show plots of relative entropy H(t)/H(0) and relative order parameter P(t)/P(1) for (a) m = 1
and (b) m = 5. Both the plots are for PR only since the corresponding plots for SR have exactly the same features. Figure 5. (a) We show plots of relative entropy H(t)/H(0) and relative order parameter P(t)/P(1) for (a) m = 1
and (b) m = 5. Both the plots are for PR only since the corresponding plots for SR have exactly the same features. To get a better understanding of disorder we now focus on the frequentist version of the probabilities {μ1, μ2,
..., μk}. Let us make N number of independent attempts to pick one node at each attempt at random with uniform
probability. Say that clusters 1, 2, …, m are picked n1, n2, ..., nm times so that ∑
=
= n
N
i
m
i
1
. The total number of
ways we could have N outcomes are Ω=
.... . N
n
n
n
! (
)!(
)! (
)! (4)
m
1
2 (4) Taking log on either side and using ni = Nμi and the Stirling’s approximation log N! = N log N − N for very
large N we obtain ∑μ
μ
μ
Ω= −
=
. =
N
NH
log
log
( )
(5)
i
m
i
i
1 (5) The total entropy is therefore equal to NH. In the case when n1 = n2 = ... = nm each cluster has the equal chance
of picking means μi = 1/N ∀ i we have the most disordered state where log Ω = N log N. Entropy and Order Parameteri The corresponding plots
for the sum rule are not shown since they have exactly the same features except the fact that tc value for m > 1 are
different. These figures clearly show that when P = 0, the entropy H is maximally high and vice versa. It means
that percolation transition, like paramagnetic to ferromagnetic transition, is accompanied by order-disorder
transition. Specific Heat and Susceptibilityi Specific Heat and Susceptibilityi α
−
~
C t
t
t
( )
(
)
c (9) The quality of data-collapse is a clear testament of the accuracy of our α = 0.98739 value. Note that the α value
of EP in BA network is slightly lower than its value in the ER network where α = 1.ii The quality of data-collapse is a clear testament of the accuracy of our α = 0.98739 value. Note that the α value
of EP in BA network is slightly lower than its value in the ER network where α = 1.ii Our next goal is to find the critical exponent β of the order parameter. Essentially, we should find the β value
also for infinite system for which P should be equal to zero over the entire regime of t < tc. This is, however, not
the case as long as we work with finite size system. Plotting P(t) as a function of t for different system size clearly
shows a sign that as N increases we find P = 0 up to an increasingly higher value of t as shown in Fig. (7a,b). The
order parameter is said to exhibit finite-size scaling if it can be expressed as φ
−
β ν
β
ν
−
~
P t N
N
t
t N
( ,
)
((
)
),
(10)
c
/
1/ (10) where φβ(x) is the universal scaling function of P. Note that unlike the finite size scaling ansatz for C(t, N), we
have a negative sign in the exponent for order parameter. It implies that if we plot P(t, N) vs z = (t − tc)N1/ν then
the value of P decreases with network size. Following the procedures in refs.37–39 we first find β/ν = 0.0468 and
once again we obersve that its value is independent of the rules and the values of m except for m = 1. We now plot
PNβ/ν vs (t − tc)N1/ν with β/ν = 0.0468 and 1/ν = 0.5195 and find that indeed all the distinct curves of Fig. (7a,b)
collapse into their respective universal curves as shown in Fig. (7c). However, even these two universal curves
collapse into a master curve if we plot A2PNβ/ν vs B2(t − tc)N1/ν − X provided we know the suitable metric factors
A2, B2 and trivial shifting factor X. Specific Heat and Susceptibilityi cific Heat and Susceptibility
we know the entropy, then finding the specific heat is just a routine matter since we know its general i
ition pi
p
y
Once we know the entropy, then finding the specific heat is just a routine matter since we know its general
definition Scientific Reports | (2019) 9:8585 | https://doi.org/10.1038/s41598-019-44446-2 6 www.nature.com/scientificreports/ =
×
. C
d
d
(control parameter)
(Entropy)
(control parameter)
(6) =
×
. C
d
d
(control parameter)
(Entropy)
(control parameter)
(6) (6) In the case of thermal phase transition, we use temperature T in the place of control parameter and Gibb’s
or Boltzmann entropy in the place of entropy. In the case of percolation, we use q = (1 − t) as the equivalent
counterpart of temperature since both entropy and order parameter behave with q in the similar fashion as their
corresponding counterpart in the thermal phase transition. On the other hand, we use Shannon entropy as the
equivalent counterpart of Gibb’s or Boltzmann entropy. The definition of specific heat for percolation therefore is =
. C t
q t
dH
dq t
( )
( )
( )
(7) =
. C t
q t
dH
dq t
( )
( )
( ) (7) Thus, differentiating H from first principles and multiplying the resulting value by the corresponding value
of q = (1 − t), we can immediately obtain C(t). We then plot C(t) in Fig. (6a,b) as a function of t for PR and SR
respectively and immediately see the sign of divergence at the critical point. Of course, the true divergence can
only be seen in the case of the system size N → ∞ which is an unsurmountable limitation of the numerical simu-
lation. One way of overcoming this is by using the finite-size scaling (FSS) hypothesis φ
−
α ν
ν
~
C t N
N
t
t N
( ,
)
((
)
),
C
c
/
1/ φ
−
α ν
ν
~
C t N
N
t
t N
( ,
)
((
)
),
(8)
C
c
/
1/ (8) where φC(z) is the universal scaling function for specific heat45. It helps us to extrapolate the values of the critical
exponents for infinite system from a set of data obtained from finite size system. One way of proving Eq. Specific Heat and Susceptibilityi (8) is
by data-collapse of distinct C(t, N) versus t for different N into one universal curve φC(z) which is our next task. / f
According to Eq. (8) the peak Ch of the C(t, N) vs t curves at t = tc increases following a power-law
α ν
~
C
N
h
/ . Plotting log(Ch) versus log(N) in Fig. (6c) we find a set of parallel lines for different m > 1 and for different rules
whose slope gives an estimate of α/ν. The same value of α/ν for all m regardless of product or sum rule suggest
that they all share the same critical exponents. Plotting C(t, N)N−α/ν versus t, see Fig. (6d), and finding that all the
peaks collapse to one point provides a clear testament of how good is this value. We checked it for m = 3, 5, 10 and
their product and sum rules. We find that all the peaks collapse at the same point with the same value of
α/ν = 0.5119. Note that the correspondng α/ν value for EP on ER networks is 0.535. We then draw a horizontal
line in the plot of C(t, N)N−α/ν versus t and measure the distance of the intercepts of each curve from the critical
point to obtain a data of t − tc versus N37,38. Plotting this data in the log − log scale gives a straight line whose slope
gives a good estimate for 1/ν value. Finally, we plot CN−α/ν vs (t − tc)N1/ν and after fine tuning the 1/ν value we
obtain a perfect data-collapse with α = 0.98739 and 1/ν = 0.519577 for both the rules as shown in Fig. (6e). Furthermore we plot A1CN−α/ν vs B1(t − tc)N1/ν in Fig. (6f) with m = 3, 5, 10 for both the rules and find that all the
scaling curves collapse into one master curve once we use appropriate metric factors A1 and B1
46. We then use the
relation
−
ν
−
~
N
t
t
(
)
c
in
α ν
~
C t
N
( )
/ and find that indeed the specific heat diverges like −
. α
−
~
C t
t
t
( )
(
)
(9)
c −
. Specific Heat and Susceptibilityi Indeed, the scaling curves for both rules and for all m except m = 1 collapse
into a master curve sharing the same critical exponents as shown in Fig. (7d). It implies that their scaling func-
tions are the same except for a multiplying metric factors A2, B2 and trivial shifting factor X. Substituting the
relation
−
ν
−
~
N
t
t
(
)
c
in
β ν
−
~
P
N
/ we get −
. β
~
P t
t
t
( )
(
)
(11)
c (11) This is exactly how the order parameter behaves near critical point in the thermal CPT as well. We once again
find that both PR and SR rules for all m > 1 share the same exponent β = 0.09007 which is slightly higher than
that in the ER network.i This is exactly how the order parameter behaves near critical point in the thermal CPT as well. We once again
find that both PR and SR rules for all m > 1 share the same exponent β = 0.09007 which is slightly higher than
that in the ER network.i Recently, we defined the susceptibility χ(t, N) for percolation as the ratio of the change in the order parameter
ΔP and the magnitude of the time interval Δt during which the change ΔP occurs. Essentially it becomes the Scientific Reports | (2019) 9:8585 | https://doi.org/10.1038/s41598-019-44446-2 7 www.nature.com/scientificreports/ Figure 6. Specific heat C versus t for (a) PR and (b) SR in the BA network with m = 5. (c) The slo
log(Ch) vs log(N), where Ch is the peak value at tc, for different m and different rules gives values o
we plot CN−α/ν vs t − tc for sum rule with m = 5 shows collapse of all the peaks with α/ν = 0.5119(
the same results for SR. (e) We plot CN−α/ν vs (t − tc)N1/ν using 1/ν = 0.519577 we find that all the
in (a,b) collapse into their own scaling curve. In (f) we plot A1CN−α/ν vs B1(t − tc)N1/ν and find tha
curves for different m and rules collapse into a master curve for suitable metric factors A1 and B1. Figure 6. Specific heat C versus t for (a) PR and (b) SR in the BA network with m = 5. Specific Heat and Susceptibilityi (c) The slopes of the plot
log(Ch) vs log(N), where Ch is the peak value at tc, for different m and different rules gives values of α/ν. In (d)
we plot CN−α/ν vs t − tc for sum rule with m = 5 shows collapse of all the peaks with α/ν = 0.5119(2). We obtain
the same results for SR. (e) We plot CN−α/ν vs (t − tc)N1/ν using 1/ν = 0.519577 we find that all the distinct plots
in (a,b) collapse into their own scaling curve. In (f) we plot A1CN−α/ν vs B1(t − tc)N1/ν and find that the scaling
curves for different m and rules collapse into a master curve for suitable metric factors A1 and B1. derivative of the order parameter P since Δt → 0 in the limit N → ∞ as Δt = 1/N. Recall that susceptibility in the
paramagnetic to ferromagnetic transition too is the derivative of the order parameter. Using ΔP = Δsmax/N in the
definition we find 8 Scientific Reports | (2019) 9:8585 | https://doi.org/10.1038/s41598-019-44446-2 www.nature.com/scientificreports/ Figure 7. We show plots of P versus t (a) for both PR and (b) for SR. In (c) we plot PNβ/ν versus (t − tc)L1/ν for
both PR and SR. We find that all the distinct plots in (a,b) collapse into their own scaling curves sharing the
same critical exponents. (d) Plots of A2PNβ/ν versus B2(t − tc)L1/ν for PR-SR collapse into a master curve if we can
choose suitable values for metric factors A2 and B2 independent of m > 1. Figure 7. We show plots of P versus t (a) for both PR and (b) for SR. In (c) we plot PNβ/ν versus (t − tc)L1/ν for
both PR and SR. We find that all the distinct plots in (a,b) collapse into their own scaling curves sharing the
same critical exponents. (d) Plots of A2PNβ/ν versus B2(t − tc)L1/ν for PR-SR collapse into a master curve if we can
choose suitable values for metric factors A2 and B2 independent of m > 1. χ
= Δ
t N
s
( ,
)
,
(12)
max (12) which is essentially the jump in the largest cluster as we add link one by one. There are two distinct ways in which
jump can takes place. Specific Heat and Susceptibilityi It may happen that an already existing largest cluster si
max may merge with a smaller cluster
and their combined size s f
max will obviously be the largest cluster. Besides, it may also happen that two smaller
clusters may merge such that their combined size s f
max may be larger than si
max. In either case the jump in the larg-
est is the difference between s f
max and si
max. f
Percolation theory is a statistical model and hence the numerical values of an observable from a single realiza-
tion are not reproducible. We thus take the average of χ value over many independent realizations under the same
condition and the resulting plots are shown in Fig. (8a) for PR with m = 5 as for Fig. (8b). This leads to assume
that it too should obey the finite-size scaling form χ
φ
−
−
. γ ν
χ
ν
~
t
t
N
N
t
t N
(
,
)
((
)
)
(13)
c
c
/
1/ (13) where φχ(ξ) is known as the universal scaling function. According to Eq. (13), the susceptibility χmax at t = tc
increases obeying a power-law χ
γ ν
~ N
h
/ and this is true for all m > 1 and for both the rules. Following the same
procedure as we have done for specific heat we find γ/ν = 0.4594 for both PR and for SR. We now plot χNγ/ν vs
(t − tc)N1/ν in Fig. (8c) and find that all the distinct curves for PR and SR with m = 5 collapse superbly into their
respective scaling curves with the same set of critical exponents γ/ν = 0.4594 and 1/ν = 0.5195. In Fig. (8d) we
plot A3χNγ/ν vs B3(t − tc)N1/ν and find that all the universal scaling curves for m = 3, 5, 10 and for the two rules Scientific Reports | (2019) 9:8585 | https://doi.org/10.1038/s41598-019-44446-2 9 www.nature.com/scientificreports/ Figure 8. Plots of susceptibility χ versus t with m = 5 (a) for PR and (b) for SR. (c) We plot χN−γ/ν vs (t − tc)N1/ν
and find distinct curves in (a) and (b) collapse into their own scaling curves. (d) We plot the same for PR-SR
with different m and that distinct scaling curves collapse into a master curve if we can choose suitable metric
factors A3 and B3. Figure 8. Specific Heat and Susceptibilityi Plots of susceptibility χ versus t with m = 5 (a) for PR and (b) for SR. (c) We plot χN−γ/ν vs (t − tc)N1/ν
and find distinct curves in (a) and (b) collapse into their own scaling curves. (d) We plot the same for PR-SR
with different m and that distinct scaling curves collapse into a master curve if we can choose suitable metric
factors A3 and B3. with different m > 1 values PR-SR belong to the same universality class regardless of the value of m > 1. Using now
the relation
−
ν
−
~
N
t
t
(
)
c
in χ
γ ν
~ N
/ we find that χ
−
γ
−
~ t
t
(
)
,
(14)
c (14) where γ = 0.884 for both PR and SR within the acceptable limit of error. It clearly shows that the susceptibility
ow diverges even without the exclusion of the largest cluster. where γ = 0.884 for both PR and SR within the acceptable limit of error. It clearly shows that the susceptibility
now diverges even without the exclusion of the largest cluster. Conclusions
h
d We have used a class of BA networks where each node is born with m links and shown that its properties are sig-
nificantly different for different m values albeit the exponent of the degree distribution remains the same. We have
used such networks as skeleton to study explosive percolation for both product and sum rule of the Achlioptas
process. Our first goal was to see the role of m and also to see the role of the product and sum rule in fixing the
critical points and the critical exponents. Our first observation is that the critical point tc decreases systematically
with increasing m starting at tc = 1 for m = 1. We have then shown that the critical exponents remain the same
for all m > 1 independent of product and sum rules. It implies that EP on the BA network belongs to one grand
universality class. Interestingly, the PR-SR universality class of EP on the BA networks is different from that of
EP on ER network although the difference in the values of their critical exponents in some cases is quite slim. It
suggests that PR-SR universality in explosive percolation is quite robust and it appears as if they are the equivalent
counterpart of the much known bond-site universality of RP on spatially embedded lattices.i Our second goal was to understand what order and disorder means in percolation and find out if percolation
in general and explosive percolation in particular is an order-disorder transition. To this end, we have found that
the behaviour of entropy and order parameter is reminiscent of what we see in the paramagnetic to ferromagnetic
transition. It suggests that explosive percolation is indeed an order-disorder transition. Another potentially inter-
esting finding is that the critical exponents α, β, γ obey the Rushbrooke inequality. Earlier, we have shown that
it also holds for EP on ER networks. These results suggest that no matter how the values of the critical exponents
change they still always obey the Rusbrooke inequality but always close to equality. So far, we have studied EP on
scale-free and random networks. It would be interesting to see if the small-world nature of the network topol-
ogy has any effect in determining the universality class and in obeying the Rushbrooke inequality. Scaling and Universality g
y
Scaling theory predicts that the various critical exponents cannot just assume values independently rather they
are bound by some scaling and hyperscaling relations. One of these scaling relations is the Rushbrooke inequality
where the exponents α, β and γ are related by (15) α
β
γ
+
+
≥
. 2
2 α
β
γ
+
+
≥
. 2
2
(15) Remarkably, many experiments and exactly solved models of thermal CPT suggest that Eq. (15) holds more
as equality than as inequality which is also supported by static Widom scaling47. Substituting our values from the
Table 1 in Eq. (15) we find α + 2β + γ = 2.05 which clearly suggests that the Rushbrooke relation holds exactly as
an inequality and approximately as an equality. Earlier we found the same results for EP on ER network and RP
on square and weighted planar stochastic lattice (WPSL)32,33,38. These studies clearly suggest the robustness of the Scientific Reports | (2019) 9:8585 | https://doi.org/10.1038/s41598-019-44446-2 10 www.nature.com/scientificreports/ Table 1. The critical exponents and Rushbrooke inequality for explosive percolation in BA and ER networks. Rushbrooke relation in percolation inependent of whether explosive or random percolation is done on networks
or on lattice.hif
Table 1. The critical exponents and Rushbrooke inequality for explosive percolation in BA and ER networks. Rushbrooke relation in percolation inependent of whether explosive or random percolation is done on networks
r on lattice.hif The classification of second order phase transition under different physical conditions into universality classes
is yet another interesting proposition. It has been put forth by Kadanoff in 1970 who suggested that two or more
systems sharing the same values of critical exponents and scaling functions are said to belong to the same uni-
versality class. It is well-known that the critical exponents of RP on spatially embedded lattice are universal in the
sense that their values depend only on the dimension of the lattice and independent of whether it is site or bond
type percolation. However, there exists just one exception as we have recently found namely that RP on WPSL
does not belong to the universality class of two dimensional lattices32,33,37. The only way WPSL is different from
all the known two dimensional lattices is that its coordination number distribution follows a power-law. Now
the question is: What happens when the skeleton is infinite dimensional network? References
1. Flory, P. J. Molecular size distribution in three dimensional polymers. I. Gelation. J. Am. Chem. Soc. 63, 3083–2090 (1941).
2. Broadbent, S. R. & Hammersley, J. M. Percolation processes I. Crystals and mazes. Proc. Cambridge Philos. Soc. 53, 629–641 (1957).
3. Stauffer, D. & Aharony, A. Introduction to Percolation Theory (Taylor and Francis, London, 1994).
4. Saberi, A. A. Recent advances in percolation theory and its applications. Phys. Rep. 578, 1–32 (2015).
5. Bastas, N., Giazitzidis, P., Maragakis, M. & Kosmidis, K. Explosive percolation: Unusual transitions of a simple model. Physica A 407,
54–65 (2014).
6. Bianconi, G. & Radicchi, F. Percolation in real multiplex networks. Phys. Rev. E 94, 060301 (2016). Scaling and Universality It is already known that RP on
ER and on the Bethe lattice belongs to the same universality class owing to the fact that both of them are infinite
dimensional skeletons48. Radicchi and Castellano on the other hand reported that the site-bond universality is
violated even in the case of RP on network49. Earlier Cohen et al. showed that RP on scale-free network, albeit
infinite dimensional, does not belong to the universality class of RP on ER and Bethe lattice34. Thus the scale-free
nature of the skeletons are different from the rest as far as random percolation is concerned. f
p
Explosive percolation, on the other hand, has been proposed only in 2009 and we witnessed a furry of activi-
ties for at least up to the first three years in order to resolve the issue of whether EP describes continuous or dis-
continuous phase transition. There has hardly been any attempt since then to classify EP into universality classes. Only recently, we have shown that explosive percolation of PR and SR type in the ER network belongs to the
same universality class. Earlier two groups, Bastas et al. and Yong et al., reported that site and bond type explosive
percolation on the same lattice do not belong to the same universality class29,50. The present work is about EP on
scale-free networks and we once again find that PR and SR belong to the same universality class but it is different
from the PR-SR universality class of EP on ER. It is, however, noteworthy to mention that the corresponding
exponents are close but they can in no way be equal. Finding the PR-SR universality in two systems as different
as the BA and ER networks suggests that PR-SR universality is quite robust. It appears that the absence of usual
site-bond universality in RP is being replaced by PR-SR universality in EP38. Conclusions
h
d In our future
endeavour, we intend to study EP in scale-free networks via mediation-driven attachment process since this has
a wide range of exponent 2 < λ ≤ 336. It would be interesting to see how much impact the exponent of the degree
distribution has in fixing the critical exponents vis-a-vis the universality class. 6. Bianconi, G. & Radicchi, F. Percolation in real multiplex networks. Phys. Rev. E 94, 060301 (2016). p
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in the result analysis and explanation, and have written the full text. The contribution of the co-author MD. Habib E Islam: Have done extensive numerical simulation, produced all the figures and contributed in the result
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An Integrated RNA and DNA Molecular Signature for Colorectal Cancer Classification
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RESEARCH RESEARCH Mohanad Mohammed1*, Henry Mwambi1 and Bernard Omolo2,3 *Correspondence:
mohanadadam32@gmail.com
1School of Mathematics, Statistics
and Computer Science, University
of KwaZulu-Natal, King Edward
Avenue, Pietermaritzburg, South
Africa Abstract Background: Colorectal cancer (CRC) is the third most common cancer among
women and men in the USA and recent studies have shown an increasing
incidence in less developed regions such as Sub-Saharan Africa (SSA). The KRAS
gene is mutated in 40% of the CRC cases and hence the RAS pathway activation
has become a major focus of drug targeting efforts. However, nearly 60% of
patients with wild-type KRAS fail to respond to RAS-targeted therapies, for
example the anti-epithelial growth factor receptor inhibitor (EGFRi) combination
therapies. Thus, there is a need to develop more reliable molecular signatures to
better predict mutation status. In this study, we develop a hybrid (DNA mutation
and RNA expression) signature and assess its predictive properties for the
mutation status and survival of CRC patients. Full list of author information is
available at the end of the article
†Equal contributor Methods: Publicly-available microarray and RNASeq data from 54 matched
formalin-fixed paraffin embedded (FFPE) samples from the Affymetrix GeneChip
and RNASeq platforms, were used to obtain differentially expressed genes
between mutant and wild-type samples. For classification, the support-vector
machines, artificial neural networks, random forests, k-nearest neighbor, na¨ıve
Bayes, negative binomial linear discriminant analysis, and the Poisson linear
discriminant analysis algorithms were employed. Results: Compared to the genelist from each of the individual platforms, the
hybrid genelist had the highest accuracy, sensitivity, specificity and AUC for
mutation status, across all the classifiers, and is prognostic for survival in patients
with CRC. Conclusions: This signature could be useful in clinical practice, especially for
colorectal cancer diagnosis and therapy. Keywords: Colorectal cancer; FFPE; Microarray; RAS pathway signatures;
RNASeq Research article Keywords: Colorectal cancer, FFPE, Microarray, RAS pathway signatures, RNASeq
Posted Date: January 8th, 2020
DOI: https://doi.org/10.21203/rs.2.20271/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Mohammed et al. *Correspondence:
mohanadadam32@gmail.com
1School of Mathematics, Statistics
and Computer Science, University
of KwaZulu-Natal, King Edward
Avenue, Pietermaritzburg, South
Africa
Full list of author information is
available at the end of the article
†Equal contributor Background Colorectal cancer (CRC) is one of the major emerging causes of mortality and mor-
bidity around the world [1]. CRC is also the third leading cause of death among
men and women [2, 3, 4, 5, 6]. There were about 1.80 million new cases and 862, 000
deaths in 2018, according to the World Health Organization (WHO) [7]. Further-
more, in 2019, CRC was reported to be the third most prevalent cancer among men
and women and an estimated 101, 420 and 44, 180 new cases of colon and rectal
cancer, respectively, and 51, 020 deaths in the USA alone [8, 2, 9]. Although the incidence rates of CRC are lower in developing countries than in
developed countries, recent studies have shown an increase in the incidence rates in Page 2 of 23 Mohammed et al. Sub-Saharan Africa [10]. Many cancer types that are relatively curable in developed
countries are detected only at advanced stages in developing countries, due to late
or inaccurate diagnoses [11]. Cancer tumor classification based on morphological
characteristics alone has been shown to have serious limitations in some studies [12]. Physicians aim to diagnose CRC as early as possible to design optimal treatment
strategies that are patient-specific. Therefore, using genetic mutation and features
of the tumor would most probably lead to better understanding and early detection
of the disease and lead to finding suitable and targeted strategies [13]. Previously, most of the cancer classification research was based on clinical features
of the tumors, which lacked accurate diagnostic ability, hence the need to develop
new methods to better address this important problem [12, 14]. Recently, DNA
microarray technology has greatly improved the classification of diseases into sub-
types, particularly cancer. This technology allows the processing of thousands of
genes simultaneously, hence providing critical information about a disease[15, 16]. Microarray gene expression data have been used widely for cancer detection, pre-
diction and diagnosis [17]. In the last decade, next-generation sequencing (NGS)
technology has emerged as an advancement in cancer and other disease research,
based on RNA sequencing methodology. NGS technology permits the measurement
of expression levels of tens of thousands of genes simultaneously. NGS platforms that
are most common include Illumina, SOLiD, Ion Torrent semiconductor sequencing,
and single-molecule real-time sequencing [18]. NGS technology has been the most attractive and dramatically been improved
over the last few years. Background This technology is high-throughput and has become pop-
ular in the detection and analysis of differentially expressed genes[18, 19]. More
recently, RNASeq data has been shown to be better than microarray data in terms
of quality and accuracy in estimating transcript abundance. However, the two
methodologies are different in design and implementation [20, 19, 21]. Although
RNASeq experiments are expensive, in contrast, they have many advantages over
microarrays. RNASeq allows detecting the variation of a single nucleotide, does not
require genomic sequence knowledge, provides quantitative expression levels, pro-
vides isoform-level expression measurements, and offers a broader dynamic range
than microarrays [20]. Moreover, RNASeq allows the detection of novel transcripts,
low background signal, and increased specificity and sensitivity [22]. However, our
view is that integrated use of data from both technologies may be the best approach,
given the available information from both technologies. Microarray and RNASeq technologies produce gene expression data in different
forms. The structure of gene expression produced using microarrays is continuous
data, while RNASeq provides a discrete type of data [23]. What is common between
the two technologies is that both generate big datasets consisting of a few sample
sizes, where each sample has a large number of genes. Many statistical and machine learning methods have been used to analyze and
extract information from massive amounts of gene expression data. These methods
include the Poison linear discriminant analysis (PLDA), negative binomial linear
discriminant analysis (NBLDA), support vector machines (SVM), artificial neural
networks (ANN), linear discriminant analysis (LDA), and random forests (RF). These methods have been used and examined in many studies based on RNASeq
and microarray data. For example, Aziz ]et al. [24] assessed the ANN performance Page 3 of 23 Mohammed et al. based on microarray data using six hybrid feature selection methods. Five gene ex-
pression datasets were used for evaluating these methods and for understanding how
these methods can improve the performance of ANN. Statistical hypothesis tests
were used to check the differences between these methods. They showed that the
combination of independent component analysis (ICA) and genetic bee colony algo-
rithm had superior performance. Salem et al. [25] proposed a new methodology for
gene expression data analysis. They combined information gain (IG) and standard
genetic algorithm (SGA) for feature selection and reduction, respectively. Their ap-
proach was tested on seven cancer datasets and then compared with the most recent
approaches. Background Their results show that the proposed approach outperformed the most
recent approaches. Jain et al. [26] presented a two-phase hybrid method for can-
cer classification using eleven microarray datasets for different cancer types. They
combined correlation-based feature selection (CFS) and improved-binary particle
swarm optimization (IBPSO). Naive Bayes with 10-fold cross-validation was used
for assessment. Results indicated that their approach had better performance in
terms of accuracy and the number of selected genes. Anders and Huber [27] conducted differential expression analysis based on the
negative binomial distribution, with variance and mean linked by local regression,
for count data. They implemented their proposed method by the DESeq R package. Zararsiz et al. [23] presented a comprehensive simulation study on RNASeq classi-
fication using PLDA, NBLDA, single SVM, bagging SVM (bagSVM), classification
and regression trees (CART), and RF. Their simulation results were applied and
compared to two miRNA and two mRNA real experimental datasets. They found
that the power-transformed PLDA, RF, and SVM were the best in classification
performance. Due to the small number of samples for gene expression data, combining inde-
pendent datasets is novel in order to increase sample size and statistical power. Taminau et al. [28] worked on the integration of gene expression analysis using
two approaches based on merging and meta-analysis. They used six gene expres-
sion datasets. Results showed that both meta-analysis and merging did well, but
merging was able to detect more differentially expressed genes than meta-analysis. Recently, combining two different gene expression data sources has been shown
to improve classification accuracy as opposed to using only one source. Castillo
and co-workers [20] introduced integration of multiple microarrays and RNASeq
platforms. They first carried out a differential expression analysis, then applied the
minimum-redundancy maximum-relevance (mRMR) feature selection approach for
further reduction of the gene-list. The top 10 genes were selected and evaluated using
four classification methods: k-Nearest neighbor (KNN), Naive Bayes (NB), RF, and
SVM. Their results showed the highest accuracy and f1-score for the KNN. Here, we
combined RNASeq and DNA expression data from colorectal cancer patients. We
obtained a hybrid gene-list from the RNASeq and microarray datasets and assessed
its classification performance based on the PLDA, NBLDA, SVM, RF, ANN, KNN,
and NB algorithms. The paper is structured as follows. Section 2 discusses the methods and the per-
formance metrics used in the study. Materials and Methods
Datasets We used publicly available microarray and RNASeq data that is also reported in
Omolo et al. [4]. The data consists of 54 matched formalin-fixed paraffin-embedded
(FFPE) samples from colorectal cancer patients. The data is available in the gene
expression omnibus (GEO) repository under the accession numbers GSE86562 and
GSE86559 for RNASeq and microarray data, respectively. The microarray gene
expression data consists of 60, 607 genes on 54 colorectal patients. KRAS muta-
tion status was used as a class variable. As a first step, the Affymetrix microarray
data was log2-transformed and quantile-normalized, and genes with more than 50%
missing values were filtered out. Thereafter, class comparison was done using the
two-sample t-test at the 0.005 significant level threshold, which yielded 165 differ-
entially expressed genes. The RNASeq dataset contained 57, 905 genes from the same colorectal cancer
patients used to generate the microarray data. This data is in the form of counts
i.e. discrete in nature. For this data, first, filtration was done to remove the genes
with more than 50% of zeros across the samples, using the counts per million (CPM)
method [29]. Genes whose CPM values are greater than 0.5 were retained. Thus,
the dimension was reduced to 17, 473 genes. Differential expression analysis was
performed using DESeq2 package in R. This step reduced the genes to 282 genes
using the 0.005 significance level threshold. The differential expression analysis tool
in DESeq2 uses a generalized linear model (GLM) of the following form: gij ∼NB(µij, αi)
µij = sjqij
log2(qij) = xj.βi,
(1) gij ∼NB(µij, αi)
µij = sjqij
log2(qij) = xj.βi, (1) (1) where gij is the counts for gene i in sample j. These counts are modeled using a
negative binomial distribution with fitted mean µij and a gene-specific dispersion
parameter αi. The fitted mean is decomposed into a sample-specific size factor sj
and a parameter qij proportional to the expected true concentration of fragments
for sample j. The coefficients βi represent the log2-fold changes for gene i for each
column of the model or design matrix X. Note that the model can be generalized
to use sample- and gene-dependent normalization factors sij. The dispersion parameter αi defines the relationship between the variance of the
observed count and its mean value. Background Section 3 shows the classification results of the
microarray, RNASeq, hybrid gene lists, and the survival analysis. Discussion and
conclusions are presented in sections 4 and 5 respectively. Page 4 of 23 Mohammed et al. Materials and Methods
Datasets That is, how far we expect the observed count
to be from the mean value, which depends both on the size factor sj and the
covariate-dependent part qij as defined above. Thus the variance function is given
by V ar(gij) = E
(gij −µij)2
= µij + αiµ2
ij. (2) (2) The steps performed by the DESeq function in DESeq2 package are estimation
of sj, and αi, and fitting negative binomial GLM for βi and Wald statistics by
nbinomWaldTest. Page 5 of 23 Page 5 of 23 Mohammed et al. Counts per million can be computed as Counts per million can be computed as Counts per million can be computed as CPMi = gi
N ∗106,
(3) CPMi = gi
N ∗106, (3) where gi denotes the counts observed from a gene of interest i and N is the number
of sequenced fragments. RNASeq and microarray data integration may help improve cancer classification
accuracy. Several studies have addressed the classification problem using RNASeq,
microarray, or a combination of both, based on heterogeneous samples ([20, 30, 31]). Our study aims to integrate homogeneous samples from the RNASeq and microarray
platforms. In this regard, we obtained the differentially expressed genes from the two
platforms based on the same set of samples. Thereafter, we used the database for
annotation, visualization, and integrated discovery (DAVID) [32], and catalogue of
somatic mutations in cancer (COSMIC) tools, to annotate the RNASeq transcripts
list. The microarray genes symbol names were obtained from the dataset in [4]. Next, the intersection, complement of the intersection, and union between the two
annotated lists were obtained. Integration was done using the intersection, complement of the intersection, and
the union of the two lists of genes. Due to the different nature of the two datasets,
RNASeq was log2 transformed and quantile- normalized in order to make both types
of data consistent with each other. Subsequently, the integration was done based on
binding the two gene-lists from the RNASeq and microarray datasets. To transform
the RNASeq data, we let Transformed Data = log2(G + 1),
(4) (4) Transformed Data = log2(G + 1), Transformed Data = log2(G + 1), where G is the RNASeq counts data matrix, and G + 1 is the RNASeq counts data
matrix with all zero counts changed to one. Quantile normalization ensures that probe intensities of each array in a set of
arrays have the same distribution. A quantile-quantile plot would help to con-
firm if two probe vectors have the same distribution (quantiles lie on the di-
agonal line) or not. This approach can be extended to n-dimensional data. Let
qk = (qk1, . . . , qkn)′, k = 1, . . . , P, be the vector of the kth quantiles for all n arrays,
and d = ( 1
√n, . . . ,
1
√n)′ be the unit diagonal. To transform from the quantiles so
that they all lie along the diagonal, we projected q on to d as below [33]: projdqk =
1
n
n
X
j=1
q1j, . . . , 1
n
n
X
j=1
qP j
. (5) (5) Classification Methods Classification Methods Several methods have been developed for classification and their performance eval-
uated in both microarray and RNASeq platforms. Below, we briefly describe seven
classification methods and how to evaluate their performances based on the inte-
gration of the two platforms. Data Integration Here, we used homogeneous data from matched-pair samples from microarray and
RNASeq technologies. Using a set-theoretic approach of taking the intersection,
complement of intersection, or union, we obtained four lists of genes from the two
platforms at the 0.005 significance level. The intersection between the two lists was
23 genes, with 401 genes being the complement of the intersection. Page 6 of 23 Mohammed et al. Poisson Linear Discriminant Analysis The PLDA classifier was proposed by Witten [34]. Witten used the Poisson log
linear model, and developed an analog of diagonal linear discriminant analysis for
sequence data. Let G denote a n × p matrix of read counts data, where n denotes the number of
observations (samples), and p the number of genes. Let Gij be the counts or reads
for gene j in sample i; it is reasonable to assume that Gij ∼Poisson(µij),
(6) (6) Gij ∼Poisson(µij), Gij ∼Poisson(µij), where µij = sigj. To avoid identifiability issues, one can require Pn
i=1 si = 1,
where si is the number of counts per sample i, and gj is the number of counts per
gene j. Suppose that we have K different classes of samples. Then we can write Gij|yi = k ∼Poisson(µijdkj),
(7) Gij|yi = k ∼Poisson(µijdkj), (7) where yi denotes the class of the ith sample (yi = 1, 2, 3, · · · , K) and dkj denotes a
measure of the level of the jth gene to be differentially expressed in class k. Let gi = (gi1, gi2, · · · , gip)′ indicate the entries of row i in the G matrix, which
are the gene expression levels of sample i. Let, G.j = Pn
i=1 Gij, Gi. = Pp
j=1 Gij,
and G.. = P
i,j Gij denote the column, row and the overall totals respectively. The
maximum likelihood estimate (MLE) for µij assuming independence is ˆµij = Gi.G.j
G.. ,
and Pn
i=1 ˆsi = 1 yields the estimates ˆsi = Gi. G.. and ˆgj = G.j.. ˆsi is the estimate of
the size factor for sample i. Maximum likelihood estimation provides the estimate
of dkj as ˆdkj =
Gckj
P
i∈ck ˆµij , where ck denotes the class of an observation. If ˆdkj > 1, then the jth gene is over-expressed relative to the baseline in the kth
class, and if ˆdkj < 1 then the jth gene is under-expressed relative to the baseline
in the kth class. If Gckj = 0 (an event that is not unlikely if the true mean for jth
gene is small), then the maximum likelihood estimate for dkj equals zero. Assume that we want to classify a new observation g∗= (G∗
1, · · · , G∗
p), and let y∗
indicate the unknown class label. By Bayes rule, P(y∗= k|g∗) ∝fk(g∗)πk,
(8) (8) P(y∗= k|g∗) ∝fk(g∗)πk, where fk is the density of a sample in class k and πk is the prior probability that an
observation belongs to class k. Then, if fk is a normal density with a class specific where fk is the density of a sample in class k and πk is the prior probability that an
observation belongs to class k. Gij ∼Poisson(µij), By Bayes’ rule, we have Pr(y∗= m|g∗
g∗
g∗) ∝fm(g∗
g∗
g∗)πm,
(13) Pr(y∗= m|g∗
g∗
g∗) ∝fm(g∗
g∗
g∗)πm, (13) Mohammed et al. Page 8 of 23 where fm is the pdf of the sample in class m, and πm is the prior probability that
a sample comes from class m. The pdf of Gij = gij in equation (12) is where fm is the pdf of the sample in class m, and πm is the prior probability that
a sample comes from class m. The pdf of Gij = gij in equation (12) is Pr(Gi,j = gij|yi = m) =
Γ(gij + φ−1
j )
gij!Γ(φ−1
j )
siλjdmjφj
1 + siλjdmjφj
!gij
1
1 + siλjdmjφj
!φ−1
j
. (14) ! (14) Thus, the discriminant score for NBLDA can be constructed from (13) and (14)
as Thus, the discriminant score for NBLDA can be constructed from (13) and (14)
as as log Pr(y∗= m|g∗
g∗
g∗) =
P
X
j=1
G∗
j[log dmj −log(1 + siλjdmjφj)]
−
P
X
j=1
φ−1
j
log(1 + siλjdmjφj) + log πm + C,
(15) (15) X
φ−1
j
log(1 + siλjdmjφj) + log πm + C, where C is a constant independent of m. The class m, which maximizes the score
in equation (15) will be assigned to the new sample g∗
g∗
g∗. NBLDA is implemented
using the R package MLSeq. Gij ∼Poisson(µij), Then, if fk is a normal density with a class specific Page 7 of 23 Page 7 of 23 Mohammed et al. mean and common covariance, PLDA classifies a new sample to class k, which
maximize equation (8). Consequently, the discriminant score of PLDA is log Pr(y∗= k|g∗) ≈
P
X
j=1
G∗
j log dkj −
P
X
j=1
s∗λjdkj + log πk + C. (9) (9) PLDA is implemented using the R package MLSeq. PLDA is implemented using the R package MLSeq. PLDA is implemented using the R package MLSeq. Negative Binomial Linear Discriminant Analysis Negative Binomial Linear Discriminant Analysis Recently, Dong et al. [22] proposed NBLDA for RNASeq data analysis. NBLDA
and Poisson linear discriminant analysis (PLDA) were considered the most suitable
classifiers for RNASeq data, due to the discrete nature of data [22, 34]. Let Gij denote the number of reads in sample i, and gene j, i = 1, 2, 3, · · · , n and
j = 1, 2, 3, · · · , p. Then Gij is assumed to follow the negative binomial distribution Gij ∼NB(µij, φj),
µij = siλj,
(10) Gij ∼NB(µij, φj),
µij = siλj, (10) where si is the size factor, used to scale gene counts for the ith sample due to
different sequencing depth, λj is the total number of reads per gene, and φj ≥
0 is the dispersion parameter. The mean and variance of the negative binomial
distribution are given by: E(Gij) = µij
V (Gij) = µij + µ2
ijφj. (11) E(Gij) = µij
V (Gij) = µij + µ2
ijφj. (11) Suppose that we have M classes. Let CM be an indicator variable such that
Cm ∈{1, 2, 3, · · · , M}. Then, the model for RNASeq data is (Gij|yi = m) ∼NB(µijdm,j, φj),
(12) (Gij|yi = m) ∼NB(µijdm,j, φj), (12) where dm,j denotes the differences among the M classes, and yi = m, m ∈
{1, 2, 3, · · · , M} denotes the class of samples i. The assumption is that all the genes
are independent. Let g∗= (G∗
1, · · · , G∗
p) be a new sample whose class is to be predicted, s∗is the
size factor, and y∗
i the class label value. Support Vector Machines The SVM method was first proposed by Boser, Guyon and Vapnik [35] at the Com-
putational Learning Theory (COLT92) ACM Conference in 1992. The method is
based on the idea of a hyperplane that lies furthermost from both classes. This
plane is known as the optimal (maximum) margin hyperplane. The hyperplane is
completely determined by a sub-set of the samples known as the support vectors
[36]. SVM has the ability to handle problems where the data are not linearly sep-
arable by transforming the data using mapping kernel functions such as the radial
basis function (RBF) kernel, polynomial function, and the linear function [37]. In
addition, SVM has can handle high dimensional data, which is clearly an important
advantage in dealing with genetic data from cancer studies. This strength makes
SVM widely appealing and applicable to real-life data analysis problems such as
handwritten character recognition, human face recognition, radar target identifi-
cation, speech identification, and, quite recently, to gene expression data analysis
[38, 39]. Suppose we have n samples and p genes. Further, assume samples belong to two
distinct outcome classes represented by +1 or −1 and a feature vector gi such that
(gi, yi) ∈G × Y
i = 1, 2, . . . n, where gi = (gi1, gi2, . . . , gip)′ is the sample profile
(vector) and yi ∈{+1, −1} is the outcome class dichotomy. The goal is to classify
the samples into one of the two classes by training the SVM which maps the input
data (using a suitable kernel function) onto a high-dimensional space (feature space)
{(Φ(gi), yi)}n
i=1. This is achieved by constructing an optimal separating hyperplane
that lies furthest from both classes. Mohammed et al. Mohammed et al. Page 9 of 23 The general form of a separating hyperplane in the space of the mapped data is
defined by wT Φ(g) + b = 0. (16) wT Φ(g) + b = 0. (16) Here, w = (w1, w2, . . . , wn)′, is the weight vector. We can rescale the w and b
such that the following equation determines the point in each class that is nearest
to the hyperplane defined by the equation |wT Φ(g) + b| = 1. (17) |wT Φ(g) + b| = 1. (17) Therefore, it should follow that for each sample i, i ∈{1, 2, . . . Support Vector Machines , n}, wT Φ(gi) + b =
≥1
if
yi = +1
≤−1
if
yi = −1. (18) (18) After the rescaling, the distance from the nearest point in each class to the hy-
perplane becomes
1
∥w∥. Thus, the distance between the two classes is
2
∥w∥, which is
called the margin. To maximize the margin, the solution of the following optimiza-
tion problem is obtained: min
w,b
∥w∥2
subject to
yi(wT Φ(gi) + b) ≥1,
i = 1, 2, . . . , n. (19) min
w,b
∥w∥2
subject to
yi(wT Φ(gi) + b) ≥1,
i = 1, 2, . . . , n. (19) (19) The square of the norm of w is considered in order to make the problem quadratic. Suppose w∗and b∗are the solutions to the optimization problem (19) above. Then
this solution determines the hyperplane in the feature space where (w∗)T Φ(g)+b∗=
0. The points Φ(gi) that satisfy the qualities yi((w∗)T Φ(gi) + b∗) = 1 are called
support vectors [36]. The SVM method is implemented using the R package kernlab
[40]. Random Forest Algorithm for Regression or Classification [41] Random Forest Algorithm for Regression or Classification [41] 1
For b = 1 to B (# random-forest trees): • Draw a bootstrap sample of size N from the training data. • Grow a random-forest tree, Tb to the bootstrapped data, by recursively
repeating the following steps for each terminal node of the tree, until the
minimum node size, nmin, is reached. • Select v genes at random from the p genes. • Pick the best gene to split on among the v based on an impurity measure. • Using the selected gene, split the node into two daughter nodes. 2
To make a prediction for a new sample, x: Let ˆCb(x) be the class prediction
of the b-th random-forest tree. Then ˆCB
rf(x) = majority vote
n
ˆCb(x)
oB
b=1 RF is implemented using the R package randomForest [47]. RF is implemented using the R package randomForest [47]. Mohammed et al. Random Forests Random forests were first introduced in 2001[41, 42]. They are an extension of
classification and regression trees, and also an improvement over bagged trees by
further modification using a random small tweak to de-correlate the trees. Growing
random forests leads to an improvement in prediction accuracy compared to single
or bagged trees [43]. We build a number of forests of decision trees on bootstrapped training samples
from the original data. A tree is obtained by recursively splitting the genes such
that at each node of the tree, a candidate gene for splitting is obtained from a
random sample of size v. A typical choice for v is such that v ≈√p, where p is the
number of candidate genes for splitting. Page 10 of 23 Mohammed et al. We then grow the trees to maximum depth. Therefore, the two-step randomization
process helps to de-correlate the trees [44]. To determine the prediction for an
unknown sample, an average over all the trees is taken for a regression problem and
a majority vote for a classification problem [41, 45, 46]. Random Forest Algorithm for Regression or Classification [41] Artificial Neural Networks Artificial neural networks (ANN) are multi-layered models that are constructed
from three layers, each layer consisting of nodes called neurons [48]. The input
layer contains nodes whose number is based on the input features. The output layer
contains nodes equal to the number of classes, and finally, the hidden layer con-
tains nodes determined by the level of tuning required. The inputs are weighted by
multiplying each input by weight as a measure of its contribution. The layers are
connected together via connection weights. These weights are determined through
stages of model fitting. The hidden nodes receive the sum weighted from the in-
put layer plus some bias. This summation is passed onto the transform function
(activation function) to generate the results. These results are called outputs and
interpreted as a class probability in our case. There are many types of architecture of ANN. Neural networks are used widely
in different fields such as prediction in time series models, economic modeling, and
medical applications [37]. In addition, ANN can be applied to the classification
problem using microarray gene expression data [48]. In this paper, we apply the
method to both microarray and RNA Sequencing gene expression data. Consider the simplest multi-layered network, with one hidden layer. Assume we
have gene expression data where n denotes the number of genes. Then the input
layer receives the n gene expression levels for a sample, each multiplied by the
corresponding weight, w(1)
ij gj, as shown in Eq 20, below: bi =
n
X
j=0
w(1)
ij gj
i = 1, 2, . . . , m, (20) Page 11 of 23 Mohammed et al. where g = (g0, g1, g2, . . . , gn)′ is a vector of input features and g0 = 1 is a constant
input feature that with weight wi0. The quantities, bi, are called activations, and
the parameters w(1)
ij
are the weights. Note that alternatively bi can be viewed as a
summary of the n genes from sample i. The superscript “(1)” indicates that this
is the first layer of the network. Each of the activations is then transformed by a
nonlinear activation function f, typically a sigmoid, as in Eq 21 below: zi = f(bi) =
1
1 + exp(−bi). Artificial Neural Networks (21) (21) The quantities zi are interpreted as the output of hidden units, so called because
they do not have values specified by the problem (as is the case for input units) or
target values used in the training (as is the case for output units). In the second layer, the outputs of the hidden units are linearly combined to give
the activations ak =
m
X
i=0
w(2)
ik zi
k = 1, 2, . . . , K. (22) (22) Again, z0 = 1 corresponds to the bias. The transformations in the second layer
of the neural network are parameterized by weights w(2)
ik
. The output units are
transformed using an activation function. Again, a sigmoid function may be used
as shown below: Again, z0 = 1 corresponds to the bias. The transformations in the second layer
of the neural network are parameterized by weights w(2)
ik
. The output units are
transformed using an activation function. Again, a sigmoid function may be used
as shown below: yk = f(ak) =
1
1 + exp(−ak). (23) (23) These equations may be combined to give the overall equation that describes the
forward propagation through the network, and describes how an output vector is
computed from an input vector, given the weight matrices as yk = f
m
X
i=0
w(2)
ik f
n
X
j=0
w(1)
ij gi
. (24) (24) ANN are implemented using the R package nnet [49]. ANN are implemented using the R package nnet [49]. ANN are implemented using the R package nnet [49]. Naive Bayes (27) (27) Class(G∗)MAP = argmax(P(Ci|G∗)). NB is implemented using the R package naivebayes. NB is implemented using the R package naivebayes. Naive Bayes The Naive Bayes classifier uses probability theory to find the most likely of the pos-
sible classes in a classification problem. The NB classifier relies on two assumptions,
namely, that each attribute is conditionally independent from the other attributes
given the class and that all the attributes have an influence on the class [50]. The
popularity of this classifier is mainly due to its simplicity, yet exhibiting a surpris-
ingly competitive predictive accuracy. The NB classifier has previously been applied
in many fields, including microarray gene expression data [37, 48]. Page 12 of 23 Mohammed et al. Mohammed et al. Consider an n x p gene expression data matrix, where n is the number of the
samples and p is the number of the genes (features). Let gkj, j = 1, 2, . . . , p, de-
note the j-th gene on the k-th sample. Let Ci be the i-th class, i = 1, 2, . . . , L. The Naive Bayes classifier uses the maximum a posteriori (MAP) classification
rule to classify these samples. The probability of the k-th sample gene infor-
mation vector, Gk = (gk1, gk2, . . . , gkp)′, is calculated and then the sample is
assigned the class with largest probability from L conditional probabilities. Let
P(C1|Gk), P(C2|Gk), . . . , P(CL|Gk) denote the set of L conditional probabilities. The NB classification depends on the Bayes rule, which states that a posterior
probability P(Ci|Gk) = P(Gk|Ci)P(Ci)
P(Gk)
∝P(Gk|Ci)P(Ci), k = 1, 2, . . . , n,
(25) (25) where P(Gk) is considered a common normalizing factor for all the L probabilities. where P(Gk) is considered a common normalizing factor for all the L probabilities. The NB classification assumes that all input features are conditionally indepen-
dent, that is, where P(Gk) is considered a common normalizing factor for all the L probabilities. The NB classification assumes that all input features are conditionally indepen-
dent that is (
)
g
p
The NB classification assumes that all input features are conditionally indepen-
dent, that is, P(gk1, gk2, . . . , gkp|Ci) = P(gk1|gk2, . . . , gkp, Ci)P(gk2, . . . , gkp|Ci)
= P(gk1|Ci)P(gk2, . . . , gkp|Ci)
= P(gk1|Ci)P(gk2|Ci) . . . P(gkp|Ci). (26) (26) Ultimately, NB classifies a new sample, G∗, according to the model with MAP
probability given the sample, as Class(G∗)MAP = argmax(P(Ci|G∗)). k-Nearest Neighbors The k-nearest neighbor classifiers (KNN) are known to be the most useful instance-
based learners. KNN is a non-parametric model [51]. If the classification is based on
Euclidean distance in a feature space, then k determines the number of neighbors
to be used. In the testing set, the new sample is assigned to the class that is most
likely among the k neighbors. Then the number of neighbors can be tuned to choose
the optimal fitted model parameters [37, 48]. The KNN uses the Euclidean distance measure to find the closest samples for
the new sample. Suppose we have two samples, each one with n genes. Denote
the two samples as S1 = (g11, g12, . . . , g1n)′ and S2 = (g21, g22, . . . , g2n)′. Then
the Euclidean distance is calculated as the square root of the sum of the squared
differences in their corresponding values. Using the Euclidean distance formula, the
distance between two points, dist(S1, S2), is given as dist(S1, S2) =
v
u
u
t
n
X
j=1
(g1j −g2j)2, dist(S1, S2) =
v
u
u
t
n
X
j=1
(g1j −g2j)2,
(28) (28) Page 13 of 23 Mohammed et al. where a large dist(S1, S2) means the two samples belong to different classes and
values near zero suggest that the samples are homogeneous. KNN is implemented
using the R package caret. Performance Metrics Several performance measures exist in the literature that can be used to assess
classification based on microarray and RNASeq gene expression data. The metrics
include accuracy, sensitivity, specificity, kappa coefficient, AUC, balanced error rate
(BER), Matthews correlation coefficient (MCC), and the F-measure (F1-score) [52,
53]. Let TP (number of positive samples correctly classified), TN (number negative
samples classified correctly), FP (number of negative samples incorrectly classified
as positive), and FN (number of positive samples incorrectly classified as negative)
denote true positives, true negatives, false positives, and false negatives, respectively. The performance measures can be expressed as follows: Results The analysis of RNASeq data using the integrated list of genes was performed
using R statistical software. Assessment of the methods was done using 10-fold
cross-validation. Here, the 54 CRC samples were divided into 10 folds randomly,
with each fold consisting of about 5 - 6 samples. Thereafter, 9 folds were used for
model-building and one fold for the testing and validation. Thus, this process was
self-iterated 10 times, and the average of the 10 iterations was used to obtain the
model performance measures. Table 1 below provides the number of genes obtained through the intersection,
complement of intersection and union of the gene-lists from differential expression
analysis (RNASeq: GSE86562, Microarray: GSE86559). Table (1)
Number of genes in the intersection, complement of intersection, and
union of the two gene-lists. Table (1)
Number of genes in the intersection, complement of intersection, and
union of the two gene-lists. Table (1)
Number of genes in the intersection, complement of intersection, and
union of the two gene-lists. Dataset
Total of DEGs
Intersection
Complement of intersection
Union
GSE86559
165
23
142
424
GSE86562
282
259
Table 2 below shows the official gene symbols and the corresponding gene names. Dataset
Total of DEGs
Intersection
Complement of intersection
Union
GSE86559
165
23
142
424
GSE86562
282
259 Table 2 below shows the official gene symbols and the corresponding gene names. Table 2 below shows the official gene symbols and the corresponding gene names. Table (2)
Official gene symbols and corresponding gene names for the 23 genes. Accuracy =
TP + TN
TP + TN + FP + FN
Sensitivity =
TP
(TP + FN)
Specificity =
TN
(TN + FP)
Kappa coefficient = Accuracy −Random Accuracy (RA)
1 −Random Accuracy (RA)
RA =
(TN + FP) × (TN + FN) + (FN + TP) × (FP + TP)
(TP + TN + FP + FN) × (TP + TN + FP + FN)
AUC = 1
2
TP
(TP + FN) +
TN
(TN + FP)
BER = 1
2
FP
(TN + FP) +
FN
(FN + TP)
MCC =
TP × TN −FP × FN
p
(TP + FP)(TP + FN)(TN + FP)(TN + FN) Accuracy =
TP + TN
TP + TN + FP + FN
Sensitivity =
TP
(TP + FN)
Specificity =
TN
(TN + FP)
Kappa coefficient = Accuracy −Random Accuracy (RA)
1 −Random Accuracy (RA)
RA =
(TN + FP) × (TN + FN) + (FN + TP) × (FP + TP)
(TP + TN + FP + FN) × (TP + TN + FP + FN)
AUC = 1
2
TP
(TP + FN) +
TN
(TN + FP)
BER = 1
2
FP
(TN + FP) +
FN
(FN + TP)
MCC =
TP × TN −FP × FN
p
(TP + FP)(TP + FN)(TN + FP)(TN + FN) RA =
(TN + FP) × (TN + FN) + (FN + TP) × (FP + TP)
(TP + TN + FP + FN) × (TP + TN + FP + FN) TP × TN −FP × FN Page 14 of 23 Mohammed et al. F1-measure =
2TP
2TP + FP + FN F1-measure =
2TP
2TP + FP + FN Results Ensemble Gene ID
Official Gene Symbol
Name
ENSG00000108511
HOXB6
homeobox B6(HOXB6)
ENSG00000169247
SH3TC2
SH3 domain and tetratricopeptide repeats 2(SH3TC2)
ENSG00000120068
HOXB8
homeobox B8(HOXB8)
ENSG00000025293
PHF20
PHD finger protein 20(PHF20)
ENSG00000136997
MYC
v-myc avian myelocytomatosis viral oncogene homolog(MYC)
ENSG00000143882
ATP6V1C2
ATPase H+ transporting V1 subunit C2(ATP6V1C2)
ENSG00000003096
KLHL13
kelch like family member 13(KLHL13)
ENSG00000131746
TNS4
tensin 4(TNS4)
ENSG00000196532
HIST1H3C
histone cluster 1 H3 family member c(HIST1H3C)
ENSG00000233101
HOXB-AS3
HOXB cluster antisense RNA 3(HOXB-AS3)
ENSG00000204104
TRAF3IP1
TRAF3 interacting protein 1(TRAF3IP1)
ENSG00000126003
PLAGL2
PLAG1 like zinc finger 2(PLAGL2)
ENSG00000120875
DUSP4
dual specificity phosphatase 4(DUSP4)
ENSG00000164070
HSPA4L
heat shock protein family A (Hsp70) member 4 like(HSPA4L)
ENSG00000111057
KRT18
keratin 18(KRT18)
ENSG00000260807
LMF1
lipase maturation factor 1(LMF1)
ENSG00000174136
RGMB
repulsive guidance molecule family member b(RGMB)
ENSG00000197818
SLC9A8
solute carrier family 9 member A8(SLC9A8)
ENSG00000187372
PCDHB13
protocadherin beta 13(PCDHB13)
ENSG00000140526
ABHD2
abhydrolase domain containing 2(ABHD2)
ENSG00000166068
SPRED1
sprouty related EVH1 domain containing 1(SPRED1)
ENSG00000182742
HOXB4
homeobox B4(HOXB4)
ENSG00000101193
GID8
GID complex subunit 8 homolog(GID8) Table (2)
Official gene symbols and corresponding gene names for the 23 genes. We performed an explanatory analysis of the RNASeq data. Figure 1 shows the
most meaningful changes at the 0.005 significance level among the genes between
the two conditions, based on the volcano plot [54]. The volcano plot shows the genes
with smaller p-values (higher −log10 values) in red. Page 15 of 23 Mohammed et al. Figure 2 illustrates the estimated dispersion of the RNASeq data using DESeq2
package, with each gene having a gene-specific dispersion parameter. Good esti-
mates of dispersion parameters lead to accurate detection of differentially expressed
genes. Underestimating the dispersion parameters might lead to false positives (i.e.,
declaring genes to be differentially expressed when they are not truly differentially-
expressed). On the other hand, overestimating the dispersion parameters might lead
to false negatives [55]. Table 3 - 6 show the performance of the gene-lists in predicting mutation status,
based on seven methods (algorithms), at the 0.005 significance level: the 282 gene-
list (Table 3); the 23 gene-list (Table 4); the 424 gene-list (Table 5); and the 401
gene-list (Table 6). It is apparent from Table 3, compared to Table 4 below, that NB, ANN, KNN,
and PLDA were improved in the common 23 genes in terms of all performance
measures, while RF and NBLDA had the same performance. SVM had a better
result on the full list of 282 genes. Results Methods
SVM
NB
RF
ANN
KNN
NBLDA
PLDA
Metric
Accuracy
0.98
0.93
0.93
0.98
0.83
-
-
Sensitivity
1
0.89
0.89
1
0.74
-
-
Specificity
0.96
0.96
0.96
0.96
0.93
-
-
Kappa
0.96
0.85
0.85
0.96
0.67
-
-
AUC
0.98
0.93
0.93
0.98
0.83
-
-
BER
0.02
0.07
0.07
0.02
0.17
-
-
MCC
0.96
0.85
0.85
0.96
0.68
-
-
F1-measure
0.98
0.92
0.92
0.98
0.82
-
- Table 5 presents the integration results using the union approach, and it is clear
that SVM, NB, RF, ANN, and KNN methods were improved compared to the case
of 282 differentially-expressed genes. These results were confirmed in Figures 4 and
6. Moreover, SVM and ANN had a higher accuracy than the other methods. Table (6)
Performance of the methods for the 401 gene-list, on the
RNASeq dataset (α = 0.005). Table (6)
Performance of the methods for the 401 gene-list, on the
RNASeq dataset (α = 0.005). Methods
SVM
NB
RF
ANN
KNN
NBLDA
PLDA
Metric
Accuracy
0.98
0.93
0.93
0.96
0.83
-
-
Sensitivity
1
0.89
0.89
0.96
0.74
-
-
Specificity
0.96
0.96
0.96
0.96
0.93
-
-
Kappa
0.96
0.85
0.85
0.93
0.67
-
-
AUC
0.98
0.93
0.93
0.96
0.83
-
-
BER
0.02
0.07
0.07
0.04
0.17
-
-
MCC
0.96
0.85
0.85
0.93
0.68
-
-
F1-measure
0.98
0.92
0.92
0.96
0.82
-
- As can be seen from the Table 6 above, the methods performed better for the
gene-list of 401 genes, compared to the 282 gene-list. Furthermore, Figures 7 confirm
these results. We compared our gene-list of 23 genes with the 18-gene RAS signature (DUSP4,
DUSP6, ELF1, ETV4, ETV5, FXYD5, KANK1, LGALS3, LZTS1, MAP2K3,
PHLDA1, PROS1, S100A6, SERPINB1, SLCO4A, SPRY2, TRIB2, and ZFP106)
(Dry et al. [56]) and found only one overlapping gene (DUSP4). It turned out that
this was also the most predictive of the 7 genes (DUSP4, DUSP6, ETV4, ETV5,
PHLDA1, SERPINB1, and TRIB2) that were discussed in Omolo et al. (2016) [4]. Additional analysis was performed to assess whether the 23 gene-list was predic-
tive of overall survival (OS). Survival analysis was performed using the mutation
status as a group variable and vital status (dead or alive) as the censoring variable. Overall, 20 deaths were recorded out of the 54 samples. The results showed that
the median OS was 1692 months for the 54 samples. Results Therefore, in general, four methods out of seven
were improved on the 23 gene-list compared to the 282 genes-list. From Figures 4
and 5, we notice NBLDA works very well in both lists of genes. Table (3)
Performance of the classification methods for the 282 gene-list,
on the RNASeq dataset (α = 0.005). Methods
SVM
NB
RF
ANN
KNN
NBLDA
PLDA
Metric
Accuracy
0.80
0.76
0.83
0.72
0.72
0.89
0.80
Sensitivity
0.89
0.59
0.78
0.78
0.67
0.81
0.81
Specificity
0.70
0.93
0.89
0.67
0.78
0.96
0.78
Kappa
0.59
0.52
0.67
0.44
0.44
0.78
0.59
AUC
0.80
0.76
0.83
0.72
0.72
0.89
0.80
BER
0.20
0.24
0.17
0.28
0.28
0.11
0.20
MCC
0.60
0.50
0.67
0.45
0.45
0.79
0.59
F1-measure
0.81
0.71
0.82
0.74
0.71
0.88
0.80 Table (3)
Performance of the classification methods for the 282 gene-list,
on the RNASeq dataset (α = 0.005). Table (4)
Performance of the classification methods for the 23 gene-list,
on the RNASeq dataset (α = 0.005). Table (4)
Performance of the classification methods for the 23 gene-list,
on the RNASeq dataset (α = 0.005). Methods
SVM
NB
RF
ANN
KNN
NBLDA
PLDA
Metric
Accuracy
0.78
0.80
0.83
0.80
0.76
0.89
0.87
Sensitivity
0.81
0.70
0.78
0.81
0.70
0.85
0.85
Specificity
0.74
0.89
0.89
0.78
0.81
0.93
0.89
Kappa
0.56
0.59
0.67
0.59
0.52
0.78
0.74
AUC
0.78
0.80
0.83
0.80
0.76
0.89
0.87
BER
0.22
0.20
0.17
0.20
0.24
0.11
0.13
MCC
0.56
0.60
0.67
0.59
0.52
0.78
0.74
F1-measure
0.79
0.78
0.82
0.80
0.75
0.88
0.87 Page 16 of 23 Mohammed et al. Table (5)
Performance of the classification methods for the 424 gene-list,
on the combined RNASeq and microarray datasets (α = 0.005). Table (5)
Performance of the classification methods for the 424 gene-list,
on the combined RNASeq and microarray datasets (α = 0.005). Results Kaplan-Meier curves were
used to graphically compare survival probabilities ( Fig 8) between the two muta-
tion groups (RAS-mutant vs wild-type). The log-rank test was performed using the
RAS mutation status as the group variable. There was no significant difference in
OS between the two groups (log-rank = 1.8, p-value = 0.2). The Cox proportional Page 17 of 23 Mohammed et al. hazards (CPH) model was then applied to assess the significance of the 23 genes
and RAS mutation status. The results show that 9 out of the 23 genes were signifi-
cantly associated with OS, including SPRED1, KLHL13, HOXB4, LMF1, HSPA4L
at the 0.05 level, and ATP6V1C2, PLAGL2, MYC, SLC9A8 at the 0.1 level (LRT
= 56.85, p-value = 0.0002) as can be seen in Tables 7). hazards (CPH) model was then applied to assess the significance of the 23 genes
and RAS mutation status. The results show that 9 out of the 23 genes were signifi-
cantly associated with OS, including SPRED1, KLHL13, HOXB4, LMF1, HSPA4L
at the 0.05 level, and ATP6V1C2, PLAGL2, MYC, SLC9A8 at the 0.1 level (LRT
= 56.85, p-value = 0.0002) as can be seen in Tables 7). Further analysis was performed on the top 9 genes using gradient boosted trees
and Shapley additive explanations (SHAP) methods to identify the top-K genes
(1 < K < 9) [57]. The SHAP approach determined the order of importance of our 9
genes. SHAP values were calculated to give the importance of a gene by comparing
what a model predicts with and without the gene. A SHAP value of 0 means that
the gene has no effect on the prediction, as shown in Fig 9. The vertical axis showed
the gene names, arranged in the order of importance, from top to bottom while the
adjacent value next to the gene name is the mean SHAP value. The horizontal axis
showed the SHAP value, which indicated how much the change was in log-odds. From the log-odds, one can obtain the probability of success. The gradient color
indicated the original value for that gene. Genes pushing the prediction higher were
coloured blue, while those pushing the prediction lower were coloured yellow. Each
point represented a row from the original dataset. Table (7)
Cox proportional hazards model for overall survival, using the 23 genes
and RAS mutation status (class) as covariates. Results (
)
Covariate
Coef
Hazard ratio (HR)
SE(Coef)
Z
Pr(> |z|)
class
2.23E+00
9.34E+00
1.41E+00
1.589
0.112
ATP6V1C2
4.72E-03
1.01E+00
2.66E-03
1.779
0.0752 . HOXB-AS3
7.96E-05
1.00E+00
1.30E-03
0.061
0.951
KRT18
-3.27E-05
1.00E+00
8.73E-05
-0.374
0.7084
RGMB
7.48E-04
1.00E+00
1.13E-03
0.662
0.5079
PLAGL2
-3.28E-03
9.97E-01
1.89E-03
-1.737
0.0824 . DUSP4
2.12E-03
1.00E+00
2.24E-03
0.946
0.3441
SPRED1
1.50E-03
1.00E+00
7.61E-04
1.969
0.0489 *
SH3TC2
6.92E-04
1.00E+00
4.42E-04
1.564
0.1178
HOXB8
-1.04E-02
9.90E-01
7.21E-03
-1.444
0.1488
ABHD2
4.67E-04
1.00E+00
4.34E-04
1.078
0.2812
TNS4
-5.58E-04
9.99E-01
3.74E-04
-1.491
0.1358
HIST1H3C
-4.67E-03
9.95E-01
3.20E-03
-1.458
0.1449
KLHL13
8.28E-03
1.01E+00
3.24E-03
2.559
0.0105 *
MYC
1.21E-03
1.00E+00
7.16E-04
1.689
0.0911 . HOXB4
9.10E-03
1.01E+00
3.60E-03
2.529
0.0114 *
HOXB6
-2.78E-04
1.00E+00
4.18E-03
-0.067
0.9469
PHF20
1.47E-03
1.00E+00
1.78E-03
0.825
0.4094
LMF1
3.19E-03
1.00E+00
1.43E-03
2.224
0.0262 *
SLC9A8
-4.98E-03
9.95E-01
2.56E-03
-1.946
0.0517 . GID8
2.16E-03
1.00E+00
2.61E-03
0.828
0.4079
HSPA4L
-6.94E-03
9.93E-01
2.78E-03
-2.497
0.0125 *
PCDHB13
-3.90E-03
9.96E-01
4.09E-03
-0.953
0.3406
TRAF3IP1
-7.63E-03
9.92E-01
4.86E-03
-1.571
0.1163
Signif. codes:
0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 Discussion The development of molecular signatures is a significant step towards understanding
the molecular mechanisms of tumorigenesis, which could help with accurate prog-
nosis and diagnosis and thus allow physicians to prescribe suitable patient-specific
therapies. Several studies have done cancer classification using either microarray or RNASeq
data only, and few studies have shown integration of both types of data, based on Page 18 of 23 Mohammed et al. heterogeneous datasets. To the best of our knowledge, no cancer classification study
has employed the integration of homogeneous datasets approach. In this study, we
integrated homogeneous microarray and RNASeq datasets, and assessed whether
such an approach could improve the classification accuracy using seven methods,
namely, SVM with radial basis function kernel, NB, RF, ANN, KNN, NBLDA, and
PLDA. We implemented the classification of the mutation status of CRC samples,
using gene-lists obtained through the intersection, complement of intersection, and
union of differentially-expressed genes from microarray and RNASeq datasets. CRC is the third most common cancer and one of the leading causes of death
around the world. The findings suggest that combining two homogeneous datasets
from different technologies could lead to an increase in CRC classification accu-
racy. The study also corroborates the findings of Castillo et al. [20], which found
that combining heterogeneous datasets from different platforms can improve the
performance of a classifier, using multiple datasets. They used data from different
technologies and platforms in order to obtain a larger sample size due to the lack
of enough RNASeq samples. Our proposed approach is different from Castillo et
al.(2019) in that we used homogeneous datasets and a balanced binary class prob-
lem. We used the 0.005 significance level to obtained the differentially expressed
genes, which is restrictive enough to control the false positive rate. A comparison of the performance of the classification methods for each gene-
list revealed that SVM yielded the highest mean accuracy (0.885), followed by
RF(0.880), ANN(0.865), NB(0.855), and KNN(0.785) across the four gene-lists. However, NBLDA performed better than PLDA as a classifier when the analy-
sis was restricted to RNASeq (count) data. Castillo et al. [20] also showed that
SVM performed second to KNN. Statnikov et al. [58] performed a comparison of 18
classification methods on 5 feature selection methods, using 8 datasets and showed
that RF had the highest accuracy (0.954). Discussion Survival analysis results showed that 9 out of the 23 genes were prognostic for
overall survival for CRC patients. Upon subjecting the 9 genes to the Shapley
additive explanations (SHAP) method to rank the genes in order of importance,
the top-5 genes to emerge were ATP6V1C2, MYC, LMF1, HSPA4L, and PLAGL2. Our findings were validated with other published molecular signatures from pre-
vious studies (e.g. Zumwalt et al. [59], He et al. [60], and Liu et al. [61]). Zumwalt
et al. [59] showed that ATP6V1C2 expression successfully distinguished between
cancerous and non-cancerous samples in CRC. In addition, He et al. [60] reported
that the expression of c-Myc, which was one of the three related human genes
encoded under MYC genes family, was observed in many human cancers and was
elevated in up to 70 - 80 % in CRC. Liu et al. [61] identified 10 lncRNAs related
with crucial outcomes in CRC and one of these 10 genes was LMF1. Zhang et
al. [62] obtained 34 genes using minimal redundancy maximal relevance (mRMR)
and incremental feature selection (IFS) methods and found that the HSPA4L
gene was the most highly expressed in CRC patients with chromosomal instability
(CIN) mechanism. Zheng et al. [63] reported that the PLAGL2 gene was vital in
increasing the effect on glioblastoma and colorectal cancer. Su et al. [64] reported
that PLAGL2 served as an oncogenic function in multiple human malignancies,
including colorectal cancer (CRC). Page 19 of 23 Mohammed et al. This study was limited by the number of homogeneous RNASeq and microarray
datasets that were available. Only one matched-pair set of 54 CRC samples was
analyzed. Future studies should extend the approach to more than one cancer type
and multiple datasets. However, the number of samples in each dataset (n = 54)
ensured that the training and validation sets were large enough for the magnitude
and statistical significance of the classification accuracies. Author details
1 g,
p
,
y
Carolina-Upstate, 800 University Way, Spartanburg, USA. 3School of Public Health, Faculty of Health Sciences,
University of the Witwatersrand, 1 Jan Smuts Avenue, Braamfontein, 2000 Johannesburg, South Africa. p
,
y
y,
p
g,
,
y
,
University of the Witwatersrand, 1 Jan Smuts Avenue, Braamfontein, 2000 Johannesburg, South Africa. University of the Witwatersrand, 1 Jan Smuts Avenue, Braamfontein, 2000 Johannesburg, South Africa. References 1. Gandomani, H.S., Aghajani, M., Mohammadian-Hafshejani, A., Tarazoj, A.A., Pouyesh, V., Salehiniya, H., et
al.: Colorectal cancer in the world: incidence, mortality and risk factors. Biomedical Research and Therapy
4(10), 1656–1675 (2017) 1. Gandomani, H.S., Aghajani, M., Mohammadian-Hafshejani, A., Tarazoj, A.A., Pouyesh, V., Salehiniya, H., et
al.: Colorectal cancer in the world: incidence, mortality and risk factors. Biomedical Research and Therapy
4(10), 1656–1675 (2017) 2. Siegel, R.L., Miller, K.D., Jemal, A.: Cancer statistics, 2019. CA: a cancer journal for clinicians 69(1), 7–34
(2019) 2. Siegel, R.L., Miller, K.D., Jemal, A.: Cancer statistics, 2019. CA: a cancer journal for clinicians 69(1), 7–34
(2019) 3. cancer research fund, W.: Colorectal cancer statistics. 3. cancer research fund, W.: Colorectal cancer statistics. https://www.wcrf.org/dietandcancer/cancer-trends/colorectal-cancer-statistics. [Online; accessed
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02-Aug-2019] (2019) 4. Omolo, B., Yang, M., Lo, F.Y., Schell, M.J., Austin, S., Howard, K., Madan, A., Yeatman, T.J.: Adaptation of
a ras pathway activation signature from ffto ffpe tissues in colorectal cancer. BMC medical genomics 9(1), 65
(2016) 5. M´armol, I., S´anchez-de-Diego, C., Pradilla Dieste, A., Cerrada, E., Rodriguez Yoldi, M.: Colorectal carcinoma:
a general overview and future perspectives in colorectal cancer. International journal of molecular sciences
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??? Conclusions In summary, data integration by taking the intersection of the individual gene-lists
from the two data types, improved the classification accuracy of CRC. However,
laboratory experiments should be conducted on this 23-gene signature to further
assess its clinical significance in CRC research. NBLDA method was the best per-
former on the RNASeq data. Results suggest that the SVM method was the most
suitable classifier for CRC across the two data types and had high accuracy before
and after the integration. Future studies should determine the effectiveness of inte-
gration in cancer survival analysis and the application on unbalanced data (where
the classes are of different sizes) as well as on data with multiple classes. Abbreviations
CRC: Colorectal cancer
SSA: Sub-Saharan Africa
EGFRi: Anti-epithelial growth factor receptor inhibitor
FFPE: Formalin-fixed paraffin-embedded
AUC: Area under the ROC curve
WHO: World Health Organization
NGS: Next-generation sequencing
PLDA: Poison linear discriminant analysis
NBLDA: Negative binomial linear discriminant analysis
SVM: Support vector machines
ANN: Artificial neural networks
LDA: Linear discriminant analysis
RF: Random forests
NB: Naive Bayes
KNN: k-Nearest neighbors
IG: Information gain
SGA: Standard genetic algorithm
CFS: Correlation-based feature selection
IBPSO: Improved-binary particle swarm optimization
bagSVM: Bagging SVM
CART: Classification and regression trees
mRMR: Minimum-redundancy maximum-relevance
GEO: Gene expression omnibus
CPM: Counts per million
GLM: Generalized linear model
DAVID: Database for annotation, visualization, and inte
COSMIC: Catalogue of somatic mutations in cancer
MLE: Maximum likelihood estimate
RBF: Radial basis function RF: Random forests NB: Naive Bayes KNN: k-Nearest neighbors IG: Information gain SGA: Standard genetic algorithm CFS: Correlation-based feature selection IBPSO: Improved-binary particle swarm optimization bagSVM: Bagging SVM CART: Classification and regression trees mRMR: Minimum-redundancy maximum-relevance GEO: Gene expression omnibus CPM: Counts per million GLM: Generalized linear model DAVID: Database for annotation, visualization, and integrated discovery DAVID: Database for annotation, visualization, and int COSMIC: Catalogue of somatic mutations in cancer MLE: Maximum likelihood estimate RBF: Radial basis function MAP: Maximum a posteriori BER: Balanced error rate MCC: Matthews correlation coefficient TP: True Positive TN: True Negative FP: False Positive FN: False Negative RA: Random Accuracy IFS: Incremental feature selection CIN: Chromosomal instability HR: Hazard ratio Page 20 of 23 Page 20 of 23 Mohammed et al. Funding g
MM was supported by the DELTAS Africa Initiative Grant No. 107754/Z/15/Z-DELTAS Africa SSACAB. The
DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)’s Alliance for
Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa’s
Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust (Grant
No. 107754/Z/15/Z) and the UK government. The views expressed in this publication are those of the author(s)
and not necessarily those of AAS, NEPAD Agency, Wellcome Trust or the UK government. DECLARATIONS Ethics approval and consent to participate
Not Applicable Consent for publication
Not Applicable Consent for publication
Not Applicable Authors’ contributions BO conceived the study. MM performed all the analyses. MM and BO drafted the manuscript. MM, HM and BO
proof-read, discussed and approved the final manuscript. Acknowledgements The authors wish to thank Prof. Bob Gagnon (GlaxoSmithKline (GSK)) for his comments on the manuscript. Availability of data and materials y
The datasets supporting the results of this article have been deposited to the public repository (GEO) under the
series accession number GSE86566. The individual datasets are accessible under the accession number GSE86559
for the microarray data and GSE86562 for the RNASeq data. The mutation and overall survival data are available
upon request from BO. Competing interests
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Perry, S.R., et al.: Plagl2 regulates wnt signaling to impede differentiation in neural stem cells and gliomas. Cancer cell 17(5), 497–509 (2010) 64. Su, C., Li, D., Li, N., Du, Y., Yang, C., Bai, Y., Lin, C., Li, X., Zhang, Y.: Studying the mechanism of plagl2
overexpression and its carcinogenic characteristics based on 3’-untranslated region in colorectal cancer. International journal of oncology 52(5), 1479–1490 (2018) Figure (6)
ROC curves based on the 424 gene-list for the RNASeq and microarray
datasets (α = 0.005). Figure Legend Figure (1)
Volcano plot of the RNASeq dataset show the 282 differentially ex-
pressed genes in red points (α = 0.005). Figure (2)
Dispersion for the RNASeq data. Figure (3)
Flow-chart of the analysis. Figure (4)
ROC curves based on the 282 gene-list for the RNASeq data (α =
0.005). Figure (5)
ROC curves based on the 23 gene-list for the RNASeq data ( α = 0.005). Figure (1)
Volcano plot of the RNASeq dataset show the 282 differentially ex-
pressed genes in red points (α = 0.005). Figure (1)
Volcano plot of the RNASeq dataset show the 282 differentially ex-
pressed genes in red points (α = 0.005). Figure (2)
Dispersion for the RNASeq data. Figure (3)
Flow-chart of the analysis. Figure (4)
ROC curves based on the 282 gene-list for the RNASeq data (α =
0.005). Figure (5)
ROC curves based on the 23 gene-list for the RNASeq data ( α = 0.005). Figure (1)
Volcano plot of the RNASeq dataset show the 282 differentially ex-
pressed genes in red points (α = 0.005). Figure (5)
ROC curves based on the 23 gene-list for the RNASeq data ( α = 0.005). Page 23 of 23 Mohammed et al. Figure (6)
ROC curves based on the 424 gene-list for the RNASeq and microarray
datasets (α = 0.005). Figure (7)
ROC curves based on the 401 gene-list (α = 0.005). Figure (8)
Kaplan-Meier curves for overall survival (in months). Figure (9)
Genes in ascending order of importance (Note: dots represent SHAP
values of specific features). Figures Figure 1
Volcano plot of the RNASeq dataset show the 282 differentially ex-pressed genes in red points (α =
0:005). Figure 1 Figure 1 Volcano plot of the RNASeq dataset show the 282 differentially ex-pressed genes in red points (α =
0:005). Volcano plot of the RNASeq dataset show the 282 differentially ex-pressed genes in red points (α =
0:005). Figure 2 Figure 2 Dispersion for the RNASeq data. Figure 3
Flow-chart of the analysis. Dispersion for the RNASeq data. Dispersion for the RNASeq data. Figure 3
Flow-chart of the analysis. Figure 3 Flow-chart of the analysis. Figure 4
ROC curves based on the 282 gene-list for the RNASeq data (α =0:005). Figure 4
ROC curves based on the 282 gene-list for the RNASeq data (α =0:005). Figure 4 ROC curves based on the 282 gene-list for the RNASeq data (α =0:005). Figure 5
ROC curves based on the 23 gene-list for the RNASeq data ( α = 0:005). Figure 5
ROC curves based on the 23 gene-list for the RNASeq data ( α = 0:005). Figure 5 ROC curves based on the 23 gene-list for the RNASeq data ( α = 0:005). Figure 6
ROC curves based on the 424 gene-list for the RNASeq and microarray datasets (α = 0:005). Figure 6 ROC curves based on the 424 gene-list for the RNASeq and microarray datasets (α = 0:005). Figure 7
ROC curves based on the 401 gene-list (α = 0:005). ROC curves based on the 401 gene-list (α = 0:005). ROC curves based on the 401 gene-list (α = 0:005). ROC curves based on the 401 gene-list (α = 0:005). Figure 8
Kaplan-Meier curves for overall survival (in months). Kaplan-Meier curves for overall survival (in months). Figure 9
Genes in ascending order of importance (Note: dots represent SHAP values of speci¦c features). Figure 9 Genes in ascending order of importance (Note: dots represent SHAP values of speci¦c features). Genes in ascending order of importance (Note: dots represent SHAP values of speci¦c features).
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https://thescipub.com/pdf/ajessp.2010.338.343.pdf
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Improving Jordan’s Law towards Sustainable Solid and Hazardous Waste Management: Lessons from USA’s Environmental Laws
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American journal of environmental sciences
| 2,010
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INTRODUCTION recycled study industry, soil conditioner industry and
bio-gas industry. In addition, this attracts researchers to
explore different factors that contribute to sustainable
solid waste investment such as: Goddard (1995);
Kinnaman (2009) and Lombrano (2009). There are enormous studies that deal with solid and
hazardous waste management issues in literature due to
the importance of taking the integrated solid and
hazardous waste management as a tool to protect the
natural
resources
from
depletion
and
maintain
sustainable development, to protect from unintended
by-product of human activities and their side-effects. In
some societies, solid and hazardous waste is no longer
considered unwanted product of our daily life activities
such as: lifestyles, industries, tourism, agricultural
activities. But it is considered a raw material in wrong
place which could be of essential need for other
activities whether for the same generators or others
within the solid and hazardous waste generated
communities or outside of such communities. If we
consider a university, for example, the study waste
which resulted from a student class could be used for
other classes or if not possible could be collected within
the university campus to be recycled. If no one is
interested to recycle study within the university campus
a broker could be brought in to collect the unwanted
study for study recycling industry. Therefore, we see
how the study waste could be a fortune for generator,
broker, or study industry. (
)
(
)
There are different roles that community could take
in order to maintain sustainable solid and hazardous
waste management regardless of economical incentive
which
is,
in
most
times,
benefiting
industry
shareholders more than public. Recycled study industry
benefited from using recycled study as raw materials
for their industry since they are able to save extra
expenses on water, energy and raw material sources and
with advances in technologies such as using water-
based ink, the quality of recycled study products are
comparable to others which are manufactured from raw
materials. On the other hand, the author feels that
public did not share the advantages of participating in
recycling programs such as lower study prices or better
study quality which, in some cases, prevent them from
participating in study recycling campaigns, even the
environment is benefited from their participation. American Journal of Environmental Sciences 6 (4): 338-343, 2010
ISSN 1553-345X
© 2010 Science Publications American Journal of Environmental Sciences 6 (4): 338-343, 2010
ISSN 1553-345X
© 2010 Science Publications American Journal of Environmental Sciences 6 (4): 338-343, 2010
ISSN 1553-345X
© 2010 Science Publications American Journal of Environmental Sciences 6 (4): 338-343, 2010
ISSN 1553-345X Improving Jordan’s Law towards Sustainable Solid and
Hazardous Waste Management: Lessons from USA’s Environmental Laws Naill M. Momani
Department of Business Administration, KAU-Jeddah, Kingdom of Saudi Arabia Abstract: Problem statement: Improper handling of solid and hazardous waste will impact the living
standards of a community in normal days and when disasters occur such misuse could disperse
response and recovery activities due to natural or man-made disasters. Approach: This study discussed
the Jordanian solid and hazardous waste management policies in light of USA equivalent ones with the
intention to improve solid and hazardous waste management policies in Jordan. Results: It was found
that establishing a council on environmental quality, special environmental court, a trust fund are the
most actions which could improve the solid and hazardous waste management policies in Jordan. Moreover, introducing environmental taxation incentives, intensifying violation penalties, increasing
public environmental awareness are strong motives which could improve the environmental conditions
in Jordan. Conclusion/Recommendations: It is important to develop the Jordanian environmental
laws in light of worldwide interest with improving environmental conditions for human being. Key words: Jordan environmental law, SWM, developing environmental laws INTRODUCTION For
instance, if university students were told that the prices
of their books will be reduced if they bring their old
ones by replacing such ones, most of them will
participate in giving back their old books with the
intention to reduce the cost. This of course if the
financial incentive is a good one, other incentive will be
through explaining the adverse consequences from
consuming such study is one of the factor which could The immense interest in economics of solid and
hazardous waste is appeared from emerging several
industries which the raw material for its product depend
mainly on generated solid and hazardous waste such as 338 Am. J. Environ. Sci., 6 (4): 338-343, 2010 escalate the global warming issue could motivate
students to participate in such recycling campaign. government body to take care of environmental matters
in Jordan. This law does not explicitly address solid and
hazardous waste management but it focuses on
hazardous
wastes
management. Recently,
the
government of Jordan Passed the Environmental
Protection Law (JEPL) No. 1 of 2003. According to this
law the first ministry of environment in Jordan of its
kind was established. This ministry is responsible for
environmental matters and hold responsible in front of
parliament for application of this environmental law. In a study to promote a community-based solid
waste management initiative in local government
within
Thailand,
Mongkolnchaiarunya
(2005)
investigated the factors which contribute to successful
“Garbage for Eggs” recycling program. In this
program, residents were encouraged to bring recycled
material to exchange for eggs at monthly exchanges in
local communities. Kaseva and Mbuligwe (2005)
found that as a result of privatization of solid waste
collection activities in Dares Salam, solid waste
collection has improved from 10% in 1994-40% of the
total waste generated in the city daily in 2001. This law contains 25 articles that handle
environmental issues in Jordan. Article No 23rd from
this law give council of ministers the authority to issue
the necessary regulations for solid and hazardous waste
management. As we noted earlier, there are different factors that
contribute to successful solid and hazardous waste
management such as economic incentives, public
involvement and stakeholders’ awareness. Some of
these factors need to be regulated through different
governmental policies so they could be implemented
effectively and efficiently given that some of the waste
management practices could affect people and
institutions outside the area where such practices are
implemented. INTRODUCTION For example, using organic waste as
source of plant nutrition will divert this part from
waste-to-energy plants main stream which will affect
the productivity of these plants. Therefore, one party is
benefited while others could bear losses. Gupta et al. (1998) found that India needs better policy and
guidelines to enable the municipal corporations to run
the waste services efficiently in his study entitled “Solid
waste management in India: option and opportunities”. The
management
of
solid
and
hazardous
regulations No. 27 of 2005 which is issued by virtue of
sub-paragraph 8 of article 23 of the previously
mentioned JEPL No. 1 of 2003. The main issues in the
solid waste regulations No. 27 of 2005 are: •
The ministry of environment has the following
duties and tasks in cooperation with other
concerned parties: •
The ministry of environment has the following
duties and tasks in cooperation with other
concerned parties: • Preparation of plans related to solid and
hazardous waste management • Specify the suitable equipment used in solid
and hazardous waste management and methods
of collection, transport, separation, storage,
recycle, treatment and disposal • Specify methods of rehabilitation of closed
landfills • Preparation of studies and data collection and
analysis
in
solid
and
hazardous
waste
management In this study we will present the existing Jordanian
environmental law and the policies which are related to
solid and hazardous waste management and compare
them with US equivalent ones with the intention to
provide suggestions to improve solid and hazardous
waste management policies in Jordan. • Carry out city/countryside street cleaning
campaign • Carry out training programs and public
awareness in the field of solid and hazardous
waste management MATERIALS AND METHODS Any party responsible for solid and hazardous
waste management should: 33
Solid and hazardous waste management policy in
Jordan (Jordanian Ministry of Environment, 2009):
There were different policies and regulations which
deal with environmental issues in Jordan. Each ministry
has its own policy to protect the environment. For
example, Ministry of Agriculture has a law that prevent
investment in agricultural lands, Aqaba Special Zone
Authority has its own policy to protect the marine live. Not until 1995, when the government of Jordan enacted
the first comprehensive environmental law No. 12,
according to which the General Corporation for
Environmental Protection was established as a •
Provide proper personnel for solid and
hazardous waste management •
Provide equipment, containers and necessary
tools
of
solid
and
hazardous
waste
management •
Monitor the process of collection and specify
the routes followed •
Provide proper sitting of containers and
maintenance and replacement •
Take the necessary actions to prevent dumping
of hazardous waste in containers and vehicles 339 Am. J. Environ. Sci., 6 (4): 338-343, 2010 accomplish these goals, the following objectives were
set forth: accomplish these goals, the following objectives were
set forth: accomplish these goals, the following objectives were
set forth: •
Record
keeping
of
quantities,
sources,
treatment and equipment used in this field •
Close monitor of works: Digging, land filling,
waste separation and activities of waste
separation contractors according to contracts •
Proper hazardous waste management
•
Waste minimization
•
Reduction in land disposal practices
•
Prohibition of open dumping
•
Encouragement
of
state
authorized
RCRA
programs
•
Encouragement of research and development
•
Encouragement
of
recovery,
recycling
and
treatment alternatives •
Proper hazardous waste management
•
Waste minimization
•
Reduction in land disposal practices
•
Prohibition of open dumping
•
Encouragement
of
state
authorized
RCRA
programs
•
Encouragement of research and development
•
Encouragement
of
recovery,
recycling
and
treatment alternatives •
Prohibit open burning and random disposal of
solid and hazardous waste •
The ministry of environment has the duty of new
landfill sitting in cooperation with concerned
parties •
The minister releases, according to secretary
general recommendation, the regulations needed to
implement the bylaws RCRA’s “cradle to grave” rules require small
businesses that generate hazardous waste to follow
stringent requirements for storage, recordkeeping, pre-
transportation
and
emergency
response
and
preparedness. MATERIALS AND METHODS Solid and hazardous waste management policy in
United States of America (USA) (US Environmental
Protection Agency, 2009): The industrial revolution in
United States of America (USA) caused environmental
degradation for natural resources. This deterioration
had reached high level in USA in 1960s. With the
increase of environmental awareness among people and
institutions National Environmental Policy Act (NEPA)
of 1969 was enacted following to this law the US
Environmental Protection Agency (US EPA) was
created in 1970. This law establish, for the first time,
broad
national
framework
for
protecting
the
environment. This law basic policy is to assure that all
branches of government give proper considerations to
the environment prior to undertaking any major federal
action that significantly affects the environment. Subtitle I of the HSWA amendments addressed the
problem of leaking Under Storage Tank (UST) systems. Subtitle I includes requirements for tank notification
interim prohibition, new tank standards, reporting and
recordkeeping requirements for existing tanks, corrective
action, financial responsibility, compliance monitoring
and enforcement and approval of state programs. In
1986, Congress passed the Superfund Amendments
Reauthorization Act (SARA) which amended Subtitle I
to provide federal funds for corrective actions on
petroleum releases from UST systems. Comprehensive
Environmental
Response,
Compensation and Liability Act (CERCLA or
Superfund):
The
Comprehensive
Environmental
Response, Compensation and Liability Act of 1980
(CERCLA) established a federal “Superfund” to clean up
uncontrolled or abandoned hazardous waste sites as well
as accidents, spills and other emergency releases of
pollutants and contaminants into the environment. The
focus is primarily on sites that were contaminated in the
past, rather than those currently being contaminated. The
latter sites are regulated under RCRA. Under CERCLA,
a system was established for obtaining funds from
Potentially Responsible Parties (PRPs). Comprehensive
C
i There are more than a dozen major statues or laws
form the legal basis for the programs of the US EPA. Given that we are trying to improve solid and
hazardous waste laws in Jordan we will list only the
ones which are related to our research. Please note that
the following summaries are derived from the US EPA. Further information can be gathered by visiting their
website at www.epa.gov. Resource Conservation and Recovery Act ( RCRA):
The Resource Conservation and Recovery Act of 1976
(RCRA) directed EPA to establish regulations that
would manage the generation, transport, treatment,
storage and disposal of hazardous wastes while
simultaneously ensuring the protection of human health
and the environment. The statute addresses the potential
for contamination from the point of waste generation to
the point of final disposal or destruction. Under CERCLA, anyone who has had any
involvement with a hazardous waste site targeted by
CERCLA could be considered a PRP and could be held
responsible for all or part of the cleanup expense. Site
operators, as well as hazardous waste transporters and
shippers, may be required later to contribute thousands of
dollars for cleanup of the disposal site used. Small
businesses that ship wastes off-site for proper and legal
disposal may be wise to verify the legitimacy and track
record of transporters and destination disposal sites used. 3
RCRA has been amended several times, most
importantly by the Hazardous and Solid and hazardous
Waste Amendments of 1984 (HSWA). Under HSWA,
RCRA became focused on waste minimization and a
national land disposal ban program. In order to 340 Am. J. Environ. Sci., 6 (4): 338-343, 2010 that pose public concerns. This practices should be
formulated in pre-defined structure so it will not be
misused by the decision makers. Therefore, we suggest
establishing a council of environment that advice and
oversee the minister decisions. The Superfund Amendments and Reauthorization
Act (SARA): The Superfund Amendments and
Reauthorization Act of 1986 (SARA) amended the
Comprehensive
Environmental
Response,
Compensation and Liability Act (CERCLA) on October
17, 1986. SARA reflected EPA’s experience in
administering the complex superfund program during
its first six years and made several important changes
and additions to the program. SARA: Article No. 11 of the JEPL No.1 of 2003 deals with
hazardous waste materials and prohibited the burying of
any dangerous waste materials into the Kingdom. Comprehensive
C
i The
perpetrator of this act shall be punished by
imprisonment for a period of not less than thirty days
and not more than three months, or by a fine of not less
than three hundred Jordanian dinars and not more than
five thousand dinars. •
Stressed the importance of permanent remedies and
innovative treatment technologies in cleaning up
hazardous waste sites •
Required superfund actions to consider the
standards and requirements found in other state and
federal environmental laws and regulations We see how the US laws enacted CERCLA as
separate act that deal with hazardous materials while in
the Jordanian equivalent one it was handled through
article within the Jordanian environmental law. •
Provided
new
enforcement
authorities
and
settlement tools Under CERCLA, there is a trust fund which is
supported by an excise tax on feedstock chemicals and
petroleum to pay for cleanup activities at abandoned
waste sites. There is no such trust fund established
under the Jordanian environmental law. It is suggested
to establish a trust fund that is supported by an excise
tax on feedstock chemicals and petroleum. •
Increased state involvement in every phase of the
superfund program •
Increased the focus on human health problems
posed by hazardous waste sites •
Encouraged greater citizen participation in making
decisions on how sites should be cleaned up and •
Increased the size of the trust fund to $8.5 billion Under CERCLA, violator should pay up to 50
million USD and if the government conducts cleanup
for the violation, violators should pay up to three times
of the cleanup cost. While in the JEPL, The violators
pay the cleanup cost plus 25%. We think this part need
to be increased so violators pay more for their
violations. For instance, Exxon oil spill in 1989 cost
Exxon Company more than 5 billions of US dollars for
a spill of oil could worth less than 10 million. In other
words, polluters could be in favor to pay the spill
removal cost with 25% if it is less than what other
contractors could ask. Also, some spills could cost
billions in dollars, so 50 million USD will not be
enough to prevent future ones. After Exxon oil some
companies established their regional response center to
help in containing marine oil spill with short notice and
not allow the spill accident to overcome the local
authorities’ capabilities. Comprehensive
C
i SARA also required EPA to revise the Hazard
Ranking System (HRS) to ensure that it accurately
assessed the relative degree of risk to human health
and the environment posed by uncontrolled hazardous
waste sites that may be placed on the National
Priorities List (NPL). RESULTS AND DISCUSSION of
environmental
environmental
Trust
taxation
violation
public
Event type and date
participants
quality
court
fund
incentives
penalties
awareness
ERA, 20-25/8/2006 training
20
7
5
4
2
1
1
EIA, 9-13/7/2006 training
17
5
4
4
2
1
1
EIA, 12-16/3/2006 training
20
6
5
3
3
2
1
Talk, 27/8/2006
18
5
3
3
2
3
2
Talk, 28/3/2006
20
7
3
2
4
3
1
Total
95
30
20
16
13
9
6 Table 1: Main applications of LCA
Drivers
Switzerland
Germany
Italy
Sweden
Product-related environmental problems
A
A
B
A
Cost-saving opportunities
A
A
A
B
Emerging green markets
A
A
B
B
Decision of the management
B
A
B
B
Perceived environmental discussions
B
A
B
B
Cost avoidance due to future liabilities
B
B
B
A
Collaborative study with ext. organizations
B
A
C
B
Meet eco label criteria B B B B
B
B
B
B
Initiatives by Research and Development
B
B
C
A
Encouragement by the parent company
C
C
A
B
New instruments for R and D
C
C
A
C
Environmental legislation
B
B
B
C
Source: Frankl and Frieder (1999) Table 2: Measures to improve environmental situations in Jordan
Council on
Special
Environmental
Intensifying
Increasing
No. of
environmental
environmental
Trust
taxation
violation
public
Event type and date
participants
quality
court
fund
incentives
penalties
awareness
ERA, 20-25/8/2006 training
20
7
5
4
2
1
1
EIA, 9-13/7/2006 training
17
5
4
4
2
1
1
EIA, 12-16/3/2006 training
20
6
5
3
3
2
1
Talk, 27/8/2006
18
5
3
3
2
3
2
Talk, 28/3/2006
20
7
3
2
4
3
1
Total
95
30
20
16
13
9
6 2: Measures to improve environmental situations in Jordan SARA required EPA to revise the Hazard Ranking
System (HRS) to ensure that it accurately assessed the
relative degree of risk to human health and the
environment posed by uncontrolled hazardous waste
sites that may be placed on the National Priorities List
(NPL). There is no such mentioning in the JEPL and we
suggest having act or article within the law which
specify how the government could deal with
uncontrolled hazardous waste sites. This will make it
easier to the government to set agenda for cleanup
process according to the law. RESULTS AND DISCUSSION There is more experience of environmental
protection within USA government given that the US
environmental law was established in 1969 while the
first Jordanian environmental law enacted in 1995. In
the Jordanian law the penalties were determined for
each violation where it was more open in the USA one. The Jordanian law gives the minister the authority in all
decisions which are related to any environmental issues. Decisions could be biased if not governed by council on
environmental quality such as the one which NEPA
allow to establish. The council on environmental
quality of USA should assist and advice the president in
the preparation of the environmental quality report, to
develop and recommend to the president national
policies that improve environment quality and to report
to the president at least once each year. In Jordan, the
minister is considered the decision makers in all
environmental issues and sometime a group of
environmental advocates could be consulted in issues The JEPL did not classify the cleanup process for
the hazardous material and especially for the abandoned
hazardous sites or spills. While under CERCLA there
are two kinds of response actions: •
Short term removals: Mean to know sources of
releasing of hazardous waste and require prompt
response p
•
Long term removals: Mean reduce dangers resulted
from release of hazardous waste, but not thread life
immediately 341 Am. J. Environ. Sci., 6 (4): 338-343, 2010 Table 1: Main applications of LCA
Drivers
Switzerland
Germany
Italy
Sweden
Product-related environmental problems
A
A
B
A
Cost-saving opportunities
A
A
A
B
Emerging green markets
A
A
B
B
Decision of the management
B
A
B
B
Perceived environmental discussions
B
A
B
B
Cost avoidance due to future liabilities
B
B
B
A
Collaborative study with ext. organizations
B
A
C
B
Meet eco label criteria B B B B
B
B
B
B
Initiatives by Research and Development
B
B
C
A
Encouragement by the parent company
C
C
A
B
New instruments for R and D
C
C
A
C
Environmental legislation
B
B
B
C
Source: Frankl and Frieder (1999)
Table 2: Measures to improve environmental situations in Jordan
Council on
Special
Environmental
Intensifying
Increasing
No. RESULTS AND DISCUSSION SARA required EPA to revise the Hazard Ranking There is enormous interest in establishing special
environmental court in Jordan and in neighboring
countries such as Kuwait which a number of Parliament
members request establishing environmental court
recently. In the First Marine Environmental Conference
which took place in Aqaba in July 2002, in which the
participants
recommended
establishing
special
environmental
court. Such
initiative
took
an
international dimension as well. For instance, the
Science Nobel Prize winner Adolf Scafell is advocating
for establishing an International Environmental Court to
deal with regional and global environmental problems. Life Cycle Assessment (LCA) is a tool that could
be used to improve the environmental situations. It is a
comprehensive and has scientific bases in order to
measure the environmental performance of product and
service systems. It covers all stages of product or
service production (cradle to grave) which includes, for
example: Extracting raw material, preparing it for use
in industry, manufacturing, distribution, use and
disposal. LCA is widely used as a mandatory study in
order to use the EU eco-labeling for products and
services which could show consumers that they are
environmentally safe one. The above discussion and research concerns were
shared
with
experts
and
environmental
group
members during special training in environmental
management issues and environmental meetings. These
trainings
include
Environmental
Risk
Assessment
(ERA)
and
Environmental
Impact
Assessment
(EIA)
ones. The
participants
are
environmental specialists with different managerial
levels who represent government and private sectors. Most training activities took place within Jordanian
Environmental Society in Jordan in 2006. During the
training, some participants were asked to suggest
potential means to improve environmental situation in
Jordan and we ranked the priorities of such means
such as shown in Table 2. Luckily, there was common
agreement which enables us to recommend applicable
and efficient ones that could play vital role in shaping
the future environmental conditions in Jordan. In a study for Frankl and Frieder (1999), as shown
in Table 1, there were different motives for companies
to implement LCA studies. Companies are ranked
according to three possible levels of importance: high,
medium and low. Each company could give several
possible answers. As we could tell from the motives
that meeting environmental legislations and eco label
criteria were ranked in medium level. 342 Am. J. Environ. Sci., 6 (4): 338-343, 2010 REFERENCES Frankl, P. and R. Frieder, 1999. LCA in industry and
business-adoption
patterns,
applications
and
implications. Springer. It is important to improve solid and hazardous
waste management laws in Jordan. There are several
alternatives that could be sought in light of social and
environmental conditions. This improvement could
facilitate quick response to deal with such waste due to
natural
or
man-made
disasters. Some
of
the
recommendations to improve solid and hazardous waste
management practices in Jordan are: Gupta, S., K. Mohan, R. Prasad, S. Gupta and A. Kansal,
1998. Solid waste management in India: Option
and opportunities. Resour. Conserv. Recycl.,
24: 137-154. DOI: 10.1016/S0921-3449(98)00033-0 Goddard, H.C., 1995. The benefits and costs of
alternative solid waste management policies. Resour. Conserv. Recycl., 13: 183-213. DOI:
10.1016/0921-3449(94)00021-V •
Intensify the penalties so there will not be a
repetition for violation and this penalty could be
determined by special environmental court Jordanian Ministry of Environment, 2009. Solid and
hazardous
waste
directorate. MOENV. http://www.moenv.gov.jo/arabicmoe/users/index.aspx •
Establish special environmental courts equipped
with trained environmental law specialists. Kaseva, M.E. and S.E. Mbuligwe, 2005. Appraisal of
solid waste collection following private sector
involvement in Dar Es Salaam city, Tanzania. Habitat
Int.,
29:
353-366. DOI:
10.1016/j.habitatint.2003.12.003 •
Establish council on environmental quality to draw
policies and advice to prime minister and hold
accountable in front of parliament •
Develop an appropriate mechanism for SWM cost
recovery based on the “polluter pays and user
pays” principle Kinnaman, T.C., 2009. The economics of municipal
solid waste management. Waste Manage.,
29:
2615-2617. DOI:
10.1016/j.wasman.2009.06.031 •
Increase public awareness which could lead to
actions so as to protect the environment Lombrano,
A.,
2009. Cost
efficiency
in
the
management of solid urban waste. Resour. Conserv. Recycl.,
53:
601-611. DOI:
10.1016/j.resconrec.2009.04.017 •
Introduce new incentive for industry to improve
their
environmental
performance
such
as
environmental taxation which is based on Life
Cycle Assessment (LCA) Mongkolnchaiarunya,
J.,
2005. Promoting
a
community-based
solid-waste
management
initiative in local government: Yala municipality. Habitat Int., 29: 27-40. DOI: 10.1016/S0197-
3975(03)00060-2 •
Establish separate law that deals with abandoned
hazardous sites and specify the cleanup priorities
and the sources of funds •
Create trust fund to clean up unidentified
responsible party to minimize potential human and
environmental risks. This trust fund is supported by
excise tax on feedstock chemicals and petroleum US Environmental Protection Agency, 2009. Laws,
regulation
and
guidance. EPA. http://www.epa.gov/lawsregs/basic/index.html 343
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English
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Cats Infected by Feline Leukemia Virus—Morpho-Quantitative Evaluation of the Thymus
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Open journal of animal sciences
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cc-by
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How to cite this paper: Melo, F.A.C., Santos, A.C., Nogueira, K., Barreto-Vianna, A.R.C. and Lima, E.M.M. (2015) Cats In-
fected by Feline Leukemia Virus—Morpho-Quantitative Evaluation of the Thymus. Open Journal of Animal Sciences, 5,
277-282. http://dx.doi.org/10.4236/ojas.2015.53032 Cats Infected by Feline Leukemia
Virus—Morpho-Quantitative
Evaluation of the Thymus Cats Infected by Feline Leukemia
Virus—Morpho-Quantitative
Evaluation of the Thymus Received 2 May 2015; accepted 3 July 2015; published 6 July 2015 Copyright © 2015 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ *Corresponding author. Open Journal of Animal Sciences, 2015, 5, 277-282
ublished Online July 2015 in SciRes. http://www.scirp.org/journal/ojas
ttp://dx.doi.org/10.4236/ojas.2015.53032
Cats Infected by Feline Leukemia
Virus—Morpho-Quantitative
Evaluation of the Thymus
Flávia Azevedo Cavalcanti de Melo, Anna Clara dos Santos, Kaique Nogueira*,
André Rodrigues da Cunha Barreto-Vianna, Eduardo Maurício Mendes de Lima
Faculty of Agronomy and Veterinary Medicine, University of Brasilia, Brasília, Brazil
Email: *kiqnogueira@gmail.com
Received 2 May 2015; accepted 3 July 2015; published 6 July 2015
Copyright © 2015 by authors and Scientific Research Publishing Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons.org/licenses/by/4.0/
Abstract
The feline leukemia virus (FeLV) is a complex disease that affects mainly the immune and hema-
tological systems of cats. And because the thymus was closely associated with the immune system,
the aim of this study was to quantify the morphological changes in the thymus of naturally infected
and uninfected animals. Thymus morphological changes were evaluated by quantifying the fol-
lowing parameters: thymocytes, fat tissue, thymic corpuscles, connective tissue, and type I and III Journal of Animal Sciences, 2015, 5, 277-282
hed Online July 2015 in SciRes. http://www.scirp.org/journal/ojas
/dx.doi.org/10.4236/ojas.2015.53032
Cats Infected by Feline Leukemia
Virus—Morpho-Quantitative
Evaluation of the Thymus
Flávia Azevedo Cavalcanti de Melo, Anna Clara dos Santos, Kaique Nogueira*,
André Rodrigues da Cunha Barreto-Vianna, Eduardo Maurício Mendes de Lima
Faculty of Agronomy and Veterinary Medicine, University of Brasilia, Brasília, Brazil
Email: *kiqnogueira@gmail.com
Received 2 May 2015; accepted 3 July 2015; published 6 July 2015
Copyright © 2015 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/
Abstract
The feline leukemia virus (FeLV) is a complex disease that affects mainly the immune and hema-
tological systems of cats. And because the thymus was closely associated with the immune system,
the aim of this study was to quantify the morphological changes in the thymus of naturally infected
and uninfected animals. Thymus morphological changes were evaluated by quantifying the fol-
lowing parameters: thymocytes, fat tissue, thymic corpuscles, connective tissue, and type I and III
collagen. This study analyzed a total of seven cats, five in the negative group (Fn) and two in the
positive (Fp) group. The areas occupied by thymocytes in FeLV negative (Fn) and positive (Fp)
animals were, respectively, 55.16% ± 6.15% and 44.81% ± 6.15% (p = 0.39); adipocytes, 42.14% ±
5.97% (Fn) and 53.42% ± 11.3% (Fp) (p = 0.34); blood vessels, 0.85% ± 0.24% (Fn) and 1.75% ±
0.52% (Fp) (p = 0.15). Whereas thymic corpuscles occupied an area of 0.68% ± 0.19% in negative
animals (Fn) only. The connective tissue covered area varied between 12.05% ± 0.68% (Fn) and
10.34% ± 0.62% (Fp) (p = 0.009). Collagen type I (p = 0.30) and type III (p = 0.59) were present in,
respectively, 1.73% ± 0.3% and 0.44% ± 0.09% in Fn, and 1.44% ± 0.36 and 0.44% ± 0.13% in Fp. The FeLV virus was not able to completely modify the thymus structure of infected animals, con-
sidering the areas occupied by thymocytes, adipose tissue, blood vessels and also type I and III
collagens. However, the connective tissue underwent a certain degree of remodeling since less
connective tissue area was observed in FeLV infected animals. The absence of thymic corpuscles in
immunosuppressed animals suggests a possible connection to thymopoiesis. Open Journal of Animal Sciences, 2015, 5, 277-282
Published Online July 2015 in SciRes. http://www.scirp.org/journal/ojas
http://dx.doi.org/10.4236/ojas.2015.53032 Domestic Cats, Morphology, Lymphoid Organ, FeLV 2. Materials and Methods Seven domestic cats (Felis catus domesticus) seen at the Veterinary Hospital of the Universidade de Brasilia
were divided into two groups and evaluated. The FeLV-positive group (Fp) had two cats (one male and one fe-
male) and the FeLV-negative (Fn) group had five cats (two males and three females). The Fp cats were positive
only for FeLV infection (not for FIV infection) and were diagnosed with anemia and leukopenia according to
Complete Blood Count (CBC) results. The Fn cats were negative for both FIV and FeLV infection and the
Complete Blood Count (CBC) results were normal. The study was approved by the Animal Use Ethics Com-
mittee of the University of Brasília (≠109508/2011). Both animals of the Fp group had FeLV infection symptoms such as decreased appetite and prostration, in
addition to severe anemia (Corpuscular Volume 9%). The infection was confirmed by the SNAP FeLV/FIV
Combo Test (INDEXX Laboratories). The animals’ age ranged between six months and three years. The thoracic lobes of the thymus of both antimeres were collected immediately after the animals’ death. To
this end, two longitudinal sections were performed along the ninth intercostal space in both antimeres to visual-
ize the corresponding thoracic lobes of the thymus. The thymic mass was considered as a single structure with-
out differentiating between the lobes, which was justified by the age of the animals. In general, some right tho-
racic lobes were already in advanced involution stage, or even absent. The thymuses were sectioned transversely along serial and parallel macroscopic plans distant 3mm average. The modified guiding method was followed to obtain IUR (isotropic, uniform and random) sections. The frag-
ments were immersed in 10% formaldehyde fixative solution for at least 24 hours, embedded in paraffin, and
subjected to conventional histological techniques. The 4μm-thick sections were obtained using a manual micro-
tome (Leica RM 2125RT). The Hematoxylin-Eosin stained slides were used to quantify the area occupied by
thymocytes, lymphoid aggregates, blood vessels and adipocytes using the STEPanizer© software with 36 hits in
the grid. The total area was calculated after counting the hits according to the Delesse principle, where A[est] is
the area occupied by the structure; R[est], the number of hits counted per structure; and Pt, total number of hits in
the grid. Abstract The feline leukemia virus (FeLV) is a complex disease that affects mainly the immune and hema-
tological systems of cats. And because the thymus was closely associated with the immune system,
the aim of this study was to quantify the morphological changes in the thymus of naturally infected
and uninfected animals. Thymus morphological changes were evaluated by quantifying the fol-
lowing parameters: thymocytes, fat tissue, thymic corpuscles, connective tissue, and type I and III
collagen. This study analyzed a total of seven cats, five in the negative group (Fn) and two in the
positive (Fp) group. The areas occupied by thymocytes in FeLV negative (Fn) and positive (Fp)
animals were, respectively, 55.16% ± 6.15% and 44.81% ± 6.15% (p = 0.39); adipocytes, 42.14% ±
5.97% (Fn) and 53.42% ± 11.3% (Fp) (p = 0.34); blood vessels, 0.85% ± 0.24% (Fn) and 1.75% ±
0.52% (Fp) (p = 0.15). Whereas thymic corpuscles occupied an area of 0.68% ± 0.19% in negative
animals (Fn) only. The connective tissue covered area varied between 12.05% ± 0.68% (Fn) and
10.34% ± 0.62% (Fp) (p = 0.009). Collagen type I (p = 0.30) and type III (p = 0.59) were present in,
respectively, 1.73% ± 0.3% and 0.44% ± 0.09% in Fn, and 1.44% ± 0.36 and 0.44% ± 0.13% in Fp. The FeLV virus was not able to completely modify the thymus structure of infected animals, con-
sidering the areas occupied by thymocytes, adipose tissue, blood vessels and also type I and III
collagens. However, the connective tissue underwent a certain degree of remodeling since less
connective tissue area was observed in FeLV infected animals. The absence of thymic corpuscles in
immunosuppressed animals suggests a possible connection to thymopoiesis. Keywords Keywords Domestic Cats, Morphology, Lymphoid Organ, FeLV F. A. C. Melo et al. 1. Introduction Feline Leukemia Virus (FeLV) belongs to the Gammaretrovirus genus, Retroviridae family and causes an im-
portant disease routinely diagnosed in domestic cats [1]. Infection usually occurs in the oropharynx due to pro-
longed direct contact, especially through mutual grooming, sharing water and feed bowls. After infection of the
oropharynx, the virus infects the lymphocytes, reaching the bone marrow [2]. The infected cells carry the viral
agent to other target tissues such as thymus, spleen and lymph nodes [3]. The FeLV infection course depends on factors such as the host immune response and disease progression. It is
known to cause a variety of neoplastic and non-neoplastic diseases in domestic cats (Felis catus), such as thymic
lymphoma, multicentric lymphoma, myelodysplastic syndromes, acute myeloid leukemia, aplastic anemia, and
immunodeficiency ([4]-[10]). The thymus is an important organ in FeLV infection because it is a lymphoid organ whose primary function is
to provide a proper environment for T lymphocytes differentiation, maturation, and selection, from the migrant
progenitor cells of the bone marrow [11]. Therefore, this study evaluated the morphological changes in the thymus, an important lymphoid organ tar-
geted by the virus, by quantifying the thymocytes, fat tissue, thymic corpuscles, connective tissue, and type I
and III collagen fibers of naturally infected and uninfected cats. The thymocytes occupied areas were 55.16% ± 6.15% and 44.81% ± 6.15% for Fn and Fp groups, respectively 2. Materials and Methods Furthermore, the samples stained with Pricrosirius red were used to determine the proportion of con-
nective tissue between groups, to differentiate type I and III collagen fibers using the polarizing filter and Image
Pro Plus 6.0® image analysis software. Photomicrographs of random fields of each fragment were obtained us-
ing an Olympus BX51 optical microscope coupled to the ProgRes® Capture Pro 2.5 image analysis and capture
software. Data are presented as means and standard deviations. The data were submitted to Kolmogorov-Smirnov nor-
mality test and compared by Mann-Whitney test, besides Pearson and Spearman correlation (GraphPad Prism
2.6 for Windows, GraphPad Software, San Diego, CA, USA), at p < 0.05 significance level. 3. Results 3. Results es occupied areas were 55.16% ± 6.15% and 44.81% ± 6.15% for Fn and Fp groups, respectively The thymocytes occupied areas were 55.16% ± 6.15% and 44.81% ± 6.15% for Fn and Fp groups, r The thymocytes occupied areas were 55.16% ± 6.15% and 44.81% ± 6.15% for Fn and Fp groups, respectively 278 F. A. C. Melo et al. F. A. C. Melo et al. (Figures 1(A)), with p = 0.34. On the other hand, adipose tissue behaved inversely to thymocytes and the occu-
pied areas were 42.14% ± 5.97% and 53.42% ± 11.3% in animals of Fn and Fp groups, respectively (Figure
1(B)). The adipose tissue formed “islands” defined by the presence of tissue extensions in both groups. The
proportion of blood vessels varied between 0.85% ± 0.24% in Fn animals and 1.76% ± 0.52% in Fp (Figure
1(C)) animals, with p = 0.15. (Figures 1(A)), with p = 0.34. On the other hand, adipose tissue behaved inversely to thymocytes and the occu-
pied areas were 42.14% ± 5.97% and 53.42% ± 11.3% in animals of Fn and Fp groups, respectively (Figure
1(B)). The adipose tissue formed “islands” defined by the presence of tissue extensions in both groups. The
proportion of blood vessels varied between 0.85% ± 0.24% in Fn animals and 1.76% ± 0.52% in Fp (Figure
1(C)) animals, with p = 0.15. Thymic corpuscles presence was 0.68% ± 0.19% in Fn cats while absent in Fp animals. The amount of connective tissue in the thymus varied between 12.05% ± 0.68% in the Fn animals and 10.34%
± 0.67% in Fp animals (Figure 1(D)), with p = 0.009. Figure 1(E) and Figure 1(F) show that type I and type III collagen fibers covered an area ranging between
1.73% ± 0.3% and 0.44% ± 0.09% in Fn animals (p = 0.30); and 1.44% ± 0.36% and 0.44% ± 0.13% in Fp cats
(p = 0.59). Type I collagen was predominant in the thymus of both groups, suggesting that this is related to the
structural arrangement of the organ. The disposition of thymocytes and adipose tissue for Fn and Fp groups is shown in Figure 2(A) and Figure
2(B), and Figure 3(A) and Figure 3(B), respectively. Thymic corpuscle for Fn group is shown in Figure 2(C). The blood vessels for Fn and Fp groups are shown in Figure 2(D) and Figure 3(C), respectively. 3. Results The connec-
tive tissue for Fn and Fp groups is shown in Figures 2(E) and Figure 3(D), respectively; lastly, the disposition
of Collagens I and III for Fn group is shown in Figure 2(E). 5. Conclusion The FeLV virus was unable structurally to modify the thymus of naturally infected animals, considering the re-
sults obtained for thymocytes, adipose tissue, blood vessels, and type I and III collagens. The statistically signif-
icant lesser amount of connective tissue found in animals infected with FeLV indicated tissue remodeling due to
the disease. The absence of thymic corpuscles in immunosuppressed animals led to believe that these structures
were linked to thymopoiesis. 4. Discussion F. A. C. Melo et al. tissues. Lima and Carneiro-Sampaio (2007) [11] stated that the amount of adipose tissue in the body increases
with age, which could explain the percent increase of adipose tissue disposition in Fp group since the animals of
this group were older than the cats in the Fn group. tissues. Lima and Carneiro-Sampaio (2007) [11] stated that the amount of adipose tissue in the body increases
with age, which could explain the percent increase of adipose tissue disposition in Fp group since the animals of
this group were older than the cats in the Fn group. Thymic corpuscles were found only after 50 days of gestation in cats [13] and between the 30th day of gesta-
tion and one year of age in dogs [14]. The time-dependency of these structures is documented in the literature,
their function, however, has not yet been clarified. The findings in feline and other species vary. However, stu-
dies evaluating VEGF (vascular endothelial growth factor) presence in human thymus corroborate the hypothe-
sis of active participation of these corpuscles in thymopoiesis [15]. In addition, the presence of the FeLV virus
suggests that thymus atrophy did not play a role in thymopoiesis, and that the decrease of thymocytes in cats of
the Fp group was probably due to the absence of thymic corpuscles. The loss of thymic epithelial cells and the
increase in both perivascular space-usually infiltrated by adipocytes-and cystic cavities were strong characteris-
tics of thymus physiological involution, which were especially expressed by thymocytes involution [16]. The connective tissue decreased in the cats of the Fp group compared to those of the Fn group. This result
suggests that there was remodeling of connective tissue due to the disease. In young animals (up to 55 days), the
thymic corpuscle capsule consists of connective tissue [13]. So, it was possible to suggest also that the thymic
corpuscles present in cats of the Fp group are closely related to the increased connective tissue in the group. The remodeling of the connective tissue only in cats of the Fp group suggests that the functional changing
process of the organ had started, but it was interrupted by the immunosuppressive activity of the disease. In oth-
er words, the convalescent state of the animals acted only as a precursor to the tissue deposition while the main-
tenance and concrete replacement could not be completed. 4. Discussion Structural changes throughout the thymic development and involution were evidenced in domestic animals [11]
[12]. This fact was evident by the organ architectural and physiological changes, in addition to the reducing
thymus. The changing structure of the thymus justified the arrangement found regarding the different component Structural changes throughout the thymic development and involution were evidenced in domestic animals [11]
[12]. This fact was evident by the organ architectural and physiological changes, in addition to the reducing
thymus. The changing structure of the thymus justified the arrangement found regarding the different component Figure 1. Percentage (%) of thymocytes (A), fat tissue (B), blood vessels (C), connective tissue (D), type I (E) and type III
(F) collagens for FeLV negative (Fn) and positive (Fp) cats. The asterisk shows significant difference between groups, ac-
cording to the Wilcoxon test results. Figure 1. Percentage (%) of thymocytes (A), fat tissue (B), blood vessels (C), connective tissue (D), type I (E) and type III
(F) collagens for FeLV negative (Fn) and positive (Fp) cats. The asterisk shows significant difference between groups, ac-
cording to the Wilcoxon test results. 279 279 279 F. A. C. Melo et al. Figure 2. FeLV-Negative Group: HE-stained photomicrograph showing adipose tissue (black arrow) and thymocytes (red
arrow) in images (A) and (B); thymic corpuscle (black arrow), in image (C) and blood vessels (black arrows) in image (D);
Picrosirius-stained photomicrograph showing connective tissue (black arrow) in image (E); Polarized light photomicrograph
showing type I collagen (black arrow) and type III collagen (red arrow) in image (F). Figure 2. FeLV-Negative Group: HE-stained photomicrograph showing adipose tissue (black arrow) and thymocytes (red
arrow) in images (A) and (B); thymic corpuscle (black arrow), in image (C) and blood vessels (black arrows) in image (D);
Picrosirius-stained photomicrograph showing connective tissue (black arrow) in image (E); Polarized light photomicrograph
showing type I collagen (black arrow) and type III collagen (red arrow) in image (F). Figure 3. FeLV-Positive Group: HE-stained photomicrograph showing adipose tissue (black arrow) and thymocytes (red
arrow) in images (A) and (B); blood vessels (black arrows) in image (C); Picrosirius-stained photomicrograph showing
connective tissue (black arrow) in image (D). Figure 3. FeLV-Positive Group: HE-stained photomicrograph showing adipose tissue (black arrow) and thymocytes (red
arrow) in images (A) and (B); blood vessels (black arrows) in image (C); Picrosirius-stained photomicrograph showing
connective tissue (black arrow) in image (D). 280 F. A. C. Melo et al. References [1]
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English
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A lncRNA fine tunes the dynamics of a cell state transition involving Lin28, let-7 and de novo DNA methylation
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eLife
| 2,017
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cc-by
| 18,097
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*For correspondence: ml440@
cam.ac.uk (MAL); lhe@berkeley.
edu (LH); austin.smith@cscr.cam.
ac.uk (AS) A lncRNA fine tunes the dynamics of a cell
state transition involving Lin28, let-7 and
de novo DNA methylation DOI: 10.7554/eLife.23468.001 DOI: 10.7554/eLife.23468.001 Received: 19 November 2016
Accepted: 24 July 2017 Received: 19 November 2016
Accepted: 24 July 2017
Published: 18 August 2017 RESEARCH ARTICLE A lncRNA fine tunes the dynamics of a cell
state transition involving Lin28, let-7 and
de novo DNA methylation A lncRNA fine tunes the dynamics of a cell
state transition involving Lin28, let-7 and
de novo DNA methylation Meng Amy Li1,2*†, Paulo P Amaral3, Priscilla Cheung2, Jan H Bergmann4, Meng Amy Li1,2*†, Paulo P Amaral3, Priscilla Cheung2, Jan H Bergmann4,
Masaki Kinoshita1, Tu¨ zer Kalkan1, Meryem Ralser1, Sam Robson3‡,
Ferdinand von Meyenn5, Maike Paramor1, Fengtang Yang6, Caifu Chen7,
Jennifer Nichols1, David L Spector4, Tony Kouzarides3, Lin He2*, Austin Smith1,8* g
y
g
g
Masaki Kinoshita1, Tu¨ zer Kalkan1, Meryem Ralser1, Sam Robson3‡, y
Ferdinand von Meyenn5, Maike Paramor1, Fengtang Yang6, Caifu Chen7,
if
i h l 1
id
S
4
id
3
i
2*
i
S
i h1 8* 1Wellcome Trust - Medical Research Council Stem Cell Institute, University of
Cambridge, Cambridge, United Kingdom; 2Division of Cellular and Developmental
Biology, Department of Molecular and Cellular Biology, University of California
Berkeley, Berkeley, United States; 3The Gurdon Institute, University of Cambridge,
Cambridge, United Kingdom; 4Cold Spring Harbor Laboratory, Cold Spring Harbor,
United States; 5Babraham Institute, Cambridge, United Kingdom; 6Wellcome Trust
Sanger Institute, Hinxton, United Kingdom; 7Integrated DNA Technologies,
Redwood, United States; 8Department of Biochemistry, University of Cambridge,
Cambridge, United Kingdom Abstract Execution of pluripotency requires progression from the naı¨ve status represented by
mouse embryonic stem cells (ESCs) to a state capacitated for lineage specification. This transition is
coordinated at multiple levels. Non-coding RNAs may contribute to this regulatory orchestra. We
identified a rodent-specific long non-coding RNA (lncRNA) linc1281, hereafter Ephemeron (Eprn),
that modulates the dynamics of exit from naı¨ve pluripotency. Eprn deletion delays the extinction of
ESC identity, an effect associated with perduring Nanog expression. In the absence of Eprn, Lin28a
expression is reduced which results in persistence of let-7 microRNAs, and the up-regulation of de
novo methyltransferases Dnmt3a/b is delayed. Dnmt3a/b deletion retards ES cell transition,
correlating with delayed Nanog promoter methylation and phenocopying loss of Eprn or Lin28a. The connection from lncRNA to miRNA and DNA methylation facilitates the acute extinction of
naı¨ve pluripotency, a pre-requisite for rapid progression from preimplantation epiblast to
gastrulation in rodents. Eprn illustrates how lncRNAs may introduce species-specific network
modulations. Present address:
†Wellcome
Trust- Medical Research Council
Stem Cell Institute, University of
Cambridge, Cambridge, United
Kingdom; ‡Bioinformatics Group,
School of Pharmacy &
Biomedical Science, University of
Portsmouth, Portsmouth, United
Kingdom Competing interests: The
authors declare that no
competing interests exist. Introduction Mouse embryonic stem cells (ESCs), in vitro counterparts of the pre-implantation epiblast, exhibit
dual properties of self-renewal and differentiation (Boroviak et al., 2015; Bradley et al., 1984;
Evans and Kaufman, 1981; Martin, 1981). These properties make them an attractive system for
investigating cell fate decision making. In the embryo, spatially and temporally coordinated signals
direct
the
rapid
and
continuous
transition
of
the
epiblast
towards
lineage
specification
(Acampora et al., 2016; Smith, 2017). In contrast, ESCs can be suspended in a ground state of plu-
ripotency, where self-renewal is decoupled from lineage specification, using two inhibitors (2i) of gly-
cogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase kinase (MEK1/2), along with
the cytokine leukaemia inhibitory factor (LIF) (Ying et al., 2008). Therefore, ESCs provides a unique Copyright Li et al. This article
is distributed under the terms of
the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited. Copyright Li et al. This article
is distributed under the terms of
the Creative Commons the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited. 1 of 24 Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 Research article Research article Developmental Biology and Stem Cells Developmental Biology and Stem Cells experimental system to explore the principles and molecular players underlying the developmental
progression of pluripotency (Kalkan and Smith, 2014). While it is increasingly clear that the ESC state is maintained by a core network of transcription
factors (Chen et al., 2008; Dunn et al., 2014; Ivanova et al., 2006), less is known about how cells
progress from this state to lineage specification (Buecker et al., 2014; Kalkan and Smith, 2014;
Smith, 2017). Loss-of-function screens have highlighted a multi-layered machinery that dismantles
the naı¨ve state transcription factor network (Betschinger et al., 2013; Leeb et al., 2014). The
latency period for transition depends on the clearance kinetics of network components (Dunn et al.,
2014). The orchestration of multiple regulators thus ensures rapid and complete dissolution of this
core network and consequent timely extinction of ESC identity upon 2i withdrawal (Kalkan and
Smith, 2014). In addition to protein coding genes, accumulating evidence suggests that non-coding RNAs can
contribute to the regulation of cell fate transitions. Introduction Within this class, long non-coding RNAs
(lncRNAs) comprise a large fraction of the transcriptome in diverse cell types and exhibit specific
spatio-temporal expression (Carninci et al., 2005; Guttman et al., 2009; Necsulea et al., 2014). The genomic distribution of lncRNAs is non-random (Luo et al., 2016). A subclass of lncRNAs are
divergently transcribed from neighbouring genes and thought to regulate proximal gene expression
in cis, either due to the process of transcription (Ebisuya et al., 2008; Engreitz et al., 2016;
Martens et al., 2004) or through local lncRNA-protein interactions that recruit regulatory complexes
(Lai et al., 2013; Lee, 2012; Luo et al., 2016; Nagano et al., 2008). However, the functions and
mode of action of the vast majority of lncRNAs remain unknown and require case-by-case experi-
mental determination. In mouse ESCs, knockdowns of a number of lncRNAs have been reported to
exert effects on the transcriptome (Bergmann et al., 2015; Dinger et al., 2008; Guttman et al.,
2011; Lin et al., 2014; Sheik Mohamed et al., 2010) and in some cases impair self-renewal
(Lin et al., 2014; Luo et al., 2016; Savic´ et al., 2014). We investigated the potential involvement of lncRNAs in transition from the naı¨ve ESC state and
identified a dynamically regulated lncRNA (linc1281) that we named Ephemeron (Eprn). We present
functional evaluation of Eprn and delineation of a downstream genetic interaction network, which is
an additional component of the regulatory machinery driving the irreversible and rapid progression
from naı¨ve pluripotency in rodent. Research article Identification of lncRNAs associated with transition from naı¨ve
pluripotency p
p
y
Post-implantation epiblast derived stem cells (EpiSCs) represent a primed state of pluripotency
developmentally downstream of naı¨ve state ESCs (Brons et al., 2007; Nichols and Smith, 2009;
Tesar et al., 2007). To identify lncRNA candidates with a possible role in ESC transition, we analysed
in silico the effect of genetic perturbation on expression of ESC and EpiSC states based on pub-
lished data. We first selected genes that are over ten-fold differentially enriched in ESCs (182 genes)
and EpiSCs (131 genes) relative to each other as molecular signatures to represent these two states
(Tesar et al., 2007). Using published data, we investigated the impact on these two signature sets
when individual lncRNAs (147 in total) and known protein coding regulators (40 in total) were
knocked down in ESCs grown in LIF/serum (Guttman et al., 2011) (Figure 1A, Figure 1—source
data 1). Serum culture supports a heterogeneous mixture of naı¨ve, primed and intermediate cells
(Chambers et al., 2007; Kolodziejczyk et al., 2015; Marks et al., 2012). Therefore, analysis in this
condition could potentially reveal regulators of the ESC and EpiSC states. The effect of each gene
knockdown was plotted based on the percentage of genes significantly altered within ESC and
EpiSC signature sets (FDR < 0.05 and fold change >2 or<0.5 over negative control defined by the
original study). We validated the approach by analysing the knockdown effects of known ESC self-
renewal regulators. As predicted, depletion of factors that maintain the ESC state, such as Stat3,
Esrrb, Sox2 and Klf4, led to a decrease in ESC and increase in EpiSC signature (Figure 1A), while
knockdown of Oct4 gave rise to a decrease in both ESC and EpiSC signatures, consistent with its
requirement in both states (Niwa et al., 2000; Osorno et al., 2012). With this system, we identified Li et al. eLife 2017;6:e23468. Identification of lncRNAs associated with transition from naı¨ve
pluripotency DOI: 10.7554/eLife.23468 2 of 24 Research article Developmental Biology and Stem Cells A
-35
-15
0
15
35
0
30
20
10
-25
25
-30
-20
-10
-5
5
% mESC signature genes (n=182)
% upregulated
% downregulated
% EpiSC signature genes (n=131)
% upregulated
% downregulated
SiStat3
SiEsrrb
SiKlf4
SiSox2
SiOct3/4
SiEphemeron
2i/LIF withdrawal
10
1
0.1
0h
12h
24.5h
36h
48h
72h
ZĞůĂƟǀĞĞdžƉƌĞƐƐŝŽŶ
B
C
ZĞůĂƟǀĞĞdžƉƌĞƐƐŝŽŶ
0
1
4
2
3
2iL
N2B27
LIF
PD
CH
LIF+PD
LIF+CH
CH+PD
E
0h (2iL)
24h AFK
5uM
5uM
24h
D
48h
72h
5uM
5uM
Nanog
Ephemeron
2kb
1kb
2iL
24h
EpiSC
MEF
2iL
24h
EpiSC
MEF
ĐƟŶ
2iL
24h
EpiSC
MEF
*
2kb
4kb
1kb
0.5kb
2kb
4kb
1kb
0.5kb
0
10
20
30
(82)
(119)
(55)
(80)
0h
24h
48h
72h
No. of cells:
ǀĞƌĂŐĞ,LJďƌŝĚŝƐĂƟŽŶ
Signal per cell
40
F
PD/LIF withdrawal
0
0.4
0.6
0.8
1.0
72h
48h
24h
8h
0h
ZĞůĂƟǀĞĞdžƉƌĞƐƐŝŽŶ
Figure 1. Dynamic expression of lncRNA Ephemeron during exit from naı¨ve pluripotency. (A) Bioinformatic analysis of potential lncRNA candidates in
naı¨ve state regulation based on published transcriptome data for lncRNA and pluripotency related gene knockdowns. Each dot represents the effect on
ESC (x-axis) and EpiSC (y-axis) gene signatures when a given gene is knocked down. (B) RT-qPCR detection of Eprn expression relative to b-actin upon
2i/LIF withdrawal. Mean ±SD, n = 3. (C) Northern blotting of Eprn, Nanog and b-actin in ESCs in 2i/LIF or withdrawn from 2i/LIF for 24 hr, EpiSCs and
MEF. * indicates a cross-hybridising RNA species since part of the probe region overlaps with LINE1-L1 and ERVK TEs. (D) RNA-FISH for Eprn upon 2i/
LIF withdrawal with quantification of average hybridisation signals per cell. Mean value of total hybridisation signals for all cells ± SD, n = 2. (E) Eprn
expression relative to b-actin upon 2i/LIF component withdrawal quantified by RT-qPCR. Cells cultured in 2i/LIF and were transferred to N2B27
containing indicated single or dual factors for 24 hr. Mean ±SD, n = 3. F, Eprn expression relative to b-actin upon PD/LIF withdrawal quantified by RT-
qPCR. Mean ± SD, n = 3. Identification of lncRNAs associated with transition from naı¨ve
pluripotency DOI: 10 7554/eLife 23468 002 C
Nanog
Ephemeron
2kb
1kb
2iL
24h
EpiSC
MEF
2iL
24h
EpiSC
MEF
ĐƟŶ
2iL
24h
EpiSC
MEF
*
2kb
4kb
1kb
0.5kb
2kb
4kb
1kb
0.5kb A
-35
-15
0
15
35
0
30
20
10
-25
25
-30
-20
-10
-5
5
% mESC signature genes (n=182)
% upregulated
% downregulated
% EpiSC signature genes (n=131)
% upregulated
% downregulated
SiStat3
SiEsrrb
SiKlf4
SiSox2
SiOct3/4
SiEphemeron A 2i/LIF withdrawal
10
1
0 1
0h
12h
24.5h
36h
48h
72h
ZĞůĂƟǀĞĞdžƉƌĞƐƐŝŽŶ
B A B C 0
10
20
30
(82)
(119)
(55)
(80)
0h
24h
48h
72h
No. of cells:
ǀĞƌĂŐĞ,LJďƌŝĚŝƐĂƟŽŶ
Signal per cell
40 ZĞůĂƟǀĞĞdžƉƌĞƐƐŝŽŶ
0
1
4
2
3
2iL
N2B27
LIF
PD
CH
LIF+PD
LIF+CH
CH+PD
E E D 0h (2iL)
24h AFK
5uM
5uM
24h
D
48h
72h
5uM
5uM F
PD/LIF withdrawal
0
0.4
0.6
0.8
1.0
72h
48h
24h
8h
0h
ZĞůĂƟǀĞĞdžƉƌĞƐƐŝŽŶ F Figure 1. Dynamic expression of lncRNA Ephemeron during exit from naı¨ve pluripotency. (A) Bioinformatic analysis of potential lncRNA candidates in
naı¨ve state regulation based on published transcriptome data for lncRNA and pluripotency related gene knockdowns. Each dot represents the effect on
ESC (x-axis) and EpiSC (y-axis) gene signatures when a given gene is knocked down. (B) RT-qPCR detection of Eprn expression relative to b-actin upon
2i/LIF withdrawal. Mean ±SD, n = 3. (C) Northern blotting of Eprn, Nanog and b-actin in ESCs in 2i/LIF or withdrawn from 2i/LIF for 24 hr, EpiSCs and
MEF. * indicates a cross-hybridising RNA species since part of the probe region overlaps with LINE1-L1 and ERVK TEs. (D) RNA-FISH for Eprn upon 2i/
LIF withdrawal with quantification of average hybridisation signals per cell. Mean value of total hybridisation signals for all cells ± SD, n = 2. (E) Eprn
expression relative to b-actin upon 2i/LIF component withdrawal quantified by RT-qPCR. Cells cultured in 2i/LIF and were transferred to N2B27
containing indicated single or dual factors for 24 hr. Mean ±SD, n = 3. F, Eprn expression relative to b-actin upon PD/LIF withdrawal quantified by RT-
qPCR. Mean ± SD, n = 3. /
f /
The following source data and figure supplements are available for figure 1: The following source data and figure supplements are available for figure 1: Source data 1. Bioinformatics analysis of all lncRNAs and protein coding genes plotted in Figure 1A. Identification of lncRNAs associated with transition from naı¨ve
pluripotency DOI: 10 7554/eLife 23468 003 Source data 1. Bioinformatics analysis of all lncRNAs and protein coding genes plotted in Figure 1A. DOI: 10.7554/eLife.23468.003 Source data 2. Expression of potential lncRNA candidates in facilitating naı¨ve state exit. DOI: 10.7554/eLife.23468.004 Figure supplement 1. Molecular characterisation of Ephemeron. DOI: 10.7554/eLife.23468.005 Figure supplement 2. Eprn expression and promoter methylation. DOI: 10.7554/eLife.23468.006 3 of 24 Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 Research article Research article Developmental Biology and Stem Cells lncRNAs that increased ESC and decreased EpiSC signatures when knocked down, suggestive of a
possible role in transition from the ESC state (Figure 1A bottom right quadrant). We examined expression profiles of these candidate lncRNAs during exit from self-renewal in
defined conditions, exploiting the Rex1::GFP (RGd2) reporter ESC cell line (Kalkan et al., 2017;
Wray et al., 2011) (Figure 1—source data 2). Upon 24 hr of 2i withdrawal, Rex1 expression status
can discriminate subpopulations of cells with distinct functional properties, with Rex1-GFP high cells
corresponding to undifferentiated ESCs and loss of GFP marking extinction of ESC identity
(Kalkan et al., 2017). Amongst the 16 candidates analysed, linc1281 (Refseq entry D630045M09Rik)
(Figure 1—figure supplement 1A) was the third highest expressed lncRNA across all time points. Notably this lncRNA showed a distinctive profile during the first 24 hr, with differential expression
observed between Rex1-GFP high and low cells (Figure 1B, Figure 1—figure supplement 1B). Due
to its dynamic and transient expression profile, we designated linc1281 as Ephemeron (Eprn). Ribo-
somal profiling analysis indicated that Eprn is indeed a non-coding RNA, with the longest predicted
open reading frame (80 amino acids) possessing a ribosome release score typical of a non-coding
sequence (Guttman et al., 2013). Eprn is located in a region of high transposable element (TE) con-
tent, with its exons comprised of 76.4% annotated TE sequences (including ERV-K, LINE L1, and
SINE B2 elements, Figure 1—figure supplement 1A). This genomic region exhibits minimal
sequence conservation in mammals (Figure 1—figure supplement 1A) and we failed to identify any
human homologue either within the syntenic region or elsewhere in the human genome. However, a
positionally conserved spliced transcript (CA504619) that shares 79% sequence identity to exon 3 of
mouse Eprn is present within the rat syntenic region (Figure 1—figure supplement 1C). Therefore,
it is likely that Eprn is conserved in rodents over 30 million years since the mouse-rat lineage
divergence. Identification of lncRNAs associated with transition from naı¨ve
pluripotency We conducted RT-qPCR, Northern blotting and RNA-FISH to evaluate expression, transcription
variants and subcellular localisation of Eprn in ESCs. Eprn showed strong induction within 12 hr of
2i/LIF withdrawal, but decreased subsequently (Figure 1B–D). In EpiSCs or mouse embryonic fibro-
blasts (MEFs), Eprn expression was below the detection limit (Figure 1C). Consistent with the UCSC
gene annotation, Northern blotting of total ESC RNA confirmed the expression of a single Eprn tran-
script over 1 kb in length (Figure 1C, Figure 1—figure supplement 1D). Transcription start and end
sites of Eprn mapped by 5’ and 3’ RACE were in agreement with the annotation (Figure 1—figure
supplement 1E,F). After 24 hr of 2i/LIF withdrawal, Eprn RNA-FISH hybridisation signals displayed
predominantly cytoplasmic localisation, but from 48 hr onwards the remaining signals were mostly in
the nucleus (Figure 1D). To explore the regulation of Eprn, two inhibitors and LIF were withdrawn singly or dually for 24
hr. In conditions lacking Gsk3 inhibitor CHIRON99021 (CH), Eprn was upregulated (Figure 1E). When transferred to non-supplemented N2B27 medium from PD/LIF, Eprn expression was main-
tained for 24 hr before declining (Figure 1F). The addition of CH to LIF/serum culture reduced Eprn
expression within 24 hr irrespective of the presence of MEK inhibitor PD0325901 (PD) (Figure 1—
figure supplement 1G,H). Therefore, Eprn is suppressed by CH in self-renewing ESCs. Through analysis of published data, we found that during early mouse development, Eprn expres-
sion peaked at E4.5 and was present in both epiblast and primitive endoderm of the mature blasto-
cyst, but absent or low in E5.5 post-implantation epiblast (Figure 1—figure supplement 2A) and
later stages between E7 and E17 (Figure 1—figure supplement 2B). Amongst adult tissues ana-
lysed, Eprn was only detected in kidney, and at a much lower level than in ESCs. We also observed
that Eprn expression is restored upon naı¨ve state resetting from EpiSCs (Guo et al., 2009;
Yang et al., 2010) (Figure 1—figure supplement 2C,D). We conclude that Eprn expression is highly
specific to ESCs and the early mouse embryo. LINE and ERVL-MaLR elements are present within the Eprn proximal promoter region (2 kb
upstream of TSS) (Figure 1—figure supplement 1A). Such repetitive elements gain DNA CpG meth-
ylation dramatically during pre- to post-implantation transition (Smith et al., 2014). Research article Identification of lncRNAs associated with transition from naı¨ve
pluripotency By examining
published data from embryos (Seisenberger et al., 2012; Wang et al., 2014) and ESC progression
in vitro (Kalkan et al., 2017), we found that CpG methylation gain at the Eprn promoter was more
extensive in the primed E6.5 epiblast (3% to 80%) than the average changes across all promoters
(9% to 35%) or the genome (24% to 70%) (Figure 1—figure supplement 2E). In contrast, no major
CpG methylation gain at Eprn promoter was present 24 hr post 2i withdrawal. These data suggest Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 4 of 24 Research article Research article Developmental Biology and Stem Cells Research article that promoter methylation does not initiate Eprn repression, but could contribute to maintain silenc-
ing in later epiblast. Molecular consequences of Ephemeron loss We performed RNA-sequencing and compared the transcriptome of wild type and Eprn KO ESCs
using three independently targeted KO ESC lines and three subclones of the parental wild type
ESCs. Sixteen genes were differentially expressed between wild type and Eprn KO cells both in PD/
LIF and after 8 hr withdrawal (Benjamini-Hochberg adjusted p<0.05, fold change >1.5 or<0.7) (Fig-
ure 2—figure supplement 2D) (Figure 2—figure supplement 2E). These include Tcf15, which has
been associated with transition from the naı¨ve state and has an inverse expression pattern compared
to naı¨ve pluripotency factors (Davies et al., 2013). Lin28a was the most differentially expressed
gene in the group, with Eprn KO cells displaying a twofold reduction in mean expression level
(Figure 2E). Although Lin28a is commonly considered as a core pluripotency factor, its expression is
actually increased when cells transition out of the naı¨ve state in vivo and in vitro (Boroviak et al.,
2015; Kalkan et al., 2017; Kumar et al., 2014; Marks et al., 2012). Attenuated downregulation of
members of the naı¨ve transcription factor network is one explanation for delayed exit from the ESC
state (Kalkan and Smith, 2014). We hypothesised that Lin28a could be a negative regulator of the
network. We examined expression of naı¨ve pluripotency transcription factors in Eprn KO cells and
found a higher level of Nanog mRNA (Figure 2E, Figure 2—figure supplement 2F). To characterise
the profile of naı¨ve pluripotency dissolution further in Eprn KO cells, a PD/LIF withdrawal time course
was monitored over 24 hr. The two-fold reduction in Lin28a mRNA in Eprn KO cells was constant
throughout this time course (Figure 2F). Conversely, Nanog transcript and protein levels remained
higher at 16 hr and 24 hr respectively (Figure 2F, see also 3E,F for protein). Mean Klf2 transcript lev-
els appeared higher in Eprn KO cells, but below statistical significance. Other members of the naı¨ve
network showed similar expression profiles in wild type and Eprn KO cells (Figure 2—figure supple-
ment 2F). Among peri-implantation epiblast markers, upregulation kinetics for Fgf5 were unchanged
in Eprn KO cells, but transcripts for Dnmt3a, Dnmt3b and Oct6 remained lower from 16 to 24 hr
(Figure 2—figure supplement 2F). Although not statistically significant, Otx2 transcripts appeared
modestly reduced throughout the time course, which could be related to the elevated expression of
Nanog (Acampora et al., 2016). Loss of Ephemeron delays exit from naı¨ve pluripotency p
y
p
p
y
Initiation of ESC differentiation in defined media upon withdrawal of self-renewal factors recapitu-
lates features of peri-implantation epiblast development (Kalkan et al., 2017). The latency of naı¨ve
state exit varies, however, according to the starting self-renewal condition (Dunn et al., 2014;
Wray et al., 2011). Higher activity of the core network in PD/LIF compared with 2i results in slower
network dissolution, reflected in later onset of RGd2 downregulation (Dunn et al., 2014). These two
conditions feature different levels of Eprn due to CH mediated suppression in 2i (Figure 1E). We
generated Eprn knockout (KO) ESCs via sequential gene targeting (Figure 2—figure supplement 1)
and examined the phenotype in each condition. In steady state self-renewal, Eprn loss did not affect
the Rex1-GFP profile in either case (Figure 2A,B). Upon transfer to N2B27, Eprn KO cells displayed
delayed downregulation of GFP compared to parental cells, measured at 24 hr from 2i culture and
40 hr from PD/LIF culture (Figure 2B). By 72 hr, however, GFP expression was fully extinguished
from either starting condition (Figure 2—figure supplement 2A). A transient delay in GFP downre-
gulation in both culture conditions was also evident upon Eprn knockdown using siRNAs (Figure 2—
figure supplement 2B). To assess the effect of Eprn depletion functionally, we conducted colony
forming assays. Cells maintained in PD/LIF were subjected to 40 hr culture in N2B27 and then
replated at clonal density in 2i/LIF to assay the persistence of ES self-renewal potential
(Betschinger et al., 2013). Eprn KO and knockdown cells both gave rise to substantially more undif-
ferentiated colonies than wild type controls (Figure 2C,D, Figure 2—figure supplement 2C). Con-
sidered together, these results indicate that Eprn deficiency impairs timely exit from naı¨ve
pluripotency. Molecular consequences of Ephemeron loss We restored Eprn expression in KO cells by inserting the Eprn genomic region under control of
the human EF1a promoter into the deleted locus (Figure 2—figure supplement 3A,B). The rescue
cells displayed a wild type exit profile as measured by GFP profile 40 hr post PD/LIF withdrawal Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 5 of 24 &ŝŐƵƌĞ Ϯ
Research article &ŝŐƵƌĞ Ϯ
Research article Developmental Biology and Stem Cells PDL
ZĞůĂƟǀĞ džƉƌĞƐƐŝŽŶ
0
0.5
1
1.5
2
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8h
16h
20h
24h
Nanog
ϮŝƚŽEϮϮϳϮϰŚ
Wͬ>ƚŽEϮϮϳϰϬŚ
0
2
4
6
8
10
12
KO1
KO2
WT1
WT2
Clonogenicity (%)
KO1
KO2
WT1
WT2
PD/LIF
EϮϮϳϰϬŚ
>ŽǁĚĞŶƐŝƚLJ
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EϮϮϳϮϰŚ &ůŽǁLJƚŽŵĞƚƌLJ
&ŝŐƵƌĞϮ
Ϯŝ
PD/L
GFP
GFP
0
Lin28a
Nanog
PDL
W>ƚŽEϮϮϳϴŚ
KO1
KO2
KO3
WT1
WT2
WT3
KO1
KO2
KO3
WT1
WT2
WT3
PDL
W>ƚŽEϮϮϳϴŚ
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0
1
2
3
4
5
6
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PDL
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0.5
1
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2
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3
ZĞůĂƟǀĞ džƉƌĞƐƐŝŽŶ
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KO
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F
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WT2=65%
KO1=80%
<KϮсϳϲй
WT1=93%
WT2=96%
KO1=95%
KO2=96%
WT1=21%
WT2=18%
KO1=34%
KO2=44%
WT1=3%
WT2=2%
KO1=24%
<KϮсϭϳй
WT=8%
<KZĞƐсϴй
KO=38%
Figure 2. Absence of Ephemeron delays exit from naı¨ve pluripotency. (A) Experimental scheme for analysing naı¨ve state exit using Rex1GFPd2 reporter
cells. (B) Rex1-GFP flow cytometry profiles of wild type and Eprn KO cells in 2i and PD/LIF and during transition from these starting conditions. Two
independent clones for wild type and Eprn KO cells were analysed. Percentage of GFP high cells were quantified. (C) Experimental scheme for colony
formation assay. (D) Colony formation assay for wild type and Eprn KO cells in 2i/LIF 40 hr post PD/LIF withdrawal. Colonies were stained with alkaline
phosphatase (AP), with representative images shown. Percentage clonogenicity was calculated by the number of AP positive colonies divided by the
total number of cells plated. Mean ± SD, n = 3. (E) Lin28a and Nanog expression relative to b-actin in three independent wild type and Eprn KO cell
lines measured by RT-qPCR. Mean ± SE, n = 3. *p<0.05, **p<0.01, student’s t-test. (F) Nanog and Lin28a expression kinetics upon PD/LIF withdrawal in
wild type and Eprn KO cells. Molecular consequences of Ephemeron loss Mean ± SD, n = 3. (G) Rex1-GFP flow cytometry profile for wild type, Eprn KO and Eprn rescue cells 40 hr post PD/LIF
withdrawal. Percentage of GFP high cells were quantified. DOI: 10.7554/eLife.23468.007
h
f ll
f
l
l bl
f
f 0
2
4
6
8
10
12
KO1
KO2
WT1
WT2
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KO1
KO2
WT1
WT2
PD/LIF
EϮϮϳϰϬŚ
>ŽǁĚĞŶƐŝƚLJ
ƌĞƉůĂƟŶŐ
WĐŽůŽŶLJ
ƐĐŽƌŝŶŐ
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KO2
WT1
WT2
ĚĞŶƐŝƚLJ
ĂƟŶŐ
WĐŽůŽŶLJ
ƐĐŽƌŝŶŐ
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2
4
6
8
10
12
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KO2
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WT2
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PD/LIF
EϮϮϳϰϬŚ
>ŽǁĚ
ƌĞƉů
D PD/LIF
EϮϮϳϰϬŚ
Ϯŝ
EϮϮϳϮϰŚ &ůŽ
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йDĂdž
,ŝŐŚ
WT1=65%
WT2=65%
KO1=80%
<KϮсϳϲй PD/L
GFP
йDĂdž
WT1=93%
WT2=96%
KO1=95%
KO2=96% D KO2 ϮŝƚŽEϮϮϳϮϰŚ
GFP
йDĂdž
WT1=21%
WT2=18%
KO1=34%
KO2=44% Wͬ>ƚŽEϮϮϳϰϬŚ
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йDĂdž
WT1=3%
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KO1=24%
<KϮсϭϳй Lin28a
PDL
W>ƚŽEϮϮϳϴŚ
KO1
KO2
KO3
WT1
WT2
WT3
KO1
KO2
KO3
WT1
WT2
WT3
0
1
2
3
4
5
6
ZĞůĂƟǀĞ džƉƌĞƐƐŝŽŶ
**
** 0
Nanog
PDL
W>ƚŽEϮϮϳϴŚ
KO1
KO2
KO3
WT1
WT2
WT3
KO1
KO2
KO3
WT1
WT2
WT3
0
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1
1.5
2
2.5
3
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** Nanog PDL
ZĞůĂƟǀĞ džƉƌĞƐƐŝŽŶ
0
0.5
1
1.5
2
4h
8h
16h
20h
24h
Nanog
24h
0
Lin28a
PDL
4h
8h
16h
20h
0.5
1
1.5
2
2.5
3
ZĞůĂƟǀĞ džƉƌĞƐƐŝŽŶ
WT
KO
F Wͬ>ƚŽEϮϮϳϰϬŚ
GFP
йDĂdž
G
WT=8%
<KZĞƐсϴй
KO=38% PDL
ZĞůĂƟǀĞ džƉƌĞƐƐŝŽŶ
0
0.5
1
1.5
2
4h
8h
16h
20h
24h
Nanog
F G 24h
0
Lin28a
PDL
4h
8h
16h
20h
0.5
1
1.5
2
2.5
3
ZĞůĂƟǀĞ džƉƌĞƐƐŝŽŶ
WT
KO F Figure 2. Absence of Ephemeron delays exit from naı¨ve pluripotency. (A) Experimental scheme for analysing naı¨ve state exit using Rex1GFPd2 reporter
cells. (B) Rex1-GFP flow cytometry profiles of wild type and Eprn KO cells in 2i and PD/LIF and during transition from these starting conditions. Two
independent clones for wild type and Eprn KO cells were analysed. Percentage of GFP high cells were quantified. (C) Experimental scheme for colony
formation assay. (D) Colony formation assay for wild type and Eprn KO cells in 2i/LIF 40 hr post PD/LIF withdrawal. Colonies were stained with alkaline
phosphatase (AP), with representative images shown. Percentage clonogenicity was calculated by the number of AP positive colonies divided by the
total number of cells plated. Mean ± SD, n = 3. The Ephemeron genetic network includes Lin28a and Nanog Based on the preceding data, we hypothesised that Lin28a could be a downstream effector of Eprn,
acting to reduce expression of Nanog. To characterise further the relationship between Eprn, Lin28a
and the naı¨ve transcription factor network, we carried out a series of genetic perturbation experi-
ments and measured both Rex1-GFP reporter dynamics and colony formation upon withdrawal from
PD/LIF. In Eprn KO cells, Nanog knockdown partially restored downregulation of Rex1-GFP 40 hr
after PD/LIF withdrawal, and colony formation was reduced to the low level observed in wild type
cells subjected to Nanog siRNA (Figure 3A). Knockdown of Klf4 had no effect on exit kinetics from
PD/LIF in either wild type or Eprn KO cells. Knockdowns of other naı¨ve transcription factors, Esrrb,
Tfcp2l1 and Klf2, accelerated exit in wild type cells but in contrast to Nanog depletion this pheno-
type was attenuated in Eprn KO cells (Figure 3—figure supplement 1B). Resistance of Eprn KO
cells to accelerated transition upon Esrrb, Tfcp2l1 and Klf2 knockdown could be attributed to ele-
vated Nanog. We therefore conducted dual knockdown experiments (Figure 3—figure supplement
1C). Simultaneous depletion of Esrrb, Tfcp2l1 or Klf2 together with Nanog largely abolished the
effect of Eprn KO on GFP downregulation (Figure 3—figure supplement 1C,D). These data are con-
sistent with Eprn acting, at least in part, via modulation of Nanog expression. We investigated whether lowered expression of Lin28a contributes to the slower exit from naı¨ve
pluripotency and the increased Nanog expression. We manipulated Lin28a dosage by either overex-
pression or knockdown in Eprn KO cells. In wild type cells, Lin28a overexpression had no significant
effect. In Eprn KO cells, however, it restored normal transition kinetics (Figure 3B). Conversely,
Lin28a knockdown phenocopied Eprn loss, delaying exit from naı¨ve pluripotency (Figure 3C). Con-
comitant knockdown of Nanog and Lin28a abolished this effect (Figure 3C). Lin28a knockdown cells
exhibited marginally elevated Nanog mRNA in PD/LIF and persistence at higher levels after 8 hr of
PD/LIF withdrawal (Figure 3D). At the protein level, Eprn null cultures displayed more cells with high
Nanog and low Lin28a expression at the 24 hr time point as quantified by co-immunostaining
(Figure 3E,F, Figure 3—figure supplement 1E). Interestingly, Lin28a was detected as concentrated
foci in the nucleus and also in the cytoplasm (observed with two independent antibodies), and both
nuclear and cytoplasmic expression were increased after PD/LIF withdrawal (Figure 3—figure sup-
plement 1F). Molecular consequences of Ephemeron loss (E) Lin28a and Nanog expression relative to b-actin in three independent wild type and Eprn KO cell
lines measured by RT-qPCR. Mean ± SE, n = 3. *p<0.05, **p<0.01, student’s t-test. (F) Nanog and Lin28a expression kinetics upon PD/LIF withdrawal in
wild type and Eprn KO cells. Mean ± SD, n = 3. (G) Rex1-GFP flow cytometry profile for wild type, Eprn KO and Eprn rescue cells 40 hr post PD/LIF
withdrawal. Percentage of GFP high cells were quantified. DOI 10 7554/ Lif
23468 007 The following figure supplements are available for figure 2: The following figure supplements are available for figure 2: Figure supplement 1. Generation of Eprn KO ESCs. DOI: 10.7554/eLife.23468.008 Figure supplement 2. Phenotypic and molecular characterisation of Eprn KO during naı¨ve state exit. DOI: 10.7554/eLife.23468.009 Figure supplement 3. Generation of Ephemeron KO rescue ESCs. Figure 2 continued on next page Figure supplement 3. Generation of Ephemeron KO rescue ESCs. 6 of 24 6 of 24 Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 Developmental Biology and Stem Cells Developmental Biology and Stem Cells Figure 2 continued Figure 2 continued
DOI: 10.7554/eLife.23468.010
Figure supplement 4. Differentiation capacity of Eprn KO ESCs. DOI: 10.7554/eLife.23468.011 (Figure 2G). Lin28a and Nanog expression levels were similar to wild type cells (Figure 2—figure
supplement 3C). To explore the differentiation capacity of Eprn KO cells, we conducted in vitro differentiation
assays directing ESCs towards EpiSCs and somatic lineages (Figure 2—figure supplement 4). Both
wild type and Eprn KO ESCs could be differentiated into EpiSCs using N2B27 supplemented with
ActivinA/Fgf2/XAV939 (Sumi et al., 2013) on fibronectin. Such in vitro differentiated EpiSCs could
be stably propagated over multiple passages, and displayed typical morphology and gene expres-
sion irrespective of genotype (Figure 2—figure supplement 4A,B). We also applied neuronal, mes-
endoderm and definitive endoderm differentiation protocols to Eprn KO ESCs and found that
lineage markers were induced, with a slight delay for mesendoderm (Figure 2—figure supplement
4C–E). Thus retarded naı¨ve state exit does not notably impair subsequent lineage commitment
capacity. Developmental Biology and Stem Cells The Ephemeron genetic network includes Lin28a and Nanog During early embryo development, expression of Lin28a and Eprn are positively corre-
lated, while Lin28a and Nanog are negatively correlated (Figure 3—figure supplement 1G)
(Ohnishi et al., 2014). These data are consistent with the proposition that Lin28a is genetically
downstream of Eprn and may facilitate exit from naı¨ve pluripotency by accelerating downregulation
of Nanog. To assess whether Eprn could regulate Lin28a or Nanog expression directly, we employed chro-
matin isolation by RNA purification (ChIRP) (Chu et al., 2011). Using this method, we were able to
selectively pull down endogenous Eprn RNA (Figure 3—figure supplement 2A). However, we did Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 7 of 24 Research article Research article Developmental Biology and Stem Cells 0
5
10
15
20
25
Clonogenicity (%)
siNeg
siLin28a+siNeg
siLin28a+siNanog
siNanog+siNeg
0
5
10
15
20
25
30
Clonogenicity (%)
WT Vector
WT Lin28a
KO Vector
KO Lin28a
WT
KO
DAPI
Nanog
Lin28a
0
2
4
6
8
10
12
14
16
Clonogenicity (%)
WT siNeg
KO siNeg
WT siNanog
KO siNanog
GFP
% of Max
% of Max
% of Max
GFP
GFP
Total Lin28a Intensity
Total Nanog Intensity
N2B27 24h
WT (n=349)
KO (n=352)
C
E
F
PD/LIF
8h
24h
A
B
siNeg
siLin28a
siNeg
siNanog
siNeg
siNanog
siNeg
siNanog
siNanog
siNeg
WT
Eprn KO
siNeg
siLin28a
D
Lin28a
Nanog
0
0.4
0.8
1.2
1.6
0
2
4
6
*
WT
Eprn KO
SiNeg=11%
SiLin28a+SiNeg=31%
SiNanog+siNeg=11%
SiLin28a+SiNanog=15%
WT SiNeg=7%
WT SiNanog=7%
KO SiNeg=21%
KO SiNanog=9%
WT Vector=30%
WT Lin28a=30%
KO Vector=37%
KO Lin28a=27%
WT=3%
KO=17%
WT=33%
KO=2%
PD/LIF
8h
24h
WT Vector
WT Lin28a
KO Vector
KO Lin28a
Figure 3. Lin28a is downstream of Ephemeron and regulates Nanog expression. (A) Rex1-GFP flow cytometry profiles (Left) and colony formation
capacity (Right) 40 hr post PD/LIF withdrawal for wild type and Eprn KO cells transfected with indicated siRNAs. (B) Rex1-GFP flow cytometry profiles
and colony formation capacity 40 hr post PD/LIF withdrawal for wild type and Eprn KO cells transfected with Lin28a expression vector. (C) Rex1-GFP
flow cytometry profile and colony formation capacity 40 hr post PD/LIF withdrawal with Nanog and Lin28a single or dual knockdowns in wild type cells. Quantification of percentage of GFP high cells were shown in (A-C). Percentage clonogenicity in (A-C) is measured by the number of AP positive
colonies divided by the total number of cells plated, with representative AP staining images shown. The Ephemeron genetic network includes Lin28a and Nanog Mean ± SD, n = 3. (D) Lin28a and Nanog
expression relative to b-actin upon PD/LIF withdrawal in Lin28a knockdown and control cells. Mean ± SD, n = 3. *p<0.05, Student’s t-test. (E)
Correlation of Nanog and Lin28a protein expression immunostaining in wild type and Eprn KO cells 24 hr post PD/LIF withdrawal. (F) Representative
images of cells co-immunostained with Nanog and Lin28a and quantified in E. DOI: 10.7554/eLife.23468.012
Th
f ll
i
fi
l
t
il bl
f
fi
3 PD/LIF
8h
24h
siNeg
siLin28a
D
Lin28a
Nanog
0
0.4
0.8
1.2
1.6
0
2
4
6
*
PD/LIF
8h
24h GFP
% of Max
A
B
WT SiNeg=7%
WT SiNanog=7%
KO SiNeg=21%
KO SiNanog=9% A D Total Lin28a Intensity
Total Nanog Intensity
N2B27 24h
WT (n=349)
KO (n=352)
E
WT=3%
KO=17%
WT=33%
KO=2% E E B GFP
% of Max
GFP
C
B
WT Vector=30%
WT Lin28a=30%
KO Vector=37%
KO Lin28a=27% 0
5
10
15
20
25
30
Clonogenicity (%)
GFP
% of Max
GFP
C
B
WT
Eprn KO
WT Vector=30%
WT Lin28a=30%
KO Vector=37%
KO Lin28a=27%
WT Vector
WT Lin28a
KO Vector
KO Lin28a WT
KO
DAPI
F F C % of Max
GFP
GFP
C
SiNeg=11%
SiLin28a+SiNeg=31%
SiNanog+siNeg=11%
SiLin28a+SiNanog=15% 0
5
10
15
20
25
Clonogenicity (%)
% of Max
GFP
C
siNeg
siNanog
siNanog
siNeg
siNeg
siLin28a
SiNeg=11%
SiLin28a+SiNeg=31%
SiNanog+siNeg=11%
SiLin28a+SiNanog=15% Figure 3. Lin28a is downstream of Ephemeron and regulates Nanog expression. (A) Rex1-GFP flow cytometry profiles (Left) and colony formation
capacity (Right) 40 hr post PD/LIF withdrawal for wild type and Eprn KO cells transfected with indicated siRNAs. (B) Rex1-GFP flow cytometry profiles
and colony formation capacity 40 hr post PD/LIF withdrawal for wild type and Eprn KO cells transfected with Lin28a expression vector. (C) Rex1-GFP
flow cytometry profile and colony formation capacity 40 hr post PD/LIF withdrawal with Nanog and Lin28a single or dual knockdowns in wild type cells. Quantification of percentage of GFP high cells were shown in (A-C). Percentage clonogenicity in (A-C) is measured by the number of AP positive
colonies divided by the total number of cells plated, with representative AP staining images shown. Mean ± SD, n = 3. (D) Lin28a and Nanog
expression relative to b-actin upon PD/LIF withdrawal in Lin28a knockdown and control cells. Mean ± SD, n = 3. *p<0.05, Student’s t-test. The function of Lin28a in ESC transition may be mediated by
suppression of let-7g Lin28a is an RNA binding protein with a well-established function in suppressing maturation of let-7
family miRNAs (Cho et al., 2012; Viswanathan et al., 2008). We investigated whether the role of
Lin28a in naı¨ve state exit is let-7 dependent. We profiled mature miRNA expression of let-7 family
members using RT-qPCR. Expression of let-7a, let-7d, let-7e, let-7g and let-7i decreased 24 hr after
2i/LIF withdrawal, coincident with the increase in Lin28a expression (Figure 4A). Mature miRNA let-
7c expression was unaffected, suggesting that let-7c expression is independent of Lin28a. This
observation is in agreement with a recent finding that let-7c-2, the major let-7c isoform expressed in
mouse ESCs, bypasses Lin28a regulation due to lack of a GGAG recognition motif in its loop region
(Triboulet et al., 2015). The Lin28a regulated let-7 miRNAs, but not let-7c, are expressed at higher
levels in ESCs in 2i/LIF than in LIF/serum (Pandolfini et al., 2016) (Figure 4—figure supplement
1A), consistent with lower Lin28a in 2i/LIF. To examine the role of Lin28a regulated let-7 miRNAs in naı¨ve state exit, we first transfected
ESCs with mature let-7g mimic. We used let-7g as a representative member since all apart from let-
7e share the same seed sequence (Figure 4—figure supplement 1B). Forced expression of let-7g in
RGd2 cells resulted in delayed GFP downregulation upon both 2i and PD/LIF withdrawal
(Figure 4B). Elevated ESC colony formation capacity post PD/LIF withdrawal was also observed
(Figure 4C). To identify downstream targets of let-7g, we curated genes that are upregulated upon
2i withdrawal in our RNA-sequencing dataset and searched for known or predicted let-7g targets
using the RNA22 tool (Miranda et al., 2006). DNA methyltransferases Dnmt3a and Dnmt3b
emerged as prime candidates, as has previously been proposed (Kumar et al., 2014). Expression of
both increases during ESC transition (Kalkan et al., 2017). Dnmt3a/3b transcript levels were lower
in Eprn KO cells than wild type control (Figure 2—figure supplement 2F). Multiple let-7g target
sites were predicted by RNA22 within the Dnmt3a 3’UTR and one site in the Dnmt3b 3’UTR
(Figure 4D, Figure 4—figure supplement 1C). ESCs were co-transfected with mature let-7g mimic
and luciferase constructs containing the entire 3’UTRs of Dnmt3a and Dnmt3b. let-7g reduced lucif-
erase expression by more than 60% relative to scrambled control (Figure 4E), suggesting that
Dnmt3a/3b transcripts are indeed let7-g targets. The Ephemeron genetic network includes Lin28a and Nanog (E)
Correlation of Nanog and Lin28a protein expression immunostaining in wild type and Eprn KO cells 24 hr post PD/LIF withdrawal. (F) Representative
images of cells co-immunostained with Nanog and Lin28a and quantified in E. DOI: 10.7554/eLife.23468.012 The following figure supplements are available for figure 3: Figure supplement 1. Characterisation of Eprn, Nanog and Lin28a genetic interaction. DOI: 10.7554/eLife.23468.013 Figure supplement 2. Eprn does not act on chromatin. DOI 10 7554/ Lif
23468 014 Figure supplement 2. Eprn does not act on chromatin. 8 of 24 8 of 24 Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 Research article Developmental Biology and Stem Cells not detect chromatin enrichment at the Lin28a or Nanog promoter regions (Figure 3—figure sup-
plement 2B–D). Indeed, no significant enrichment genome-wide was observed in wild type com-
pared to Eprn KO cells (Figure 3—figure supplement 2E). Thus we found no evidence that Eprn
functions by chromatin association (Rinn and Guttman, 2014). The H3K4me3 modification was reduced at the Lin28a promoter in Eprn KO cells, in line with
reduced transcription (Figure 3—figure supplement 2F). One explanation for anti-correlated
expression could be direct negative regulation of Lin28a by Nanog. We inspected two published
Nanog
chromatin
immunoprecipitation
(ChIP)
sequencing
datasets
(Chen
et
al.,
2008;
Marson et al., 2008) but observed no localisation of Nanog at the Lin28a locus (Figure 3—figure
supplement 2G). Furthermore, we did not observe Lin28a upregulation in Nanog knockdown cells
(Figure 3—figure supplement 2H). Therefore, Nanog does not appear to be a direct upstream reg-
ulator of Lin28a. The function of Lin28a in ESC transition may be mediated by
suppression of let-7g To test specificity of this repression, we generated
two Dnmt3b 3’UTR luciferase reporter constructs with the let-7 seed recognition site mutated (Fig-
ure 4—figure supplement 1D). These mutant reporters escaped repression by the let-7g
mimic (Figure 4—figure supplement 1D). Dnmt3a and Dnmt3b methylate the Nanog promoter during naı¨ve state
exit Epiblast progression is associated with genome-wide de novo methylation during pre-to post-
implantation development (Auclair et al., 2014). This phenomenon is recapitulated when naı¨ve ESCs
are withdrawn from 2i (Kalkan et al., 2017). Previous studies demonstrated hypomethylation of the
Nanog promoter in mouse ESCs compared to lineage committed cells (Farthing et al., 2008;
Yu et al., 2007). We speculated that impeded de novo DNA methylation could allow perdurance of
Nanog expression at the onset of naı¨ve state exit. To investigate this hypothesis, we carried out
bisulfite sequencing analysis across the Nanog proximal promoter region, 1 kb upstream of the TSS, Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 9 of 24 Research article Developmental Biology and Stem Cells 2i/LIF
N2B27 24h
EpiSCs
let-7a
let-7b
let-7c
let-7d
let-7e
let-7f
let-7g
let-7i
-5
-3
-4
-2
Relative Expression
Log10- CT
A
B
Dnmt3a
TAA
6.7 kb
let-7g
10832389
2945
3575
Dnmt3b
TAA
1.3 kb
102
D
C
E
% of Max
GFP
PD/LIF - N2B27 40h
% of Max
GFP
2i - N2B27 24h
0
Scramble
let-7g
4
8
10
14
0
60
Scramble
Dnmt3a
let-7g
Scramble
let-7g
20
40
80
100
120
% Repression of Luc/RLuc
3’UTR
Dnmt3b
3’UTR
Scramble=40%
let-7g=65%
Scramble=2.6%
let-7g=6%
GFP
2i
PD/LIF
GFP
Scramble=17%
let-7g=17%
Scramble=42%
let-7g=42%
F
PD/LIF
PD/LIF
PD/LIF
PD/LIF
PD/LIF
PD/LIF
24h
24h
24h
24h
24h
24h
Relative Expression
Relative Expression
Relative Expression
Relative Expression
Relative Expression
Relative Expression
Otx2
Lin28a
Dnmt3b
Fgf5
Oct6
Dnmt3a
Scramble
let-7g
% Clonogenicity
% of Max
% of Max
PD/LIF
24h
Relative Expression
let-7g
Scramble
let-7g OE
0.1
1.0
10.0
100.0
PD/LIF
PD/LIF
PD/LIF
PD/LIF
PD/LIF
PD/LIF
24h
24h
24h
24h
24h
24h
Relative Expression
Relative Expression
Relative Expression
Relative Expression
Relative Expression
Relative Expression
Nanog
Klf4
Tbx3
Klf2
Esrrb
Tfcp2l1
0.0
0.4
0.8
1.2
1.6
0.0
0.4
0.8
1.2
1.6
0.0
2.0
4.0
6.0
8.0
0.0
0.4
0.8
1.2
1.6
0.0
0.4
0.8
1.2
1.6
0.0
0.4
0.8
1.2
1.6
0.0
0.4
0.8
1.2
1.6
0.0
0.4
0.8
1.2
1.6
0.0
40.0
80.0
100.0
0.0
0.5
1.0
1.5
2.0
0.0
10.0
20.0
30.0
40.0
2.5
0.0
2.0
4.0
6.0
Figure 4. Lin28a function is mediated via members of let-7 miRNAs. (A) Mature let-7 family microRNA expression quantified by RT-qPCR in 2i/LIF, 24 h
post 2i/LIF withdrawal, and EpiSCs. (B) Rex1-GFP flow cytometry profile upon forced expression of mature let-7g mimic during transition from 2i and
PD/LIF. Dnmt3a and Dnmt3b methylate the Nanog promoter during naı¨ve state
exit DOI: 10.7554/eLife.23468.015
The following figure supplement is available for figure 4: 2i/LIF
N2B27 24h
EpiSCs
let-7a
let-7b
let-7c
let-7d
let-7e
let-7f
let-7g
let-7i
-5
-3
-4
-2
Relative Expression
Log10- CT
A
C B
GFP
2i
Scramble=17%
let-7g=17%
% of Max
% f M B PD/LIF
GFP
Scramble=42%
let-7g=42%
% of Max Dnmt3a
TAA
6.7 kb
let-7g
10832389
2945
3575
Dnmt3b
TAA
1.3 kb
102
D
E
0
60
Scramble
Dnmt3a
let-7g
Scramble
let-7g
20
40
80
100
120
% Repression of Luc/RLuc
3’UTR
Dnmt3b
3’UTR A D E % of Max
GFP
PD/LIF - N2B27 40h
Scramble=40%
let-7g=65%
GFP % f M
% of Max
GFP
2i - N2B27 24h
Scramble=2.6%
let-7g=6% C -5
-3
-4
-2
Log10- CT
C
0
cramble
let-7g
4
8
10
14
Scramble
let-7g
% Clonogenicity PD/LIF
PD/LIF
PD/LIF
24h
24h
24h
Relative Expression
Relative Expression
Relative Expression
Nanog
Klf4
Tbx3
Klf2
Esrrb
Tfcp2l1
0.0
0.4
0.8
1.2
1.6
0.0
0.4
0.8
1.2
1.6
0.0
0.4
0.8
1.2
1.6 PD/LIF
PD/LIF
24h
24h
Relative Expression
Relative Expression
Relative Expression
Otx2
Lin28a
Fgf5
Oct6
0.0
40.0
80.0
100.0
0.0
10.0
20.0
30.0
40.0 S
F
PD/LIF
24h
Relative Expression
let-7g
Scramble
let-7g OE
0.1
1.0
10.0
100.0 PD/LIF
24h
p
Dnmt3b
Dnmt3a
0.0
2.0
4.0
6.0
8.0 F PD/LIF
24h
Relative Expression
Otx2
0.0
0.4
0.8
1.2
1.6 PD/LIF
24h
Dnmt3b
0.0
2.0
4.0
6.0 PD/LIF
24h
Relative Expression
Relative Expression
Lin28a
0.0
0.5
1.0
1.5
2.0
2.5 PD/LIF
PD/LIF
PD/LIF
24h
24h
24h
Relative Expression
Relative Expression
Relative Expression
0.0
0.4
0.8
1.2
1.6
0.0
0.4
0.8
1.2
1.6
0.0
0.4
0.8
1.2
1.6 Figure 4. Lin28a function is mediated via members of let-7 miRNAs. (A) Mature let-7 family microRNA expression quantified by RT-qPCR in 2i/LIF, 24 hr
post 2i/LIF withdrawal, and EpiSCs. (B) Rex1-GFP flow cytometry profile upon forced expression of mature let-7g mimic during transition from 2i and
PD/LIF. Quantification of percentage of GFP high cells was shown. (C) Colony formation assay in 2i/LIF of cells with forced expression of let-7g mimic
and control 40 hr post PD/LIF withdrawal. Colonies were stained with alkaline phosphatase (AP). Percentage clonogenicity was calculated by the
number of AP positive colonies divided by the total number of cells plated. Mean ± SD, n = 3. (D) Predicted target sites of let-7g in 3’UTRs of Dnmt3a
and Dnmt3b by RNA22. Dnmt3a and Dnmt3b methylate the Nanog promoter during naı¨ve state
exit Quantification of percentage of GFP high cells was shown. (C) Colony formation assay in 2i/LIF of cells with forced expression of let-7g mimic
and control 40 hr post PD/LIF withdrawal. Colonies were stained with alkaline phosphatase (AP). Percentage clonogenicity was calculated by the
number of AP positive colonies divided by the total number of cells plated. Mean ± SD, n = 3. (D) Predicted target sites of let-7g in 3’UTRs of Dnmt3a
and Dnmt3b by RNA22. (E) Dual luciferase assay measuring repression by let-7g mediated through 3’UTRs of Dnmt3a and Dnmt3b. Fold repression of
Luc/Rluc relative to scramble was plotted. Mean ± SD, n = 3. (F) Relative expression normalised to b-actin of naı¨ve and peri-implantation epiblast
associated genes in ESCs with forced expression of let-7g mimics. Mean ± SD, n = 3. Dnmt3a and Dnmt3b methylate the Nanog promoter during naı¨ve state
exit (E) Dual luciferase assay measuring repression by let-7g mediated through 3’UTRs of Dnmt3a and Dnmt3b. Fold repression of
Luc/Rluc relative to scramble was plotted. Mean ± SD, n = 3. (F) Relative expression normalised to b-actin of naı¨ve and peri-implantation epiblast
associated genes in ESCs with forced expression of let-7g mimics. Mean ± SD, n = 3. DOI: 10.7554/eLife.23468.015 a members of let-7 miRNAs. (A) Mature let-7 family microRNA expression quantified by RT-qPCR in 2i/LIF, 24 hr The following figure supplement is available for figure 4: Figure supplement 1. let-7 family mature miRNA expression, sequence and predicted let-7g sites in the 3’UTRs of Dnmt3a and Dnmt3b. DOI: 10.7554/eLife.23468.016 10 of 24 Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 Research article Research article Developmental Biology and Stem Cells after siRNA knockdown of Dnmt3a/3b singly or together (Figure 5A). We observed a marked reduc-
tion of CpG methylation in the 1 kb to 761 bp region (region 1) 40 hr after PD/LIF withdrawal
(Figure 5A). Cells transfected with scrambled control siRNA exhibited 40% CpG methylation at
scored sites, whereas Dnmt3b depleted cells displayed 18% CpG methylation and Dnmt3a or
Dnmt3a/b double knockdown cells showed only around 8%. Effects were less obvious in the 538
bp to +18 bp region (region 2), which was barely methylated at this time point. These data suggest
that Dnmt3a and Dnmt3b have overlapping roles in mediating de novo methylation at the Nanog
proximal promoter. Eprn KO cells also exhibited reduced methylation at the Nanog promoter, with
region one again showing a more prominent reduction (Figure 5—figure supplement 1A). To explore the role of de novo DNA methylation in ESC transition, we investigated functional
consequences of Dnmt3a and Dnmt3b depletion. We created Dnmt3a and Dnmt3b single and com-
pound knockouts in RGd2 ESCs using CRISPR/Cas9. Using two guide RNAs (gRNAs), we generated
deletions of highly conserved PC and ENV motifs (motifs IV and V) within the catalytic domain for
both Dnmt3a and Dnmt3b, recapitulating the previously characterised Dnmt3b and Dnmt3b mutant
gene structures (Okano et al., 1999) (Figure 5—figure supplement 1B). Dnmt3a and Dnmt3b sin-
gle and double KO cells exhibited delayed Rex1-GFP downregulation (Figure 5B). Colony formation
capacity of the single and double KO cells 40 hr post PD/LIF withdrawal confirmed slower extinction
of ESC identity (Figure 5C). Interestingly, however, as with Eprn KO, the delay in GFP downregula-
tion did not endure (Figure 5—figure supplement 1C). Dnmt3a and Dnmt3b methylate the Nanog promoter during naı¨ve state
exit Absence of Dnmt3a and Dnmt3b singly or
together was associated with transient perdurance of Nanog expression (Figure 5D). At 8 hr after
PD/LIF withdrawal, Nanog mRNA in Dnmt3a and/or Dnmt3b mutants was equivalent to wild type
cells in PD/LIF, whereas wild type cells had downregulated Nanog expression by 50% (Figure 5D). We also observed elevated expression of Tfcp2l1, Klf2, Klf4 and Tbx3 in Dnmt3a/3b single or com-
pound KO cells (Figure 5—figure supplement 1E). The promoters of these genes are methylation
refractory in the 2i withdrawal time course (Kalkan et al., 2017). Therefore, the elevated expression
should be secondary to some other factor(s) such as increased Nanog. Dnmt3a/3b compound KO
also resulted in impeded upregulation of peri-implantation markers Fgf5, Oct6 and Otx2 at 24 hr
post PD/LIF withdrawal (Figure 5—figure supplement 1F). These data indicate that de novo DNA
methylation facilitates timely progression from the ESC state. Importantly, however, methylation by
Dnmt3a/3b is not essential for the exit from naı¨ve pluripotency. Discussion Mouse ES cell self-renewal is robust due to recursive wiring of a core transcription factor network
(Dunn et al., 2014; Martello and Smith, 2014; Young, 2011). Rapid developmental progression
from such a resilient state is achieved through parallel mechanisms. In this study, we find that a
lncRNA, Ephemeron, participates in the timely dissolution of naı¨ve identity. Genetic interactions link
Eprn with known players in post-transcriptional and epigenetic regulation (Figure 5E). Eprn lies
upstream of Lin28a/let-7g and Dnmt3a/3b, and ultimately contributes to timely downregulation of
the pivotal naı¨ve transcription factor Nanog. Eprn depletion reduces Lin28a expression, although the
molecular mechanism underlying this effect remains unclear. Lower Lin28a stabilises expression of
the let-7 miRNAs whose targets include de novo DNA methyltransferases Dnmt3a and Dnm3b. Resulting decreased Dnmt3a/3b reduces Nanog proximal promoter CpG methylation, correlating
with transiently perduring expression. This lncRNA/miRNA/DNA methylation module provides an
additional layer in the multi-layered machinery that enforces transition from naı¨ve to formative pluri-
potency (Acampora et al., 2016; Jang et al., 2017; Kalkan et al., 2017; Kalkan and Smith, 2014;
Smith, 2017). Eprn promotes ESC transition and is suppressed by Gsk3 inhibition during ground state self-
renewal in 2i or 2i/LIF. ESCs cultured without Gsk3 inhibition can self-renew in the presence of PD/
LIF or LIF/serum. In these conditions they express Eprn and higher levels of Lin28a. The lack of overt
consequence is presumably due to the dominant self-renewal environment provided by Stat3 activa-
tion and MEK inhibition that sustain expression of Nanog and other naı¨ve factors. Nonetheless, loss
of Eprn in PD/LIF resulted in elevated Nanog and delayed transition kinetics. We observed a Mendelian ratio of homozygous Eprn mutant mice from heterozygous intercrosses
(5:18:7, wild type: heterozygous: homozygous offspring). Therefore, in common with Lin28a
(Shinoda et al., 2013), Eprn is dispensable for development of laboratory mice. Some protein- Li et al. eLife 2017;6:e23468. Discussion (D) Expression of Nanog relative to b-actin in Dnmt3a and Dnmt3b single
Figure 5 continued on next page
Research article
Developmental Biology and Stem Cells Research article Developmental Biology and Stem Cells siDnmt3b
siDnmt3a/3b
18%
3%
8%
1% siNeg
siDnmt3a
siDnmt3b
siDnmt3a/3b
40%
5%
9%
2%
18%
3%
8%
1%
Region 2
Region 1
Nanog promoter region
122,706,641
122,706,730
122,706,776
122,706,804
122,707,027
122,707,066
122,707,077
122,707,134
122,707,156
122,707,266
122,707,288
122,707,482
122,707,491
122,707,573
122,707,583
Region 2(556 bp)
122,706,584
122,706,552
122,706,544
Region 1 (260 bp)
122,707,565
TSS
A siNeg
siDnmt3a
40%
5%
9%
2%
gion 2
ion 1 A C 2i - N2B27 24h
WT=18%
Dnmt3aKO=24%
Dnmt3bKO=20%
DKO=23%
100
101
102
103
104
GFP
% of Max
0
20
40
60
100
80
PD/LIF- N2B27 40h
100
101
102
103
104
GFP
% of Max
0
20
40
60
100
80
B
WT=27%
Dnmt3aKO=35%
Dnmt3bKO=29%
DKO=44% 2i - N2B27 24h
WT=18%
Dnmt3aKO=24%
Dnmt3bKO=20%
DKO=23%
100
101
102
103
104
GFP
% of Max
0
20
40
60
100
80
B 4
PD/LIF- N2B27 40h
100
101
102
103
104
GFP
% of Max
0
20
40
60
100
80
WT=27%
Dnmt3aKO=35%
Dnmt3bKO=29%
DKO=44% 0
5
10
15
20
25
WT
Dnmt3a KO
Dnmt3b KO
Dnmt3a/3b DKO
Clonogenicity (%)
C B WT
Dnmt3a KO
Dnmt3b KO
Dnmt3a/3b DKO Dnmt3a KO Dnmt3b KO Dnmt3a/3b DKO 0
0.5
1
1.5
2
Relative Expression
WT
3aKO
3bKO
PDL
8h
24h
3a/3bDKO
WT
3aKO
3bKO
3a/3bDKO
WT
3aKO
3bKO
3a/3bDKO
D
Naive TF
Network
New
Network
Ephemeron
Lin28a
let-7
Nanog
Naive pluripotency
dƌĂŶƐŝƟŽŶ>ĂƚĞŶĐLJ
&ŽƌŵĂƟǀĞWůƵƌŝƉŽƚĞŶĐLJ
PD
Lif
CH
Naive TF
Network Nanog
Dnmt3a/3b
Nanog
Ephemeron Loss
Lin28a Loss
džƚĞŶĚĞĚdƌĂŶƐŝƟŽŶ>ĂƚĞŶĐLJ
let-7g Gain
Dnmt3a/3b Loss
E
Nanog 0
0.5
1
1.5
2
Relative Expression
WT
3aKO
3bKO
PDL
8h
24h
3a/3bDKO
WT
3aKO
3bKO
3a/3bDKO
WT
3aKO
3bKO
3a/3bDKO
D
Nanog D Naive TF
Network
New
Network
Ephemeron
Lin28a
let-7
Nanog
Naive pluripotency
dƌĂŶƐŝƟŽŶ>ĂƚĞŶĐLJ
&ŽƌŵĂƟǀĞWůƵƌŝƉŽƚĞŶĐLJ
PD
Lif
CH
Naive TF
Network Nanog
Dnmt3a/3b
Nanog
Ephemeron Loss
Lin28a Loss
džƚĞŶĚĞĚdƌĂŶƐŝƟŽŶ>ĂƚĞŶĐLJ
let-7g Gain
Dnmt3a/3b Loss
E E Dnmt3a/3b Nanog Nanog džƚĞŶĚĞĚdƌĂŶƐŝƟŽŶ>ĂƚĞŶĐLJ džƚĞŶĚĞĚdƌĂŶƐŝƟŽŶ>ĂƚĞŶĐLJ Figure 5. Loss of Dnmt3a and Dnmt3b delays naı¨ve state exit associated with transient persistence of Nanog expression. (A) Bisulfite sequencing
analysis of Nanog proximal promoter CpG island DNA methylation in Dnmt3a and Dnm3b single and dual knockdown cells at 40 hr post PD/LIF
withdrawal. The positions of cytosines analysed (mm10) are indicated on the left panel. Discussion DOI: 10.7554/eLife.23468 11 of 24 0
5
10
15
20
25
WT
Dnmt3a KO
Dnmt3b KO
Dnmt3a/3b DKO
Clonogenicity (%)
WT
Dnmt3a KO
Dnmt3b KO
Dnmt3a/3b DKO
siNeg
siDnmt3a
siDnmt3b
siDnmt3a/3b
40%
5%
9%
2%
18%
3%
8%
1%
Region 2
Region 1
Nanog promoter region
122,706,641
122,706,730
122,706,776
122,706,804
122,707,027
122,707,066
122,707,077
122,707,134
122,707,156
122,707,266
122,707,288
122,707,482
122,707,491
122,707,573
122,707,583
Region 2(556 bp)
122,706,584
122,706,552
122,706,544
Region 1 (260 bp)
122,707,565
TSS
A
C
2i - N2B27 24h
WT=18%
Dnmt3aKO=24%
Dnmt3bKO=20%
DKO=23%
100
101
102
103
104
GFP
% of Max
0
20
40
60
100
80
PD/LIF- N2B27 40h
100
101
102
103
104
GFP
% of Max
0
20
40
60
100
80
B
0
0.5
1
1.5
2
Relative Expression
WT
3aKO
3bKO
PDL
8h
24h
3a/3bDKO
WT
3aKO
3bKO
3a/3bDKO
WT
3aKO
3bKO
3a/3bDKO
D
Naive TF
Network
New
Network
Ephemeron
Lin28a
let-7
Nanog
Naive pluripotency
dƌĂŶƐŝƟŽŶ>ĂƚĞŶĐLJ
&ŽƌŵĂƟǀĞWůƵƌŝƉŽƚĞŶĐLJ
PD
Lif
CH
Naive TF
Network Nanog
Dnmt3a/3b
Nanog
Ephemeron Loss
Lin28a Loss
džƚĞŶĚĞĚdƌĂŶƐŝƟŽŶ>ĂƚĞŶĐLJ
let-7g Gain
Dnmt3a/3b Loss
E
WT=27%
Dnmt3aKO=35%
Dnmt3bKO=29%
DKO=44%
Nanog
Figure 5. Loss of Dnmt3a and Dnmt3b delays naı¨ve state exit associated with transient persistence of Nanog expression. (A) Bisulfite sequencing
analysis of Nanog proximal promoter CpG island DNA methylation in Dnmt3a and Dnm3b single and dual knockdown cells at 40 hr post PD/LIF
withdrawal. The positions of cytosines analysed (mm10) are indicated on the left panel. Black and while circles represent methylated and unmethylated
cytosine respectively. (B) Rex1-GFP flow cytometry profiles of Dnmt3a and Dnmt3b single and dual KO cells withdrawn from 2i or PD/LIF for 24 and 40
hr respectively. Percentage of GFP high cells were quantified. (C) colony formation capacity 40 hr post PD/LIF withdrawal for Dnmt3a and Dnmt3b
single and compound KO cells. Percentage clonogenicity was measured by the number of AP positive colonies formed divided by the total number of
cells plated, with representative AP staining images shown. Mean ± SD, n = 3. Discussion The presence of Eprn exclusively in rodent may be associ-
ated with rapid embryonic progression from pre-implantation epiblast to gastrulation (Rossant and
Tam, 2017), which necessitates acute extinction of the naı¨ve pluripotency programme (Smith, 2017). LncRNAs are more tolerant to TE integration than protein coding genes, which could drive more
rapid evolution (Kelley and Rinn, 2012; Necsulea et al., 2014). Non-coding transcripts harbouring
TE sequences are enriched in ESCs and early embryo development in both mouse and human
(Fort et al., 2014; Go¨ke et al., 2015; Kelley and Rinn, 2012) and in several instances have been
proposed to regulate pluripotency (Durruthy-Durruthy et al., 2016; Fort et al., 2014; Lu et al.,
2014). Eprn is comprised of 76.4% TEs, compared to the average of 41.4% TE composition in the
mouse genome and 33% reported for mouse multi-exon lincRNA sequences (Kelley and Rinn,
2012). The aligned sequence between Eprn and the rat transcript from the syntenic region includes
ERVK LTR and SINE B2 elements. These sequences have been preserved for over 30 million years
since the mouse-rat lineage divergence. Lin28a is known as a human somatic cell reprogramming factor (Yu et al., 2007). However,
Lin28a is expressed at a lower level in ground state mouse ESCs (Marks et al., 2012) and pre-
implantation epiblast, than in post-implantation epiblast and EpiSCs (Boroviak et al., 2015). The
expression pattern is consistent with our evidence that up-regulation of Lin28a at the onset of mouse
ESC differentiation functions to facilitate transition from the naı¨ve state. During human iPSC genera-
tion, Lin28a may promote acquisition of primed pluripotency, the endpoint for current human
somatic cell reprogramming. Lin28a itself is a target of let-7 miRNAs (Kumar et al., 2014;
Melton et al., 2010) and the reciprocal negative feedback loops can act as a bimodal switch. Our
findings are consistent with the recent report that loss of Lin28a reduced ESC heterogeneity in
serum/LIF, favouring a more naı¨ve state (Kumar et al., 2014). This effect was attributed to let-7g. We note, however, that Lin28a can post-transcriptionally regulate the expression and/or translation
of many RNAs independently of let-7 (Cho et al., 2012; Zhang et al., 2016) that could also contrib-
ute to ESC transition. De novo methyltransferases Dnmt3a/3b have previously been proposed as targets of let-7g
(Kumar et al., 2014). Our results show that loss of Dnmt3a and Dnmt3b, individually and in combi-
nation, delays naı¨ve state exit. Discussion Black and while circles represent methylated and unmethylated
cytosine respectively. (B) Rex1-GFP flow cytometry profiles of Dnmt3a and Dnmt3b single and dual KO cells withdrawn from 2i or PD/LIF for 24 and 40
hr respectively. Percentage of GFP high cells were quantified. (C) colony formation capacity 40 hr post PD/LIF withdrawal for Dnmt3a and Dnmt3b
single and compound KO cells. Percentage clonogenicity was measured by the number of AP positive colonies formed divided by the total number of
cells plated, with representative AP staining images shown. Mean ± SD, n = 3. (D) Expression of Nanog relative to b-actin in Dnmt3a and Dnmt3b single
Figure 5 continued on next page Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 12 of 24 Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 Research article R Research article Developmental Biology and Stem Cells Developmental Biology and Stem Cells Developmental Biology and Stem Cells Figure 5 continued
and compound KO cells quantified by RT-qPCR. Mean ± SD, n = 2. (E) Schematic representation of the inferred Eprn pathway. Legends for Figures and
Source Data. g
and compound KO cells quantified by RT-qPCR. Mean ± SD, n = 2. (E) Schematic representation of the inferred Eprn pathway. Legends for Figures and
Source Data. DOI 10 7554/ Lif
23468 017 DOI: 10.7554/eLife.23468.017 The following figure supplement is available for figure 5: Figure supplement 1. Phenotypic and molecular characterisation of Dnmt3a/3b KO in naı¨ve state exit. DOI: 10.7554/eLife.23468.018 coding genes that exhibit demonstrable loss-of-function phenotypes in ESC self-renewal or transition
also show no early embryo phenotype (Leeb et al., 2014; Martello and Smith, 2014). Our interpre-
tation is that ESCs provide a sensitised platform for identifying components whose functions may be
compensated during in vivo development. coding genes that exhibit demonstrable loss-of-function phenotypes in ESC self-renewal or transition
also show no early embryo phenotype (Leeb et al., 2014; Martello and Smith, 2014). Our interpre-
tation is that ESCs provide a sensitised platform for identifying components whose functions may be
compensated during in vivo development. The majority of lncRNAs are not phylogenetically conserved (Necsulea et al., 2014). Due to their
rapidly evolving nature, it is thought that lncRNAs are likely to acquire species or lineage-restricted
functions and several examples have recently been described (Durruthy-Durruthy et al., 2016;
Paralkar et al., 2014; Rani et al., 2016). Discussion Naı¨ve ESCs (Ficz et al., 2011; Habibi et al., 2013; Leitch et al.,
2013) and pre-implantation epiblast (Monk et al., 1987; Sanford et al., 1987) have low expression
of Dnmt3a/3b and display global DNA hypomethylation. However, the post-implantation epiblast
rapidly
acquires
global
DNA
methylation
and
this
process
is
dependent
on
Dnmt3a/3b
(Auclair et al., 2014). A similar acute trend is observed upon naı¨ve ESC withdrawal from 2i
(Kalkan et al., 2017). Early de novo methylation may have functional consequences for specific naı¨ve
pluripotency associated factors, such as Nanog, enduring rapid downregulation. It is noteworthy,
however, that the Dnmt3a/3b KO phenotype is transient and ESC identity still collapses. Therefore,
although de novo DNA methylation facilitates ESC transition it is not mandatory for the exit from
naı¨ve pluripotency. Human naı¨ve pluripotency shares molecular and cellular features with mouse, consistent with
conserved core pluripotency programme in mammals (Guo et al., 2017, 2016; Smith, 201 Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 13 of 24 Research article Research article Developmental Biology and Stem Cells Developmental Biology and Stem Cells Takashima et al., 2014; Theunissen et al., 2014). However, species-specific differences are evident. Notably, Gsk3 inhibition has less impact on human naı¨ve state maintenance (Guo et al., 2017;
Theunissen et al., 2016). This is partly explained by the lack of ESRRB expression in human pluripo-
tent cells (Blakeley et al., 2015; Martello et al., 2012; Takashima et al., 2014; Theunissen et al.,
2014), but absence of Eprn may be an additional factor that reduces requirement for Gsk3
inhibition. In summary, we have mapped a genetic interaction module consisting of a novel lncRNA, proteins
and miRNAs that is integrated into the multi-pronged molecular machinery that propels mouse ESCs
towards lineage competence. The fine-tuning effect of Eprn could be representative of lncRNA
actions in the regulation of molecular networks and illustrative of their potential contribution to spe-
cies diversification. Cell culture ESCs were cultured on 0.1% gelatin in 2i/LIF medium (homemade N2B27 base medium, supple-
mented with 1 mM PD0325901, 3 mM CHIR99021, and 20 ng/ml LIF) as described (Ying et al., 2008). For gene targeting, ESC were maintained with serum containing medium supplemented with 2i/LIF
as above (KO-DMEM high glucose, 15% FCS, 2 mM L-Glutamine, NEAA, 1 mM Sodium Pyruvate
(Thermo Fisher Scientific), 100 mM b-Mercaptoethanol (Sigma Aldrich, St. Louis, MO, USA). Cor-
rectly targeted clones were transferred to N2B27 based 2i/LIF medium for expansion and experi-
mentation. The RGd2 reporter wild type subclones and Eprn KO ESC clones are of V6.5 origin
(RRID:CVCL_C865). An independent wild type RGd2 reporter line is of E14 origin (RRID:CVCL_
C320). All cell lines are mycoplasma negative by PCR screening in house. Targeting, expression and gRNA vector construction Targeting, expression and gRNA vector construction
BAC RP24-353A19 (C57BL/6J background) was obtained from Wellcome Trust Sanger Institute for
constructing the Eprn knockout targeting vectors by recombineering using bacterial strain EL350
(Lee et al., 2001). Floxed drug resistant cassettes containing hygromycin B phosphotransferase
gene (Hygro) or Blasticidin S-resistance gene (Bsd) were PCR amplified using chimeric primers miniU
and miniD (Supplementary file 1A) harbouring 80 bp mini-homologies to the genomic region flank-
ing Eprn locus. The purified PCR fragments loxP-PGK-Hygro-bghpA-loxP and loxP-PGK-Bsd-bghpA-
loxP were used to replace the entire genomic region of Eprn locus with the drug resistant cassettes. The retrieval homology arms were PCR amplified using primers ReUF and ReUR for upstream and
ReDF and ReDF for downstream mini-arms (Supplementary file 1B). The mini arms were subse-
quently cloned into pBS-MC1-DTA vector by 3-way ligation using restriction enzymes, SpeI, HindIII
and XhoI. The mini-arm containing vector was linearised by HindIII and used to retrieve the targeting
vectors from the modified BACs giving rise to the final targeting vectors HygroTV and BsdTV vectors from the modified BACs, giving rise to the final targeting vectors, HygroTV and BsdTV. Lin28a overexpression vector was constructed by PCR cloning mouse Lin28a from cDNA using
forward
primer
AATTGTCGACATGGGCTCGGTGTCCAACCAGCAGT
and
reverse
primer
AA
TTGCGGCCGCTCAATTCTGGGCTTCTGGGAGCAG and cloned into pENTR2B vector. It was subse-
quently cloned into PiggyBac-based expression vector using Gateway LR clonase (Thermo
Fisher Scientific, Waltham, MA, USA, 11791020) to generate pCAG-Lin28a-pA:PGK-hygro-pA
plasmid The gRNA design was conducted using online CRISPR gRNA design tool https://www.dna20. com/eCommerce/cas9/input. The chosen gRNAs were based on minimal off-target scores. Deletions
were designed to recapitulate the original Dnmt3a and Dnmt3b KO mutations (Okano et al., 1999),
excising the highly conserved PC and ENV motifs (motifs IV and V) within the catalytic domain. The
gRNAs were generated by annealing the indicated oligos (Supplementary file 2A), which were sub-
sequently ligated into pX458 vector (Addgene) digested with BbsI. The constructs were sequence
validated before transfection. Research article Naı¨ve pluripotency exit assays p
p
y
y
ESCs were plated at 1 104/cm2 in 2i without LIF or PD/LIF. The next day, cells were carefully
washed with PBS before switching to N2B27 medium. Rex1-GFP profile was analysed at indicated 14 of 24 Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 Research article Research article Developmental Biology and Stem Cells time points in at least two independent experiments using a Cyan or Fortessa FACs analyser. Live
dead discrimination was performed using TO-PRO-3 (Thermo Fisher Scientific, T3605). For clonal
assay, post 24 hr or 40 hr 2i or PD/LIF withdrawal respectively, 300–500 cells were plated per well of
a 12 well plate coated with Laminin (1:100 dilution, Sigma Aldrich, L2020) and cultured in 2i/LIF for 6
days. Alkaline Phosphatase staining (Sigma Aldrich, 86R-1KT) was conducted to visualise ES colonies. AP-stained plates were imaged using an Olympus IX51, DP72 camera with CellSens software and
subsequent colony counting was conducted manually using ImageJ software. EpiSC derivation from ESCs and EpiSC resetting p
p
g
ESCs were plated at 1 104/cm2 in 2i/LIF on a gelatin coated plate. The next day, cells were washed
with PBS before medium switch to N2B27 medium supplemented with 20 ng/ml Activin A and 12
ng/ml Fgf2 together with 2 mM XAV939 (Sigma Aldrich, X3004), A/F/X. Cells were then passaged to
fibronectin coated plate in A/F/X medium. EpiSCs were passaged for at least seven times before
gene expression analysis and resetting. For EpiSC resetting, EpiSCs were stably transfected with
GY118F construct by piggyBac transposition (Yang et al., 2010). 1 104 cells were plated in a one
well of a 12 well plate in A/F/X, the next day, 2i plus GCSF was supplied to initiate resetting. siRNA, miRNA mimics and plasmid transfection siRNA, miRNA mimics and plasmid transfection
siRNAs and miRNA mimics were obtained from Qiagen and the catalogue numbers are listed in
Supplementary file 3. Transfection was performed using Dharmafect 1 (Dharmacon, Lafeyette, CO,
USA, T-2001–01) in a reverse transfection protocol with the final siRNA or miRNA mimics concentra-
tion to be 10 nM. Two siRNA combination were used per transfection for each target gene
knockdown. Plasmid transfection was performed using Lipofectamine 2000 (Thermo Fisher Scient
11668027) following the manufacturers protocol. For piggyBac based stable integration, a piggy
transposon and hyperactive PBase (hyPBase) ratio of 3:1 was used. Plasmid transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific,
11668027) following the manufacturers protocol. For piggyBac based stable integration, a piggyBac
transposon and hyperactive PBase (hyPBase) ratio of 3:1 was used. Generation of Dnmt3a and Dnmt3b KO ESCs with CRISPR/Cas9
A pair of gRNA containing plasmids based on px458 backbone (Ran et al., 2013) were transfected
using Fugene HD (Promega, Madison, WI, USA, E2311). 100 ng of each plasmid were transfected
with 0.6 ul Fugene HD (1:3 ratio) to 2 105 ESCs in suspension in 2i/LIF medium overnight. The
next day, the media was refreshed and 48 hr post transfection, 1,000 GFP high cells were sorted
into a well of a six well plate for colony formation. Individual colonies were picked and genotyping
was conducted from extracted genomic DNA by triple primer PCR to identify clones with designed
deletion (Supplementary file 2B). For Dnmt3a KO, deletion resulted in genotyping PCR product
shift from 760 bp representing the wild type allele to 1132 bp. For Dnmt3b KO, deletion resulted in
shift from 344 bp representing the wild type allele to 653 bp. Only homozygous mutants were cho-
sen for subsequent experimentation. Differentiation assays For neuronal differentiation, ESCs were plated at 1 104/cm2 in N2B27 medium on laminin (1:100
in PBS) for up to 4 days for gene expression analysis. For mesendoderm differentiation, cells were
plated at 0.6 104/cm2 in N2B27 based medium containing 10 ng/ml ActivinA, 3 mM CHIR99021 on
Fibronectin for up to 4 days for gene expression analysis. For definitive endoderm differentiation,
cells were plated at 1.5 104/cm2 in N2B27 based medium containing 20 ng/ml ActivinA, 3 mM
CHIR99021, 10 ng/ml FGF4, 1 mg/ml Heparin, 100 nM PI103. On day 2, the media was switched to
SF5 based medium containing 20 ng/ml ActivinA, 3 mM CHIR99021, 10 ng/ml FGF4, 1 mg/ml Hepa-
rin, 100 nM PI103 and 20 ng/ml EGF2. Per 100 ml SP5 basal medium, it contains 500 ml N2, 1 ml B27
without VitaminA supplement, 1% BSA, 1 ml L-glutamine and 100 ml b-mercaptoethanol. Detailed
protocols can be found in Mulas et al (Mulas et al., 2017). Southern blotting g
Genomic DNA of individually picked ESC clones was extracted and digested with XmnI, size-frac-
tionated on a 0.8% agarose gel and transferred to Hybond-XL blotting membrane (GE Healthcare,
Chicargo, IL, USA, RPN20203) using standard alkaline transfer methods. The 5’ and 3’ external 15 of 24 Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 Research article Research article Research article probes were generated by PCR with primer sequences shown in Supplementary file 4A. Southern
blot hybridization was conducted as described previously (Li et al., 2011). probes were generated by PCR with primer sequences shown in Supplementary file 4A. Southern
blot hybridization was conducted as described previously (Li et al., 2011). RNA extraction, reverse transcription and Real-time PCR RNA extraction, reverse transcription and Real-time PCR
Total RNA was isolated using Trizol (Thermo Fisher Scientific, 15596026) or RNeasy kit (Qiagen, Hil-
den, Germany, 74136) and DNase treatment was conducted either after RNA purification or during
column purification. cDNA was transcribed from 0.5 ~ 1 ug RNA using SuperScriptIII (Thermo Fisher
Scientific, 18080044) and oligo-dT priming. Real-time PCR was performed using StepOnePlus
machine (Applied Biosystems) with Fast Sybrgreen master mix (Thermo Fisher Scientific, 4385612). Target gene primer sequences are shown in Supplementary file 6. Expression level were normalised
to Actinb. Technical replicates for at least two independent experiments were conducted. The
results were shown as mean and standard deviation calculated by StepOnePlus software (Applied
Biosystems). The cDNA library for E7-E17 embryos and adult tissues were purchased from Clontech
(Mouse Total RNA Master Panel, 636644). Northern blotting g
10 mg of purified RNA was resolved by denaturing formaldehyde agarose gel electrophoresis with
MOPS buffer. RNA was transferred to Hybond-XL membrane in 1xSSC buffer overnight by capillary
transfer. RNA was UV cross-linked to the membrane and pre-hybridised with Expresshyb (Clone-
Tech, Mountainview, CA, USA, 636831) for 2 hr at 65˚C. The DNA probe was generated by PCR (pri-
mers are shown in Supplementary file 4B) and 25 ng of probe DNA was labelled with [32P]-dCTP
using Radprime DNA labeling system (Thermo Fisher Scientific, 18428–011). The free-nucleotide was
removed from labelled probe using G-50 column (GE Healthcare, 27-5330-01), and was heat-dena-
tured followed by snap cooling. The probe was added to the pre-hybridised membrane and incu-
bated overnight at 65˚C in a rolling incubator. Membrane was washed with wash buffer containing
0.1 x SSC and 0.1% SDS 3 times at 65˚C with 10 min intervals. The membrane was placed in a phos-
phoimager and exposed for at least overnight at 80˚C before scanned using Typhoon 9410 phos-
phoimager system (GE Healthcare). Mature miRNA expression profiling Total RNA was extracted using Trizol. 1 ug RNA was reverse transcribed using Taqman MicroRNA
Reverse Transcription Kit (Thermo Fisher Scientific, 366596). Mature miRNA expression was analysed
using Taqman Array Rodent MicroRNA A + B Cared Set V3.0 (Thermo Fisher Scientific, 444909). RNA-FISH RNA-FISH was conducted using ViewRNA ISH Cell Assay for Fluorescence RNA In Situ Hybridization
system (Thermo Fisher Scientific, QVC0001) with modifications and imaged on a DeltaVision Core
system (Applied Precision), as described in Bergmann et al. (2015). The probe set used for Ephem-
eron was VX1-99999-01. 5’ and 3’ RACE 5’ RACE was conducted using 5’-Full RACE Core Set (Takara, Kusatsu, Japan, #6122) following man-
ufacture’s protocol. The sequences for RT-primer and nested PCR primers A1, A2, S1, and S2 are
shown in Supplementary file 5A. 3’ RACE was conducted by using a polyT RT-primer with a unique
sequence tag to synthesis cDNA. The 3’ end region was PCR amplified using a primer specific to the
RT-primer and a gene specific primer. The primers are shown in Supplementary file 5B. Both 5’ and
3’ RACE PCR products were cloned into plasmids using Zero blunt TOPO PCR cloning kit
(Thermo Fisher Scientific, 451245) for subsequent sequencing. ChIP The experimental procedure was conducted as described previously (Betschinger et al., 2013). 2 ug
of H3K4me3 antibody (Diagenode, Ougre´ e, Belgium pAb-003–050) and IgG control (Santa Cruz,
Dallas, TX, USA, sc-2345) was used for 4 106 cells per ChIP. qPCR was performed with primers
shown in Supplementary file 8. Luciferase assay Luciferase assay
The Entire 3’UTR of both Dnmt3a and Dnmt3b were PCR cloned downstream of the firefly luciferase
coding region into pGL3 vector. For Dnmt3a 3’UTR, forward primer AATTGGCCGGCCGGGACA
TGGGGGCAAACTGAAGTAG and reverse primer AATTGGATCCGGGAAGCCAAAACATAAAGATG
TTTATTGAAGCTC
were
used
for
PCR
cloning. For
Dnmt3b
3’UTR,
forward
primer
AA
TTGGCCGGCCTTCTACCCAGGACTGGGGAGCTCTC
and
reverse
primer
AATTGGATCCTTA Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 16 of 24 Research article Research article Developmental Biology and Stem Cells TAGAGAAATACAACTTTAATCAACCAGAAAGG were used for PCR cloning. To generate mutant
Dnmt3b reporter constructs, let-7g binding site was mutated to include BsrGI (mutation V1) and
EcoRI (mutation V2) sites by PCR cloning. Each firefly luciferase construct (500 ng) together with
Renilla luciferase construct (10 ng) were con-transfected with either let-7g mimic or scrambled con-
trol (20 nM). The firefly and Renilla luciferase activity was determined by dual luciferase assay (Prom-
ega, E1960) 48 hr post-transfection. Immunostaining Cells were fixed in 4% paraformaldehyde for 10 min at room temperature and were blocked with
blocking buffer (5% semi-skimmed milk with 0.1% Triton in PBS) for 2 hr at room temperature. Pri-
mary antibodies were diluted in blocking buffer and incubated at 4˚C overnight. Primary antibody
was carefully washed away with 0.1% Triton in PBS three times with 10 min incubation between each
wash. Secondary antibody diluted in blocking buffer (1:1000) was incubated at room temperature
for 1 hr followed by 3 washes with 0.1% Triton in PBS. Nuclei were counterstained with DAPI. Pri-
mary antibodies used were Nanog (eBioscience, 14–5761, RRID:AB_763613, 1:200) and Lin28a (Cell
signalling, 3978, RRID:AB_2297060, 1:800; 8706, RRID:AB_10896850, 1:200). Images from random
fields were taken with Leica DMI3000 and the images from different fields at each time point were
combined and analysed using CellProfiler software (Broad Institute, RRID:SCR_007358) to conduct
nuclear and cytoplasmic compartmentalisation and total fluorescent intensity for each sub-cellular
compartments as well as the whole cell for each cell was extracted for correlation analysis. Nanog promoter DNA methylation analysis g p
y
y
Genomic DNA was extracted using GenElute Mammalian Genomic DNA miniprep kit (Sigma
Aldrich, G1N70-1KT). 500 ng purified genomic DNA was treated with sodium bisulfite to convert all
unmethylated
cytosine
residues
into
uracil
residues
using
Imprint
DNA
modification
Kit
(Sigma Aldrich, MOD50-1KT) according to the manufacturer’s protocol. Nanog proximal promoter
regions (Region 1 and 2 as indicated in Figure 5a) were amplified using a nested PCR approach with
KAPA HiFi Uracil + Readymix (KapaBiosystems/Roche, Basel, Switzerland, KK2801). The PCR condi-
tion for both nested rounds of PCR is as follows: denaturation at 98˚C for 5 min followed by 10
cycles of gradient PCR, 98˚C for 15 s, 62˚C (starting annealing temperature) for 15 s with annealing
temperature reduced by 1˚C per cycle and 72˚C for 1.5 min. Followed by this, a 35 cycles of 98˚C for
15 s, 58˚C for 15 s and 72˚C for 1.5 min were conducted. 2 ml first round PCR product was used as
template for the nested PCR. All primer sequences are shown in Supplementary file 9. The PCR
products were verified and purified by gel electrophoresis and subsequently subcloned by TOPO
cloning. Reconstructed plasmids were purified and individual clones were sequenced (Eurofins). Chromatin isolation by RNA purification (ChIRP) The antisense oligo probes were selected with GC content in the range of 40–50% in regions of the
Eprn exons without repetitive sequences (Figure 1—figure supplement 1A). The probes sequences
are in shown in Supplementary file 7. CHIRP was conducted following published protocol
(Chu et al., 2011). The data is available at the NCBI Gene Expression Omnibus (accession number:
GSE90574). The link to the data is as follows: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=
GSE90574. Eprn promoter CpG methylation analysis p
p
p
y
y
Using published genome-wide bisulpite sequencing data (Kalkan et al., 2017; Seisenberger et al.,
2012; Wang et al., 2014), Eprn promoter region was defined as the 2 kb region upstream of the
TSS and the percentage of CpG methylation within the region was quantified. For promoter aver-
age, percentage of CpG methylation around the 2 kb promoter region of each annotated gene was
quantified and averaged for all values. For genome average, percentage of CpG methylation of all
50 kb tiling windows was quantified and averaged all values. Transcriptome sequencing and analysis p
q
g
y
Total RNA was isolated with RNeasy RNA purification. Ribo-zero rRNA depleted RNA was used to
generate sequencing libraries for wild type and Ephemeron knockout cells in PD/LIF and 8 hr with-
drawal from PDL from three independent cell lines. Single end sequencing was performed and the
reads were mapped using NCBI38/mm10 with Ensembl version 75 annotations. RNA-seq reads were 17 of 24 Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 Research article Research article Developmental Biology and Stem Cells aligned to the reference genome using tophat2. Only uniquely mapped reads were used for further
analysis. Gene counts from SAM files were obtained using htseq-count with mode intersection non-
empty, -s reverse. Differential gene expression analysis was conducted using Bioconductor R pack-
age DESeq2 version 1.4.5. DESeq2 provides two P-values, a raw P-value and a Benjamini-Hochberg
P-value (adjusted p value). An adjusted p-Value threshold of 0.05 was used to determine differential
gene expression (95% of the results are not false discoveries, error rate 0.05 = 5%). The data is avail-
able at the NCBI Gene Expression Omnibus (accession number: GSE90574, https://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE90574). Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 Acknowledgements We thank Kosuke Yusa and Graziano Martello for comments on the manuscript. We are grateful to
Carla Mulas for assisting the miRNA expression plot, Yiping Zhang for lncRNA candidate prediction
analysis and Rosalind Drummond for technical support. We thank Heather Lee for providing Dnmt3a
and Dnmt3b siRNAs and Wolf Reik for support. We thank Nicholas Ingolia for useful discussion on
lncRNA ribosomal footprinting. We also thank Peter Humphreys and Andy Riddell for technical sup-
port for imaging analysis and flow cytometry respectively. AS is supported by Medical Research
Council (G1100526/1), Biotechnology and Biological Sciences Research Council (BB/M004023/1),
European Commission (HEALTH-F4-2007-200720 EUROSYSTEM), and Wellcome Trust (091484/Z/
10/Z). LH is supported by National Cancer Institute (R01 CA139067, 1R21CA175560-01) and Califor-
nia Institute of Regenerative Medicine (RN2-00923-1), American Cancer Society (123339-RSG-12-
265-01-RMC), Tobacco-related Disease Research Program (21RT-0133). DLS is supported by NIGMS
42694 and NCI 5PO1CA013106-Project 3. TK is supported by programme grants from Cancer
Research UK (C6/A18796) and European Research Council CRIPTON Grant (268569) and core grants
from the Wellcome Trust (092096) and Cancer Research UK (C6946/A14492). The Cambridge Stem
Cell Institute receives core funding from the Wellcome Trust and the Medical Research Council. MAL
was a Siebel postdoctoral fellow at the University of California, Berkeley and a Sir Henry Wellcome
postdoctoral fellow (096125/Z/11/Z). AS is a Medical Research Council Professor. Additional information Funding
Funder
Grant reference number
Author
Wellcome
Sir Henry Wellcome
Postdoctoral Fellowship
Meng Amy Li
California Institute for Regen-
erative Medicine
Research Grant
Lin He
Wellcome
Research Grant
Austin Smith
Medical Research Council
Research Grant
Austin Smith
National Institute of General
Medical Sciences
Research Grant
David L Spector
National Cancer Institute
Research Grant
David L Spector
Lin He
Cancer Research UK
Research Grant
Tony Kouzarides
European Research Council
Research Grant
Tony Kouzarides Li et al. eLife 2017;6:e23468. DOI: 10.7554/eLife.23468 18 of 24 Research article Research article Biotechnology and Biological
Sciences Research Council
Research Grant
Austin Smith
European Commission
Research Grant
Austin Smith
American Cancer Society
Research Scholar Award
Lin He
Tobacco-Related Disease Re-
search Program
Research Grant
Lin He
The funders had no role in study design, data collection and interpretation, or the decision to
submit the work for publication. Developmental Biology and Stem Cells Developmental Biology and Stem Cells Additional files
Supplementary files pp
y
. Supplementary file 1. Primers for generating Eprn Targeting vector and genotyping. DOI: 10.7554/eLife.23468.019 . Supplementary file 2. Primers for generating gRNAs vectors and genotyping for Dnmt3a/
knockouts. . Supplementary file 2. Primers for generating gRNAs vectors and genotyping for Dnmt3a/3b
knockouts. DOI 10 7554/ Lif
23468 020 . Supplementary file 3. siRNAs and mature miRNA mimics used in this study. DOI: 10.7554/eLife.23468.021 . Supplementary file 3. siRNAs and mature miRNA mimics used in this study. DOI: 10.7554/eLife.23468.021 . Supplementary file 4. Primers for generating Southern and Northern blotting probes by PCR. DOI: 10.7554/eLife.23468.022 . Supplementary file 4. Primers for generating Southern and Northern blotting probes by PCR. DOI: 10.7554/eLife.23468.022 . Supplementary file 5. Primers for RACE and nested PCR amplification. DOI: 10.7554/eLife.23468.023 Author contributions MAL, Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investiga-
tion, Visualization, Methodology, Writing—original draft, Writing—review and editing; PPA, Formal
analysis, Investigation, Methodology, Writing—review and editing; PC, MK, MP, JN, Investigation,
Methodology; JHB, FvM, Formal analysis, Investigation, Methodology; TKa, Resources, Data cura-
tion; MR, Formal analysis, Visualization; SR, Data curation, Formal analysis, Visualization; FY, Formal
analysis, Investigation; CC, Resources, Dr. Chen provided miRNA expression profiling assays; DLS,
LH, Supervision, Funding acquisition, Writing—review and editing; TKo, Supervision, Funding acqui-
sition; AS, Supervision, Funding acquisition, Writing—original draft, Writing—review and editing Author ORCIDs
Meng Amy Li,
http://orcid.org/0000-0002-6619-9919
Austin Smith,
http://orcid.org/0000-0002-3029-4682 Author ORCIDs Author ORCIDs
Meng Amy Li,
http://orcid.org/0000-0002-6619-9919
Austin Smith,
http://orcid.org/0000-0002-3029-4682 Additional files
Supplementary files
. Supplementary file 1. Primers for generating Eprn Targeting vector and genotyping. DOI: 10.7554/eLife.23468.019
. Supplementary file 2. Primers for generating gRNAs vectors and genotyping for Dnmt3a/3b
knockouts. DOI: 10.7554/eLife.23468.020
. Supplementary file 3. siRNAs and mature miRNA mimics used in this study. DOI: 10.7554/eLife.23468.021
. Supplementary file 4. Primers for generating Southern and Northern blotting probes by PCR. DOI: 10.7554/eLife.23468.022
. Supplementary file 5. Primers for RACE and nested PCR amplification. DOI: 10.7554/eLife.23468.023
. Supplementary file 6. Primers for Real-time quantitative RT-PCR. DOI: 10.7554/eLife.23468.024
. Supplementary file 7. ChIRP probes used in this study. DOI: 10.7554/eLife.23468.025
. Supplementary file 8. Primers used for Lin28a promoter ChIP PCR. DOI: 10.7554/eLife.23468.026
. Supplementary file 9. Primers used for Nanog promoter DNA methylation analysis. DOI: 10.7554/eLife.23468.027
Major datasets
The following dataset was generated:
Author(s)
Year Dataset title
Dataset URL
Database, license,
and accessibility
information
Li MA, Amaral PP,
Cheung P, Berg-
mann JH, Kinoshita
M, Kalkan T, Ralser
2016
A lncRNA/Lin28/let7 axis coupled
to DNA methylation fines tunes
the dynamics of a cell state
transition
http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi? acc=GSE90574
Publicly available at
the NCBI Gene
Expression Omnibus
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https://openalex.org/W2315205004
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https://zenodo.org/record/1496974/files/article.pdf
|
English
| null |
An experimental study of poison oak
|
Experimental biology and medicine
| 1,912
|
public-domain
| 8,567
|
AN EXPERIMENTAL STUDY OF POISON OAK*
EDWARD VON ADELUNG, M.D.
OAKLAND, CAL. While it is true that poison oak and its sister plants have rarely, if
ever, caused a death, it is also true that a large majority of people living
in the temperate zones find their enjoyment of the country seriously
curtailed by the menace of these noxious plants. And besides this
restriction of healthful pleasures, there is an unestimated monetary loss
to laborers and construction companies operating in infested regions,
which I am sure is not inconsiderable. Yet in spite of these well-known
facts, very little has been attempted, and still less accomplished, to abate
this wide-spread evil. SKETCH OF PLANT The plants specially referred to are poison oak (Rhus
diversiloba),
poison ivy (R. toxicodendron) and poison sumach (R. venenata), which
form a widely distributed group. The poison ivy1 is found in abundance
throughout the United States
as far west
as eastern Texas, eastern
Kansas and Minnesota, and in greater or less abundance throughout the
less arid regions of the west, with the exception of California and the
western parts of Oregon and Washington, where it appears to be entirely
replaced by the poison oak. The plants are very similar to each other,
each being a shrub that trails along the ground, or climbs fences, trees
or brush, or in many instances stands erect independently. It does not
frequent the higher mountains. The irregularly lobulated and dentated
leaves of the poison oak are a beautiful red when young, and an equally
attractive russet when old, rendering them enticing to the uninitiated. The fine hairs that are found on the leaves, especially along the margins,
are supposed by some to carry the poison when wafted by breezes, thus
explaining poisoning at a distance. The berries and blossoms are green
and extremely poisonous. But the berries turn white as they ripen, lose
their poison and are often eaten by birds, which are the means of dis¬
tributing the plants over so wide a territory. *From the Hearst Laboratory of Pathology and Bacteriology, University of
California.
b
i
d f
bli
ti
Th A
hi
25
1912 *Manuscript
submitted for publicationinTheArchives. 25, 19
1 Chestnut: Farmer's Bulletin No
86
U
S
Dept Agriculture ,
p
g
2. Pfaff: Jour. Experimental Medicine, 1897, (2), 181. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 THE TOXIC
PRINCIPLE NON-VOLATILE While poison oak, poison ivy and poison sumach differ botanically,
their juices seem to be chemically identical. Franz Pfaff,2 of the Harvard Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 EDWARD
VON
ADELUNG 149 Medical School, in his excellent monograph, states that the poison is
identical in the ivy and in the oak. That the dermatitis caused by either
plant is identical clinically, has been recognized for a long time. Several
attempts have been made to isolate chemically the poisonous principle of
the plants. This problem is of special importance because it underlies
much of the clinical and biologic work. biologic
The earliest attack on this difficult problem was made in 1779 by
van Mons,3 who concluded that the poison was a "gaseous hydrocarbon,"
emanating from the plant only at night or on cloudy days. Lavini,
writing thirty-three years later (1825), also believed the poison to be a
gas exhaled at night, but thought it was not a hydrocarbon. Another
thirty-three years then elapsed before Khittell (1858) analyzed the plant
and derived his "rhustannic acid."
But he said that there was also a
volatile, toxic alkaloid. In 1865, Maisch isolated his "toxicodendric acid,"
again a volatile substance. Then followed two Japanese, Ishimatsu and
Yoshida (1882 and 1883), who also concurred that the toxic agent is
volatile. The chemical problem really had but little light thrown on it
until Pfaff approached the problem from the clinical viewpoint and
showed that Maisch's "toxicodendric acid" is entirely inert; indeed,
nothing but acetic acid, and that contrary to all previous impressions,
the toxin is actually
a non-volatile substance which he extracted and
named "toxicodendrol" on account of its oily appearance. Of this sub¬
stance, as little as 1/1,000 mg. suffices to produce the typical dermatitis.2
All parts of the plant yield toxicodendrol, the leaves and green berries,
however,
more in proportion to weight. The best and most recent
chemical study of the toxin is than of W. A. Syme,4 whose dissertation on
"Some Constituents of the Poison Ivy," was accepted by Johns Hopkins
University in granting him the degree of Ph.D. in 1906. His analysis
shows that the poison is
a complex substance of
a glucosidal nature,
yielding, on analysis, gallic acid, fisetin and rhamnose. The poison was
again shown to be non-volatile, even when mixed with acetic acid (with
which it is naturally associated in the plant), or with alcohol. 3. L. E. Warren has compiled
an extensive and excellent bibliography in
The Midland Druggist and Pharmaceutical Review, 1910, xliv, Nos. 3, 4 and 5.
4. W. A. Syme: Johns Hopkins Univ. Bulletin, 1906, "Some Constituents of
the Poison Ivy Plant." Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 THE TOXIC
PRINCIPLE NON-VOLATILE y
p
),
Thus it is seen that for a hundred and eighteen years, from van Mons
to Pfaff, the opinion prevailed that the toxin was volatile
—
a natural
conclusion in the presence of the common observation that persons are
poisoned while simply passing near the plant, without actual contact. The importance of Pfaff's work, confirmed and enlarged by Syme, is
therefore evident. Before dismissing the subject of the nature of the
poison, it may be noted that a bacterial theory had its supporters, but its
proponent (Burrill, 1882) admitted (1895) that proof was lacking. That THE
ARCHIVES
OF
INTERNAL
MEDICINE 150 the poison is not bacterial is to-day very clear, although bacteria often
play an important rôle as agents in secondary infection. POISONING WITHOUT CONTACT The toxic principle having been shown to be non-volatile, the prac¬
tical question immediately presents itself, how does poisoning at
a
distance occur? That persons are poisoned without direct contact with
the plant is too common an observation to be denied. The explanation
is doubtless the mechanical transportation of the poison, as happens when
the pollen, or the plant hairs, or other dust from the plant is carried by
air-currents. Or, perhaps
as commonly, the transporting agents
are
simply clothes, or tools or animals, which, after brushing against the
plant,
are able to transfer the poison to susceptible persons. Such
explanations are easily acceptable when one remembers how minute
a
quantity suffices to induce inflammation. In order to show that the
poison is not volatile the following experiment was made : Experiment 1.—Young, fresh poison oak leaves vere crowded into a glass tube,
one end of which was closed with cotton. Holding this end against the skin of a
susceptible person, air was blown through the tube. The spot struck by the air
current was then covered with zinc oxid plaster to retain what poison might have
been deposited, and after forty-eight hours the spot
was inspected. Were the
poison volatile, dermatitis should have resulted. The result was negative. On the other hand, I was severely poisoned by the dust raised while
handling the dried plants in the laboratory. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 THE OCCURRENCE
OF THE ERUPTION Because the spots of dermatitis often break out successively in widely
separated areas of the skin, the impression has gained credence that the
poison is distributed by the blood-stream—a matter of considerable
importance in its bearing on the pathology and treatment of the disorder. Light
was thrown
on this question by Pearson (1880), who showed
experimentally that
serum from the
vesicles
is not toxic. I
have
repeatedly tried in vain to produce dermatitis by rubbing in the serum
from vesicles. Chestnut1 also concludes that the dermatitis is purely
local, and is never distributed by the blood. The following experiment
is deemed conclusive on this point : Experiment !¡.—I frequently poisoned limited skin areas and prevented any
spread of the toxin by covering it immediately with zinc-oxid plaster. Under these
conditions a dermatitis never occurs in any other than the exact spot where the
toxin is applied. It is easily conceived, however, that in natural exposures several
elements would tend towards the occurrence of dermatitis in crops rather
than at all points simultaneously. Before all the poison has penetrated,
some of it might easily be transferred to other areas, thus producing a
new crop. Again, the poison might be transferred to new areas from
clothes or hair. Furthermore, it is not unlikely that where the skin is
thin, as on the face, wrists, etc., the time necessary for penetration—the
latent period—is shorter than where the skin is thicker—again account¬
ing for succession rather than for simultaneity. PHYSIOLOGICAL PATHOLOGY The exact physiological nature of the poisoning by rhus juice has
never been studied. This is not surprising when one realizes that the
exact nature of the toxin is still not fully known. The chemical problems
must first be solved before the physiologist can offer any explanation of
why this peculiar glucoside should cause inflammation. That there are
variations in general or local susceptibility in different individuals and
in the same individual at different times, cannot be doubted. But the
exact property of cells or fluids of the body that determines susceptibility
to rhus is as yet a mystery. SMOKE FROM BURNING PLANTS IS POISONOUS Another fact of interest, amply sustained by popular personal testi¬
mony, is that the smoke of burning rhus plants is especially toxic. This
is somewhat surprising after reading Pfaff's statement2 that "toxico-
dendrol is decomposed easily by heat."
The degree of heat required is
not mentioned. On this point the following experiment was made : Experiment 2.—Fresh green leaves were placed in a test-tube closed with cotton,
and submerged (except the open end) in boiling water for one hour. Dermatitis
was readily secured by contact with the boiled leaves, thus showing that the
poison is not destroyed by 100 C. for one hour. The literature presents no evidence on this point. The above experi¬
ment supports the popular belief that smoke from burning rhus (espe¬
cially if the plant is green) carries the poisonous juice of the plant. It
is probable that in a heated state the juice more readily penetrates the
epidermis and causes toxic inflammation. Experiment 3.—In order to make this test more positive, dried rhus leaves were
crowded into a glass tube, to one end of which was attached a rubber mouth piece. The glass was then held in the flame until the leaves burned. While blowing
gently through the tube, the smoky current was caused to impinge on the skin Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 EDWARD
VON
ADELUNG 151 of a susceptible person. The spot was then covered with zinc oxid plaster to retain
what poison may have been deposited. Characteristic dermatitis developed in
twenty-four hours. 5. W. W. Ford: "Antibodies to Glucosides with Special Reference to Rhus Toxi-
codendron," Journal Infectious Diseases, 1907, iv, No. 4.
6. L. E. Warren: "The Poisonous Principle of Rhus," Pharmaceutical Jour.,
Oct. 30, Nov. 6, 1909. RELATION TO SWEAT REACTION Misled by Maisch's statement that the poison was an acid, I made a
series of observations some years ago on sweat reactions as determined by
litmus paper, to see whether acidity or alkalinity of the sweat bears any
relation to susceptibility to the poison. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 THE
ARCHIVES
OF
INTERNAL
MEDICINE 152 Experiment 5.—A number of sweat reactions were taken and recorded, each
person being asked whether he was susceptible to the poison. Of each class—acid,
alkaline, and neutral—there
were
some susceptible and
some immune. Among
them
was one person with strongly acid sweat who was extremely susceptible. But there was another equally susceptible whose eaction was strongly alkaline. So that no relation to sweat reaction could be deiermined even though account
was taken of the physiological change of reaction during exercise. VACCINATION AND IMMUNITY Considerable popular testimony is available upholding the possibility
of vaccination against rhus by ingestion of the plant or its derivatives. There are those who state that the tincture of the fluid extract taken
internally prevents attacks on subsequent contact with the plant. Like¬
wise, many persons claim to have gained immunity by chewing or eating
small quantities of the rhus leaves. This prophylactic measure is said to
have been found valuable by the engineers of the Union Pacific Eailroad
Company while the line was being built through poison oak country. Similar testimony is obtained from residents in the Adirondack Moun¬
tains. And I am told by an eye witness that Indians and other residents
of New Mexico habitually eat the leaves each spring to avoid poisoning
during the summer. Experimental confirmation or scientific observation
of these folk-sayings is, however, entirely lacking. But they open the
large question of acquired immunity. Ford5 states that there is no
difficulty in showing that many persons are quite resistant to the action
of rhus toxin. On the other hand, the same writer expresses the opinion
that the so-called natural immunity is really an acquired immunity. He
raises the question, obviously difficult to answer, whether "the cases of
supposed natural immunity do not occur in individuals in whom as chil¬
dren the effects from handling the plant have gradually worn off, the
original dermatitis having been so insignificant as to have escaped notice,
or being so many years distant as to have been forgotten."
He concludes
that complete natural immunity, when put to experimental test, is
exceedingly rare. Warren" likewise thinks that "the belief in absolute
immunity is a delusion." Desiring to settle this question by experimental
evidence I made the following tests: Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 EXPERIMENTS ON IMMUNES days
Test 4: Applied same to forearm and covered with plaster. Eesult nega¬
tive after three days. y
Test 4: Applied same to forearm and covered with plaster. Eesult nega¬
tive after three days. days. Test 5 : Applied active tincture rhus to forearm, covered with zinc oxid
plaster. Eesult negative after several days. days
Test 5 : Applied active tincture rhus to forearm, covered with zinc oxid
plaster. Eesult negative after several days. f l plaster. g
y
Test 6: Believing him immune, he was sent to gather a sack of leaves. Eleven days later he noticed spots of dermatitis with itching and his face
became swollen. Ehus dermatitis was markedly developed on face and arms. p
g
y
Test 6: Believing him immune, he was sent to gather a sack of leaves. Eleven days later he noticed spots of dermatitis with itching and his face
became swollen. Ehus dermatitis was markedly developed on face and arms. Thus of seven "immunes" tested, all responded to the poison. This
indicates universal susceptibility to this toxin. Thus of seven "immunes" tested, all responded to the poison. This
indicates universal susceptibility to this toxin. EXPERIMENTS ON IMMUNES Experiment 6.—A. B. claims he acquired immunity through several
severe
attacks. A fresh rhus leaf
was applied to each forearm and held in place by
plaster. Both applications caused marked dermatitis. p
pp
Experiment 7.—E. L. claims natural immunity from birth. A fresh rhus leaf
was applied to each arm and held in place by plaster. Slight dermatitis was
caused on each arm. 153 EDWARD
VON
ADELUNG Experiment 8.—D. S. claims acquired immunity except from the smoke. A
fresh leaf
was applied to each arm and held in place by plaster. Both spots
showed dermatitis. Experiment 9.—J. C. claims natural immunity from birth. She therefore
offered to gather rhus leaves for me. While bringing them in she held them as
one does
a bouquet, against her chest. The waist being cut low permitted the
leaves to touch the skin. Numerous mild spots of dermatitis developed. Experiment 9.—J. C. claims natural immunity from birth. She therefore
offered to gather rhus leaves for me. While bringing them in she held them as
one does
a bouquet, against her chest. The waist being cut low permitted the
leaves to touch the skin. Numerous mild spots of dermatitis developed. p
p
Experiment 10.—E. F. claims immunity from birth. She accompanied J. C. gathering leaves. But she developed no dermatitis. Believing her immune, she
was engaged to gather a larger quantity of leaves. After this exposure of about
three hours among the February leaves, she developed numerous troublesome spots. g
y
p
p
Experiment 11.—L. A. claims immunity from birth. Tested with tincture of
rhus on forearms, he developed red itching spots. g
y
p
p
Experiment 11.—L. A. claims immunity from birth. Tested with tincture of
rhus on forearms, he developed red itching spots. p
g
p
Experiment 12.—A. E. claims immunity from birth. He
never
has
been
poisoned, though exposed. p
g
p
Experiment 12.—A. E. claims immunity from birth. He
never
has
been
poisoned, though exposed. ,
g
p
Test 1: Eubbed fresh green leaves
on forearm. Eesult negative, though
observed for one week. p
g
p
Test 1: Eubbed fresh green leaves
on forearm. Eesult negative, though
observed for one week. Test 2 :
Put active tincture on arm. Eesult negative after several days. i
E
l g
y
Test 3 :
Applied alcohol solution of pure toxin to the wrist. Eesult nega¬
tive after three days. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 7. Rhus toxicodendron. ANIMAL IMMUNITY Whether immunity can be induced experimentally in animals is an
important problem that was attacked by W. W. Ford5 (1907). His
experiments
were done
on rabbits and guinea-pigs, using fresh fluid
extract of Rhus toxicodendron. He states that after subcutaneous injec¬
tion of a toxic dose, there elapses a latent period of seven or eight days
in which the animal loses weight and dies at the end of another week of
nephritis. Incidentally, a slough is formed at the site of injection. The
estimated toxic dose for a 250 gm. guinea-pig was 0.25 c.c, and for an
800 gm. rabbit, 1 c.c. On the basis of these toxic doses, he gave repeated
small doses at intervals of
a few days, and claims to have immunized
animals so that they resisted successfully five or six times their fatal dose. y
y
Such immunized animals showed
no nephritis when killed. The
serum from these immunized animals protected other animals completely
against fatal doses of fluid extract of rhus. Thus Ford appears to have
produced active and passive immunity in these animals. These results,
however, are inconclusive because of the unfortunately small number of
experiments, and because of the material used—the fluid alcoholic extract,
which contains, besides an indefinite amount of rhus toxin, various unde- Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 THE
ARCHIVES
OF
INTERNAL
MEDICINE 154 termined extractives of the plant, some glycerin, and as much as 58 per
cent, of alcohol. Furthermore, in a personal communication Ford states
that he was unable to get the same results a second time, attributing
the failure to impotency of the fluid extract. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 COMMERCIAL FLUID
EXTRACT
OF RHUS An attempt was made to repeat Ford's work on animals, but the doses
he found to be toxic were without effect. The material used was Wyeth's
fluid extract of Rhus radicane.7
The result is seen in Table 1. Table 1.—Subcutaneous Injections
of Guinea-Pigs
with Wyeth's Fluid
Extract of Rhus Radicans, Containing 58 Per Cent Alcohol Table 1.—Subcutaneous Injections
of Guinea-Pigs
with Wyeth's Fluid
Extract of Rhus Radicans, Containing 58 Per Cent Alcohol Table 1.—Subcutaneous Injections
of Guinea-Pigs
with Wyeth's Fluid
Extract of Rhus Radicans, Containing 58 Per Cent Alcohol Ttfn
Weight
Wyeth's
TimeOb-
p?g "Ä
' ™" Ext·
sfTed>
Re81llt
°
c.c. Days
1
585
5
5
In 24 hours sick; dead 5 days. 5
240
3
1/6
Drunk 5 minutes; dead 1 day. 9
440
1
16
Negative; gained weight steadily ; no induration Re81llt Although the larger doses kill guinea-pigs, controls with the corre¬
sponding amount of alcohol alone, died, and exhibited the same indura¬
tion and necrosis at the injection site as described by Ford (see Guinea-
pigs 10, 18, 20 and 19, Table 2). Table 2.—Guinea-Pigs Receiving Subcutaneous Injections of Alcohol Table 2.—Guinea-Pigs Receiving Subcutaneous Injections of Alcohol
No. Weight, Alcohol,
Per Water,^J^"
Eesult
Pig Gms. c.c. Cent,
c.c
8
280
1.0
95
6
210
3.0
95
4
335
3.0
95
10
440
1.5
95
18
410
1.0
60
18
420
2.0
60
Day3
19
Induration and necrosis. Drunk; dead in few minutes. Drunk ; dead in few minutes. 17
Stupid; large necrosis; died. 2
Site softened, wt.=420 gm. 10
Large necrosis;
wt.=250
gm.;
died
no nephritis. 20
310
2.0
60
2.0
9
Drunk; necrosis; wt.=250 gm.; died
infected;
no nephritis. 19
360
2.0
98
1.0
11
Drunk; necrosis; wt.=220 gm.; died
infected; no nepuritis. Tables 3 and 4 seem to indicate that the alcohol solution of rhus toxin
is somewhat more toxic than the same amount of alcohol alone. However,
the short time intervening between the injection and death argues in
favor of the alcohol alone being responsible for the death. This is referred
to later. EDWARD
VON
ADELUNG 155 Table 3.—Besults of Intravenous Injections of 95 Per Cent. Alcohol in
Eabbits Table 3.—Besults of Intravenous Injections of 95 Per Cent. Alcohol in
Eabbits
Babbit
No. 10
6
10
4
5
7
Weight,
Gms. 2140
1050
2040
2560
1500
780
Alcohol,
95 Per Cent. 0.5
1.0
1.0
1.0
1.0
2.0
Time Observed
Days
4
7
2
1
1
Eesult
Negative
Negative
Negative
Negative
Negative
Died Table 4.—Eestjlts of Intravenous Injections of Alcoholic Solution of Ehus
Toxin in Eabbits Babbit
Weight,
No. Gms. 6
1015
6
1160
2240
2550
2560
2045
2580
8
1900
*Additional. 10
4
4
9
11
Toxin,
Gms. Table 1.—Subcutaneous Injections
of Guinea-Pigs
with Wyeth's Fluid
Extract of Rhus Radicans, Containing 58 Per Cent Alcohol 0.0125
0.0125
0.0125
0.0125
0.01625
0.025
0.025
0.05
Alcohol, 98 Time Observed
Per Cent., c.c. Days
Eesult
0.5
0.5
0.5
0.5
0.65
1.0
0.5
2.0
46
Negative
Negative
Negative
Negative
Died immediately
Died immediately
Negative
Died immediately Not desiring to work further with a substance containing an unknown
quantity of the poison, besides alcohol, glycerin and other undetermined
substances in considerable quantities, the plant was collected and the
pure toxin extracted after the method recommended by Syme:
Fresh
leaves of poison oak
are thoroughly extracted with absolute alcohol. After filtration, lead acetate in water is added till precipitation is com¬
plete. The precipitate is then washed well and dried at a low tempera¬
ture, when it is extracted in Soxhlet extractors for at least ten hours. The ether extract is then mixed up with water and treated with hydrogen
sulphid, to separate the lead. The water and ether are then separated,
the ether filtered, and well washed, after which it is evaporated at a low
temperature. The presence of the toxin is then confirmed by testing for
glucoside (Molisch's test) and by application to the skin, the physiological
test. This toxin was carefully weighed and dissolved in absolute alcohol,
so that 1 c.c. contained 0.0125 of pure toxin. INTRAVENOUS ADMINISTRATION OF SUSPENSION Noting that alcoholic menstruums, such as the fluid extract, produce
marked induration at the site of subcutaneous injection, thus rendering
problematic the amount of toxin entering the general system, it was deter¬
mined to introduce the toxin intravenously. But both alcohol and ether
when employed in workable quantities, intravenously, cause clotting of
the blood, obliterating the vessels, and resulting in rapid death from heart Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 THE
ARCHIVES
OF
INTERNAL
MEDICINE 15G clots. After studying various other possible
solvents for the toxic
glucosid,3 a suspension was finally settled on. It was prepared by taking
the desired dose of toxin dissolved in absolute alcohol, and mixing it
with freshly sterilized distilled water (0.5 ,to 2 c.c). This yields a sus¬
pension of the toxin so fine that it does not separate after standing for
several months. The addition of normal saline solution, however, does
cause it to precipitate inside of forty-eight hours. That this suspension
is still toxic was proved by tests on my arm. Such a suspension is readily
and safely introduced into the blood-stream without causing clotting or
death in the doses used. Table 5.-—Intravenous Injections of Rabbits with Suspension of
Rhus Toxin j
p
Rhus Toxin
.a
Weight, Toxin,
Alcohol, Water. Tlme 0J°~
„
,,
,3
. „
s
'
r,
'
served,
Result
3 o
Gms. Gms. c.c. c.c. ^
„„
'
d ¡2¡
Days
10
2360
.003125
.125
.375
5
Negative
10
2280
.003125
.125
.375
6*
Negative
Iff
2180
.0125
.5
.5
5*
Negative
10
2260
.0125
.5
.5
8*
Negative
12
1650
.025
1. 2. 30
Negative
16
2410
.0228
1. 2. 30
Gained weight
16
2675
.0228
1. 1. 11*
Dead; pleuro-pneumonia; no
nephritis. 15
2300
.0456
2. 2. 23
Negative
13
2170
.0684
3. 2. 30
Site necrosed
18
1700
.0456
2. 1. . . Died immediately
*Days additional. The suspension
was also given to guinea-pigs, subcutaneously,
as
shown in Table 6, without deaths from the toxin, though it caused
induration and necrosis at the site of injection. 8. The toxin is insoluble in glycerin or in rabbit serum. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 COMPARISON OF DOSAGE In my extraction of the rhus plants, I found that the plant yields in
pure toxin about 1/1,000 of its weight. One c.c. of the fluid extract used
represents 1 gm. of the plant. From these data it is assumed that 1 c.c. fluid extract contains 0.001 pure toxin. But the fluid extract is made by
extracting the plant with 65 per cent, alcohol, whereas I used 95 per
cent., which extracts much more toxin. The disparity of toxicity was
also clearly demonstrated by tests on my arm. So that while the above
dosage appears small, the toxicity as compared with the fluid extract is
satisfactory. Thus while Ford found 0.25 c.c. fluid extract fatal for a
250 gm. guinea-pig, the dose in terms of pure toxin, 0.0031 gm.,
or
.0000123 gm. toxin per gram weight of pig, is very much smaller than I
was able to give—0.03875 gm. or .0001385 gm. toxin per gram weight of
pig, as illustrated by Guinea-Pig 15, Table 6. And even in terms of fluid
extract, if one might assume that the extract Ford used was of equal
toxicity to mine, I was able to give a larger dose, as illustrated by Guinea-
Pig 9, Table 1. The doses I used were not fatal, the deaths occurring
from intercurrent diseases, and showing no nephritis. INTRAVENOUS ADMINISTRATION OF SUSPENSION Table 6.—Subcutaneous Injection of Guinea-Pigs with Suspension of
Rhus Toxin
o
-w
3
o "ö
&
Üo 2
.S" 2
H
S
m "
Result
bo
'v M
% S
o *i
ci
b S3
Oh
>
^
>
a
12
470
.0125
.5
.75
21
Slight induration
13
330
.0125
.5
.75
21
Slight induration
14
500"
.025
1.0
1.5
17
Induration and necrosis
15
280
.03875
1.5
3.5
12
Induration and necrosis
16
300
.0228
1.0
1. 33
nephritis. Necrosis. Died
17
430
.0456
2. 1. 29
No nephritis. Necrosis. Died. 8. The toxin is insoluble in glycerin or in rabbit serum. Table 6.—Subcutaneous Injection of Guinea-Pigs with Suspension of
Rhus Toxin Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 157 EDWARD
VON
ADELUNG CUTANEOUS TESTS From clinical observation of human cases, and from the internal
administration to animals,2 the toxin appears to have a selective action
for epithelial cells. For this reason it is customary to experiment on the
cutaneous surfaces of animals. Observations of this character indicate
clearly that individuals of the same species vary widely in their suscep¬
tibility to rhus poisoning, as witnessed by the following experiments on
rabbits and a monkey (Table 7). Of ten rabbits tested by applying rhus
poison to shaved spots on the outer surface of their ears, seven failed to
develop any dermatitis at all. Although the pure toxin
was applied
thoroughly to the thin skin of the monkey, the test was absolutely nega¬
tive, observed for one month. Table 7.—Besults of Applying Ehus Toxin to the Skin of Animals Table 7.—Besults of Applying Ehus Toxin to the Skin of Animals Time Observed
Rabbit No. Materials Used
Days
Result
1
Fluid extract . 90
Negative
3
Pure toxin . 18
Negative
5
Puretoxin. 14
Negative
4
10 per cent, tinct. 14
Negative
2
Green leaves . 2
Positive
6
Pure toxin . 14
Positive
4
Pure toxin . 5
Positive
11
Alcohol sol. toxin . 14
Negative
18
Alcohol sol. toxin . 14
Negative
17
Alcoholic sol. toxin. 14
Negative
Monkey. Pure toxin (left eyelid and chin) ... 30
Negative Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 158 THE
ARCHIVES
OF
INTERNAL
MEDICINE THE ANTIGENIC PROPERTIES OF RHUS TOXIN The possibility of producing an antitoxic serum is so attractive that
special study was made as to the possibility of artificial immunization of
animals. My experiments on this point indicate clearly that the toxin
is not in itself fatal for rabbits and guinea-pigs, and, therefore, no anti¬
toxic action of an immune serum could be tested on these animals. It is,
however, well known, that the Bordet-Gengou fixation reaction serves to
detect antibodies in an immune serum and the demonstration of such
substances was sought for in rabbits that had received several doses of the
toxin. The test was made with the sera of three animals, treated
as
follows : Table 8.—Rabbit 19. Immunization. Intravenous Injections of Suspension
of Rhus Toxin
Date
Aug. 6
Aug. 9
Aug. 10
Aug. 14
Aug. 16
Aug. 20
Aug. 27
Weight,
Gms. 1920
1980
2040
2020
1970
2030
0.0228
0.0228
Results
Toxin,
98 Per Cent. Water
Gms. Alcohol, c.c. c.c. 0.0228
1
1
Normal
. .. .. Edema of
ear
0.0228
1
1
1
Large necrosis of
ear
Bled to death for serum
Table 9.—Rabbit 15. Immunization. Intravenous Injection of Suspension
of Rhus Toxin
Date
Aug. 10
Aug. 14
Aug. 16
Aug. 20
Aug. 25
Aug. 27
Weight,
Gms. 2190
2260
2230
2180
Toxin,
Gms. 0.0228
0.0228
0.0228
0.0228
98 Per Cent. Alcohol, c.c. 1
1
1
1
Water
c.c. 1
1
1
1
Results
Normal
Large necrosis ear
Littered six
Bled to death for serum
Table 10..—Rabbit 13. Date
Aug. 10
Aug. 14
Aug. 15
Aug. 16
Aug. 20
Aug. 24
Weight,
Gms. 2190
2160
2150
2140
2160
2180
Intensive Immunization. Intravenous Injection of
Suspension of Rhus Toxin
Toxin,
Gms. 0.0456
0.0228
0.0228
0.0228
98 Per Cent. Alcohol, c.c. 2
1
1
1
Water
c.c. 2
1
1
1
Results
Normal
Normal
Edema of ears
Large necrosis ear
Bled to death for serum Table 8.—Rabbit 19. Immunization. Intravenous Injections of Suspension
of Rhus Toxin Table 9.—Rabbit 15. Immunization. Intravenous Injection of Suspension
of Rhus Toxin Date
Aug. 10
Aug. 14
Aug. 16
Aug. 20
Aug. 25
Aug. 27
Weight,
Gms. 2190
2260
2230
2180
Toxin,
Gms. 0.0228
0.0228
0.0228
0.0228
98 Per Cent. Alcohol, c.c. 1
1
1
1
Water
c.c. 1
1
1
1
Results
Normal
Large necrosis ear
Littered six
Bled to death for serum
Table 10..—Rabbit 13. Date
Aug. 10
Aug. 14
Aug. 15
Aug. 16
Aug. 20
Aug. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 THE ANTIGENIC PROPERTIES OF RHUS TOXIN ;
pe
Alexin=Normal guinea-pig serum, 18 hours old, 10 per cent, in normal saline. It is seen that these hemolytic tests failed to demonstrate the presence
of antibodies by the Bordet-Gengou method in the sera of rabbits that
had received repeated doses of the toxin. The conclusion suggested is
that attempts to demonstrate antibodies in rabbits treated with rhus toxin
are futile. This does not exclude the possibility of tissue immunity. Nevertheless, contrary to the reported experience of others, I am still
as sensitive to poison oak as ever, though I have been severely poisoned,
accidentally, on man3r occasions, and have poisoned limited areas experi¬
mentally a great many times. THE ANTIGENIC PROPERTIES OF RHUS TOXIN 24
Weight,
Gms. 2190
2160
2150
2140
2160
2180
Intensive Immunization. Intravenous Injection of
Suspension of Rhus Toxin
Toxin,
Gms. 0.0456
0.0228
0.0228
0.0228
98 Per Cent. Alcohol, c.c. 2
1
1
1
Water
c.c. 2
1
1
1
Results
Normal
Normal
Edema of ears
Large necrosis ear
Bled to death for serum With the sera from these supposedly immunized animals, viz. : Eabbits
19, 15 and 13, the fixation test was done as indicated in the experiment
shown in Table 11, which includes appropriate controls. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 EDWARD
VON
ADELUNG 159 Table 11.—Results of Fixation Tests with Serum from Immunized Antmals*
Tube
Rhus An-
No. tigen,
c.c. Immune Serum
56°, c.c. 4
"x"
5
"y"
7
Saline
8
"x"
10
11
12
13
14
"y"
"z"
"x"
"y"
"z"
15
"y"
16
"z"
17
Saline
18
Saline
No. 19,
No. 10,
No. 19,
0.3
0.3
0.3
Norm. Rab. 0.3
Norm. Rab. 0.3
Norm. Rab. 0.3
No. 19,
Saline
Saline
Saline
No. 15,
No. 15,
No. 15,
No. 13,
No. 13,
No. 13,
No. 15,
No. 13,
0.3
0.3
ff.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
c.c. Hemolysis
Alexin,
0.1
+ 25 minutes
Alexin,
0.1
-f- 15 minutes
Alexin,
0.1
+ 15 minutes
Alexin,
0.1
-f- 25 minutes
Alexin,
0.1
-(- 15 minutes
Alexin,
0.1
-f-
15 minutes
Alexin,
0.1
+ 15 minutes
Saline
0.1
0
Saline
0.1
0
Saline
0.1
0
Alexin,
0.1
-f- 15 minutes
Alexin,
0.1
-f- 15 minutes
Alexin,
0.1
-j- 15 minutes
Alexin,
0.1
+
18 hours
Alexin,
0.1
-f-
1 hr. 15 min. Alexin,
0.1
-j-
1 hour
Alexin,
0.1
+
15 minutes
Alexin,
0.1
+1 hour Table 11.—Results of Fixation Tests with Serum from Immunized Antmals* *Incubated 37° one hour; then added to each tube 1 c.c. 5 per cent, saline sus¬
pension sheep corpuscles, sensitized; incubated 1% hours. *Incubated 37° one hour; then added to each tube 1 c.c
pension sheep corpuscles, sensitized; incubated 1% hours. p
p
p
,
;
Note:
Antigen=Suspension of Rhus diversiloba toxin in normal saline, made
from saturated alcoholic solution of toxin, in following proportions: ,
g p p
"x"=5 per cent, saturated alcoholic solution with normal saline. y
5 p
,
"z';=.05 per cent, saturated alcoholic solution with normal saline. IMMUNEN SERUM While it is recognized that a person, though immune, might not yield
antitoxin in his serum, still, in connection with the previous experiments
in animal immunity, it was thought worth while to test therapeutical]}'
the serum of a person who shows marked resistance to the toxin. Blood
was therefore taken from the "Immune" heretofore referred to as "E. F.,"
Experiment 10. Experiment 13.—
Test 1 : A mixture of equal parts of water, tincture rhus, and blood from
E. F., an immune, was applied to my arm and knee. Dermatitis developed at
each place. ^
Test 2: A mixture of equal parts of E. F.'s blood and tincture rhus was
applied to my arm. Dermatitis developed. p
Test 1 : A mixture of equal parts of water, tincture rhus, and blood from
E. F., an immune, was applied to my arm and knee. Dermatitis developed at
each place. ^ p
Test 2: A mixture of equal parts of E. F.'s blood and tincture rhus was
applied to my arm. Dermatitis developed. pp
y
p
Test 3: Having smeared E. F.'s blood on my arm, tincture of rhus was
then applied on the same area. Dermatitis resulted. pp
p
Test 3: Having smeared E. F.'s blood on my arm, tincture of rhus was
then applied on the same area. Dermatitis resulted. From these tests it appears that blood from an immune contains nothing able
to inhibit the toxicity of rhus. app ed
From these tests it appears that blood from an immune contains nothing able
to inhibit the toxicity of rhus. y
Experiment Ik {Ammonia Water).—Equal parts of ammonia and tincture rhus
were mixed and tested on the skin. The mixture is toxic, from which it is evident
that ammonia does not destroy the poison. y
p
Experiment 15 (Peroxid of Hydrogen).—Because it is a strong oxidizer, it was
presumed that hydrogen peroxid would have some curative property. But when
tested by adding it in equal quantity to the rhus tincture, it failed to inhibit the
toxicity, and when tested, with control, on a patch of dermatitis it was found inert. y
p
Experiment 16 (Aristol).—The left of two artificial patches of dermatitis was
treated during five days with a solution of aristol in cotton-seed oil. The control
received no treatment. Both were scratched. Result: The untreated patch recov¬
ered first. THERAPEUTIC TESTS In addition to the above investigations into the nature and action of
the toxin, some observations and experiments were made testing ther¬
apeutic and prophylactic
measures. Two methods were employed in
studying therapy ; some persons were poisoned in the usual accidental way
by exposure to the plant in the country, and in other cases the dermatitis Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 THE
ARCHIVES
OF
INTERNAL
MEDICINE 160 was purposely induced9 by applying the toxin, either pure or in some
menstruum. The latter method is preferred because symmetrical spots
can be chosen, and the usual variables in individuals eliminated. In cases
of accidental poisoning the worse half of the body was selected for testing
the therapeutic agent, the milder half being used for control. The
prophylactic measures were tried on myself. The clinical observations
detailed below serve to illustrate the methods employed, and to indicate
the value of some treatments. 9. In all human skin tests the author was the subject, except where otherwise
mentioned; and the toxin always from Rhus diversiloba, except where otherwise
stated. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 IMMUNEN SERUM Experiment 11.—The
same test was made on the left of two spots produced
by green leaves. The aristol in oil was applied five times in four days and pro¬
tected by gauze held in place by plaster. Result:
No difference could be noted. Aristol therefore appears to be of no value. Experiment 18 (Castor Oil).—A mixture of equal parts of 10 per cent, tincture
of rhus and castor oil was rubbed on the arm. In thirty-six hours there was a
slight itching, but no eruption. g
g
p
Experiment 19 (Cedar Oil).—A similar mixture with cedar oil was applied to
the arm. After thirty-six hours, a slight eruption appeared which later developed
into a fair patch of itching dermatitis. EDWARD
VON
ADELUNG 161 Experiment 20
(Cotton Seed Oil).—A similar mixture with cotton seed oil
was applied to the thin skin at the wrist. After eight days no dermatitis had
appeared. pp
Experiment 21.—Cotton seed oil in which green leaves had been soaked for
twenty-four hours was applied to the arm. The iesult was
a slight dermatitis. This, repeated on another person, gave the same result. p
g
Experiment 22.—Some cotton seed oil in which green leaves had been heated
was applied to the arm. After four days, itching began, and in five days,
a
slight eruption. g
p
Experiment 2S.—A mixture of equal parts of tincture of rhus and cotton seed
oil was applied to the arm. No dermatitis was detected in eight days. These results raised the question whether cotton-seed oil did not com¬
bine chemically with the toxin, destroying the toxicity. To test this
point, the mixture of oil and tincture was allowed to stand a few days
when the tincture formed a layer above the oil. This supernatant fluid
was toxic, producing dermatitis. Experiment 2!¡.—A mixture of equal parts of tincture rhus and of tincture of
green soap was applied to the arm and protected by gauze. After twenty-four
hours only a slight eruption was noted. y
g
p
Experiment 25.—A mixture of tincture of rhus, 25 parts, and tincture of green
soap, 5 parts, was applied to arm and protected with gauze. Only slight dermatitis
resulted. Experiment 26.—After allowing tincture of green soap to dry on a spot, tinc¬
ture of rhus was applied. A control spot was made with the rhus alone. Both
spots
were protected with gauze. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 IMMUNEN SERUM abso ute y
Experiment 31/.—One of two patches of dermatitis was painted with potassium
permanganate in the above strength. The treated patch healed earlier than the
control. Experiment 35 (Magnesium Sulphate).—Chemical Tests:
A fresh rhus leaf
macerated with
a saturated solution of magnesium sulphate remained toxic,
as
proven by testing on my arm. Experiment 35 (Magnesium Sulphate).—Chemical Tests:
A fresh rhus leaf
macerated with
a saturated solution of magnesium sulphate remained toxic,
as
proven by testing on my arm. p
y
g
y
Experiment 36.—Saturated solution of magnesium sulphate (Squibb's), added
in equal quantity to tincture rhus, does not inhibit the toxicity, for dermatitis
results when the mixture is applied to the skin. THERAPEUTIC TESTS Though magnesium sulphate fails to destroy the toxicity of rhus when
mixed with its tincture, or its juice, it nevertheless possesses definite
therapeutic value. Experiment 37.—Test 1 :
In one of two patches of dermatitis, saturated solu¬
tion of magnesium sulphate was rubbed three times at hour intervals. By the
next morning the treated patch was better than the control. Three more similar
applications were made during the forenoon and by noon a very marked improve¬
ment was noted. The treated patch recovered earlier than the control. d
i i patch
Test 2: To one of two patches of dermatitis, saturated solution of magnesium
sulphate (Squibb's)
was applied
on gauze, covered with rubber tissue, and
a
bandage. A similar dressing was applied to the control, using water in place of
magnesium. After twenty-four hours, while the first patch was not healed, it was
free from itching, was not tender, and not edematcus, thus contrasting with the
control, which remained tender for six days. p
Test 2: To one of two patches of dermatitis, saturated solution of magnesium
sulphate (Squibb's)
was applied
on gauze, covered with rubber tissue, and
a
bandage. A similar dressing was applied to the control, using water in place of
magnesium. After twenty-four hours, while the first patch was not healed, it was
free from itching, was not tender, and not edematcus, thus contrasting with the
control, which remained tender for six days. IMMUNEN SERUM The control took well, while the soaped spot
showed a slight dermatitis. Thus it appears that soap deters the poison of rhus. g
pp
p
p
Experiment 21 (Ichthyol Collodion).—The worse of two patches of dermatitis,
three days old, was painted with a 5 per cent, ichthyol collodion daily. In twenty-
four hours distinct improvement was noted in the treated patch, and this patch
recovered earlier. This was confirmed in treating hospital cases. g
p
Experiment 28 ( Hyposulphite of Sodium ).—Tests with this substance were also
negative. It failed to inhibit the toxicity when added in large proportion to the
tincture of rhus; and it failed to show curative effect on dermatitis patches com¬
pared with controls. p
Experiment 29 (Iodid of Potassium).—This substance in strong solution, added
to an equal part of tincture rhus, failed to inhibit the toxicity as tested on the skin. q
p
y
Experiment 30 (Tincture of Iodin).—Full officinal strength tincture of iodin
when mixed with an equal quantity of tincture of rhus destroys the poison; for
when this mixture is tested on my arm no dermatitis results. If, however, the
strength is reduced to less than 5 per cent, of the mixture, by addition of water
or alcohol, the toxicity is not destroyed completely. y
y
p
y
Experiment 31.—Tincture of iodin also has curative property. To one of two
patches of dermatitis, officinal tincture of iodin
was applied. The application
burned. But the treated patch recovered earlier than the control. Itching quickly
subsided and healing followed. The spot remained discolored, and tender, pre¬
sumably from iodin burning. y
g
Experiment 32.—The right of two patches of dermatitis was rubbed with 90
drops of water mixed with 10 drops of tincture of iodin. The control was rubbed
with alcohol. The application of iodin caused
a burning sensation, not
severe. In twenty-four hours the rhus dermatitis had disappeared, but was replaced by
an iodin burn. The control ran a normal course. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 THE
ARCHIVES
OF
INTERNAL
MEDICINE 162 Experiment 33
(Potassium Permanganate).—A mixture of equal parts of
potassium permanganate (0.56 gm. in 120 c.c.) with tincture rhus, when tested
on the arm was found to be absolutely non-toxic. absolutely
Experiment 31/.—One of two patches of dermatitis was painted with potassium
permanganate in the above strength. The treated patch healed earlier than the
control. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 HOSPITAL CASES A. B., male, aged 23, was poisoned four days previously. Whole face
was
edematous and the right eye closed. On Oct. 25, 1911, in the afternoon, hot appli¬
cations of 2 per cent, permanganate were begun, but applied only to the right side
of the face. In thirty hours the right eye was in good condition. The left side
recovered tardily. y
B. C, male, March, 1912, face and hands severely poisoned. The right side
of the face and the right arm were treated with the hot permanganate, while the
opposite side was treated with hot standard photographer's solution of sodium
hyposulphite. In addition, both arms were bandaged in their respective solutions. After twenty-four hours the patient stated that the permanganate side felt dis¬
tinctly better than the other. The right side recovered the earlier. t ct y
g
E. U., male, aged 25, was poisoned four days previously. Both arms showed
marked edema, vesicles and pustules. He had already applied cold permanganate
six or seven times. Hot permanganate was applied frequently by the nurse. There
was no improvement in forty-eight hours. When the solution was changed to hot
mercuric chlorid, and bandaging begun with the same, recovery began. y
g
E. U., male, aged 25, was poisoned four days previously. Both arms showed
marked edema, vesicles and pustules. He had already applied cold permanganate
six or seven times. Hot permanganate was applied frequently by the nurse. There
was no improvement in forty-eight hours. When the solution was changed to hot
mercuric chlorid, and bandaging begun with the same, recovery began. g g
g
y
g
This case illustrates the uselessness of permanganate when the vesicles become
infected. ld
Th
h l
f
ll g g
g
y
g
This case illustrates the uselessness of permanganate when the vesicles become
infected. A. E., male, aged 45. Dermatitis one day old. The whole face was swollen. The eyes half closed by edema of both lids. The right
ear was much swollen. Thirty hours after the dermatitis began, hot permanganate saturated solution was
applied to all
areas. The application burned severely, especially the eyelids. Recovery occurred in five days, though this man had always been two weeks getting
well. A weaker solution of permanganate would have been better. TREATMENT Inasmuch as the poison, after alighting on the skin, takes some time
to penetrate, the first thing to do is to prevent penetration by a soap and
hot-water bath of the whole body, including the hair. No article of
clothing should be donned that has been exposed to the poison. Itching
is readily relieved by water
as hot as can be borne, and is usually a
pleasant treatment, and of itself assists cure. The remedies previously
discussed may then be used. If pustules have formed, the condition of
bacterial infection must be recognized and will alter treatment, as illus¬
trated by hospital case, E. U. PROPHYLAXIS I have frequently protected myself against poisoning, as have others,
when not in direct contact with the plant, by simply washing the exposed
surfaces within a few hours after exposure, using soap and hot water. When, however, I am to be thoroughly exposed, as in gathering the leaves
or handling the dried plants, I prefer the protection of cotton-seed oil
on hands, arms and face, gloves, a bath for the whole body as soon after
exposure as possible, and a change of clothing. HOSPITAL CASES Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 EDWARD
VON
ADELUNG 163 There are two objections to the use of potassium permanganate. It
produces a mahogany brown stain, at times difficult to remove. However,
a 1 per cent, solution of oxalic acid is usually sufficient to disperse the
stain. It should not be forgotten that this acid, internally, is a violent
poison, and should be used with great caution. The other objection is
that after its use, followed by oxalic acid, the skin is left severely cracked,
which is especially disagreeable on the face and hands. This is met by
soothing ointments or oils. I gladly acknowledge my lasting obligation to Dr. F. P. Gay, Dr. G. Y. Rusk,
and Dr. Chas. . Bennett of the University of California for their valuable advice
and help. CONCLUSIONS The toxic principle of rhus, while non-volatile, can poison at a dis¬
tance by means of mechanical carriers. It is not destroyed by subjection
for one hour to 100 C, and is carried, potent, by the smoke from burning
rhus plants. The dermatitis is purely a local affection, and is not spread
by the blood or lymph, or by the serum of the blebs. The sweat reaction
has no relation to susceptibility. Absolute immunity in man is improb¬
able, and experimental immunity in animals is not yet demonstrated. A permanent aqueous suspension of the alcoholic solution of the toxin
can be prepared and remains toxic; this is precipitated by salt. As
much as 0.025 gm. toxin can be given intravenously in aqueous suspen¬
sion to a 2,000 gm. rabbit without fatal effect; and as much as 0.03875
gm. can be given to a 280 gm. guinea-pig subcutaneously without fatal
effect. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015 164 THE
ARCHIVES
OF
INTERNAL
MEDICINE Inasmuch as animal's cannot be killed by the pure toxin, it is impos¬
sible to demonstrate antitoxic effect by the serum of animals that have
received repeated doses of the toxin. The Bordet-Gengou fixation reac¬
tion failed to demonstrate the presence of antibodies in the
sera of
animals so treated. The simplest prophylactic measure against rhus poisoning is to wash
well with soap and hot water as soon after exposure as possible. The
protection of the skin by anointing with cotton-seed oil before exposure,
and washing this off within
a few hours with soap and water, renders
prophylaxis fairly certain. The following remedies have therapeutic
value : hot water, ichthyo] collodion, potassium permanganate, magnesium
sulphate and tincture of iodin. Downloaded From: http://archinte.jamanetwork.com/ by a New York University User on 05/15/2015
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https://openalex.org/W4390113438
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https://www.researchsquare.com/article/rs-3758965/latest.pdf
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A Universal Validation Method for Mitigating Machine Learning Extrapolation Risk
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A Universal Validation Method for Mitigating Machine
Learning Extrapolation Risk
Fangyou Yan
(
yanfangyou@tust.edu.cn
)
Tianjin University of Science and Technology
Mengxian Yu
Tianjin University of Science and Technology
Yin-Ning Zhou
Shanghai Jiao Tong University
Qiang Wang
Tianjin University of Science and Technology
Article
Keywords:
Posted Date: December 22nd, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3758965/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License.
Read Full Licen
Additional Declarations: There is NO Competing Interest. A Universal Validation Method for Mitigating Machine
Learning Extrapolation Risk
Fangyou Yan
(
yanfangyou@tust.edu.cn
)
Tianjin University of Science and Technology
Mengxian Yu
Tianjin University of Science and Technology
Yin-Ning Zhou
Shanghai Jiao Tong University
Qiang Wang
Tianjin University of Science and Technology
Article
Keywords:
Posted Date: December 22nd, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3758965/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License
Additional Declarations: There is NO Competing Interest. Main Machine learning (ML) has made impressive achievements in substance discovery, data analysis, and image processing
over the past decades, accelerating advances in fields as numerous as earth & life1–3, communications &
transportation4–10, and chemistry & medicine11–18. Spotlight to the field of chemistry, ML provides experimentalists with
advice on selecting target molecules for synthesis by predicting physicochemical properties11–15, biological effects16–18,
and reaction routes19–21. Although ML models are still not a complete substitute for expert intuition22, they are
sufficiently sophisticated to recognize complex patterns beyond the reach of expert intuition to provide decision-making
advice for major challenges in science and engineering, as multiple algorithms and different architectures for ML
solutions emerge.23–25 Grasping the trustworthiness of the decision-making advice given by ML models remains challenging.26–28 Influencing
the trustworthiness of model decision-making involves the whole process of modeling, i.e., not only the preparation of
data but also the process of algorithm selection, hyper-parameterization, etc.29 The accurate prediction of previously
unknown things and the generation of reasonable decisions by ML models are derived from the data information
available during development. As such, model uncertainty arising from the range of data and its distribution may lead to
models making unconvincing (high-risk) decisions. For example, Li et al.30 discovered that ML models trained on
Materials Project 2018 may have severely degraded performance when predicting new compounds for Materials Project
2021, which was attributed to the changes in the distribution of the dataset. Model uncertainty can be estimated using
cross-validation and external validation tools.29,31,32 External validation is performed on data not involved in modeling. Cross-validation divides the training set according to various data partitioning schemes (e.g., random, leave-one-out,
cluster, or time-split33) to evaluate the performance of the model in future applications. Worth considering, the property
distribution of molecules in the training set may not be identical to the distribution of molecules encountered in the
future, i.e., molecules encountered in the future may be outside of the domain-of-applicability of the model. In time-split
cross-validation, the model is trained on data generated before a certain date and tested on a retained dataset generated
after that date. Thus, the time-split cross-validation method is deemed to be closer to the evaluation of new discoveries,
i.e., prospective validation29,34. Due to data availability constraints, it may be difficult to obtain data that conforms to
this approach in certain cases. Article License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License
Additional Declarations: There is NO Competing Interest. Page 1/17 Page 1/17 Abstract Machine Learning (ML) can provide decision-making advice for majo
rapid development has led to advances in fields like chemistry & med
transportation. Grasping the trustworthiness of the decision-making
especially when applying them to samples outside the domain-of-ap
situation (i.e., complete extrapolation-failure) that would occur in mo
algorithms is confirmed, and the root cause is revealed. Further, a un
the extrapolation validation (EV) method, is proposed, which is not re
architecture in its applicability. The EV method quantitatively evaluat
ML methods and digitalizes the extrapolation risk arising from variat
Meanwhile, the EV method provides insights and solutions for evalua
prediction and selecting trustworthy ML methods. Machine Learning (ML) can provide decision-making advice for major challenges in science and engineering, and its
rapid development has led to advances in fields like chemistry & medicine, earth & life, and communications &
transportation. Grasping the trustworthiness of the decision-making advice given by ML models remains challenging,
especially when applying them to samples outside the domain-of-application. Here, an untrustworthy application
situation (i.e., complete extrapolation-failure) that would occur in models developed by ML methods involving tree
algorithms is confirmed, and the root cause is revealed. Further, a universal extrapolation risk evaluation scheme, termed
the extrapolation validation (EV) method, is proposed, which is not restricted to specific ML methods and model
architecture in its applicability. The EV method quantitatively evaluates the extrapolation ability of 11 popularly applied
ML methods and digitalizes the extrapolation risk arising from variations of the independent variables in each method. Meanwhile, the EV method provides insights and solutions for evaluating the reliability of out-of-distribution sample
prediction and selecting trustworthy ML methods. Mathematical model extrapolation testing To observe the extrapolation ability of ML models, developed models for data with deterministic functional relationships
(i.e., linear univariate, linear multivariate, and nonlinear multivariate) by 11 ML methods, including multiple linear
regression (MLR), least absolute shrinkage and selection operator (LASSO), ridge regression (Ridge), support vector
machine (SVM), gaussian process regression (GPR), multilayer perceptron (MLP), adaptive boosting (AdaBoost),
extreme gradient boosting (XGBoost), RF, K-nearest neighbor (KNN) and gradient boosting decision tree (GBDT) To observe the extrapolation ability of ML models, developed models for data with deterministic functional relationships
(i.e., linear univariate, linear multivariate, and nonlinear multivariate) by 11 ML methods, including multiple linear
regression (MLR), least absolute shrinkage and selection operator (LASSO), ridge regression (Ridge), support vector
machine (SVM), gaussian process regression (GPR), multilayer perceptron (MLP), adaptive boosting (AdaBoost),
extreme gradient boosting (XGBoost), RF, K-nearest neighbor (KNN) and gradient boosting decision tree (GBDT)
algorithms. A test (B) set and a test (F) set are set up to evaluate the extrapolation performance of ML models, where the
test (B) set is dominated by dependent variables below the minimum value of the dependent variable in the training set,
and the test (F) set is dominated by dependent variables above the maximum value of the dependent variable in the
training set. Moreover, a test (I) set in which the dependent variable is included in the range of the dependent variable of
the training set is used to validate the interpolation ability of the models. Based on the results of models established by the initial hyperparameters of the 11 ML methods (Figs. 1a, b, and c,
Supplementary Information Tables S1 ~ S3), it is found that the regressors involving tree algorithms (i.e., RF, KNN, XGB,
AdaBoost, and GBDT) perform excellent predictability in the value domain of the training set, which is confirmed by the
squared correlation coefficients (R2) of training set and test (I) set being close to 1. However, facing target values outside
the value domain of the training set, their predicted vs. observed values behave as horizontal straight lines, with the fact
that the R2
test(B) and the R2
test(F) are both 0. This suggests that ML models involving tree algorithms have great
interpolation ability but may not have extrapolation ability. Since hyperparameters have non-negligible effects on model
performance, the hyperparameters of the models established by 10 methods other than MLR are optimized. Even for the
optimal models (Figs. Main While cross-validation and external validation provide an important tool for testing the potential utility of ML workflows,
nevertheless, they are unable to distinguish between the predictions for in-domain and out-of-domain samples, which
makes it hard to provide a quantitative evaluation of the extrapolation ability of a ML model. The consequences would Page 2/17 Page 2/17 be inconceivable if ML model extrapolation performance degradation even extrapolation-failure occurs in artificial
intelligence (AI)-driven applications, especially in high-risk scenarios such as self-driving cars, automated financial
transactions, and smart healthcare. Hence, a method for quantitatively evaluating the extrapolation ability of a model is
desired to reasonably circumvent the extrapolation risk. Here, 11 ML methods are tested for out-of-domain samples prediction results on datasets with linear univariate, linear
multivariate, and nonlinear multivariate functional relationships. Based on the extrapolation results, the involvement of
the tree algorithm is suspected as the prime culprit in extrapolation-failure of the ML model. Subsequently, the potential
reasons are explored by using the RF method as an example. To quantitatively evaluate the extrapolation ability, an
extrapolation validation (EV) method is proposed. The EV method is applied to ML models with data from deterministic
functional relationships, and the quantitative structural property relationship models for glass transition temperature
(Tg) of polyimide (PI) in the macromolecular field as a real-world application example. Revealing the reason for extrapolation-failure To gain insights into the reason for the poor extrapolation ability of regressors involv
developed for data from the linear univariate functional relationship is visualized, wh
(DTs), each of depth 4 (Fig. 2, Supplementary Information Figs. S4 and S5). Each nod
dichotomy point in the decision-making process. For any input value lower than the d
training set, each node is determined to be “True”, so the predicted value of each DT i
For any input value higher than the domain of the definition of the training set, each n
the predicted value of each DT is the maximum of its value domain. The predicted va
of the predicted values of all DTs. The example model, with only one independent var
value of 979.9619 and a minimum predictive value of 417.0969, the potential predict
[417.0969,979.9619] (Supplementary Information Figs. S4). Thus, the range of poten
independent variables is a closed interval in the case of ML methods involving tree al
independent variables, the dependent variable is a combined transformation of value
Further, the value domain constituted by the combined transformations of the potent
values of all the independent variables is a closed interval, which may be the reason f
models developed by ML methods involving tree algorithms. To gain insights into the reason for the poor extrapolation ability of regressors involving tree algorithms, an RF model
developed for data from the linear univariate functional relationship is visualized, which contains 10 Decision Trees
(DTs), each of depth 4 (Fig. 2, Supplementary Information Figs. S4 and S5). Each node can be considered as a
dichotomy point in the decision-making process. For any input value lower than the domain of the definition of the
training set, each node is determined to be “True”, so the predicted value of each DT is the minimum of its value domain. For any input value higher than the domain of the definition of the training set, each node is determined to be “False”, so
the predicted value of each DT is the maximum of its value domain. The predicted value of the RF model is the average
of the predicted values of all DTs. The example model, with only one independent variable, has a maximum predictive
value of 979.9619 and a minimum predictive value of 417.0969, the potential predicted value range is
[417.0969,979.9619] (Supplementary Information Figs. S4). Mathematical model extrapolation testing 1d, e, and f, Supplementary Information Tables S4 ~ S9), the R2
test(B) and R2
test(F) of the ML
models involving tree algorithms are still 0, which rules out the correlation between extrapolation disability and
hyperparameters selection. Furthermore, the predicted values of the optimal models of the MLR, LASSO, GPR, and MLP
methods are almost close to the observed values. Nevertheless, Ridge and SVM models using data from linear or
nonlinear multivariate functional relationships have large prediction errors when observed values are far away from the
domain of values in the training set, as evidenced by their R2
test(B) failing to reach 1 (Figs. 1e and f). During hyperparameter conditioning of the regressors involving tree-based algorithms (Supplementary Information Figs. S1 ~ S3), all predictions in the test (B) and test (F) sets are close to the maximum and minimum values in the training
set, respectively. Particularly noted is that AdaBoost, RF, and XGBoost models exhibit piecewise functional data Page 3/17 Page 3/17 relationships as the hyperparameters are varied, therefore, conjecturing that this may be the reason for the extrapolation-
failure of the model developed by ML methods of involving tree algorithms, i.e., having constant output values for a
certain range of input values. Revealing the reason for extrapolation-failure Thus, the range of potential predicted values for any of the
independent variables is a closed interval in the case of ML methods involving tree algorithms. When having multiple
independent variables, the dependent variable is a combined transformation of values within these closed intervals. Further, the value domain constituted by the combined transformations of the potential maximum to minimum predicted
values of all the independent variables is a closed interval, which may be the reason for extrapolation-failure for the
models developed by ML methods involving tree algorithms. Extrapolation Validation (EV) Method (1)
ED =
∑
i
(
)
1
ni
∑
j
ei,j
∑
j
ai,j
ei,j =
⎧
⎪
⎪
⎪
⎪
⎪
⎨
⎪
⎪
⎪
⎪
⎪
⎩
min
i (xtrain
i,j ) −xtest
i,j , xtest
i,j < min
i (xtrain
i,j )
xtest
i,j −max
i
(xtrain
i,j ), xtest
i,j > max
i
(xtrain
i,j )
0, others
ai,j =
⎧
⎪
⎪
⎪
⎪
⎪
⎪
⎨
⎪
⎪
⎪
⎪
⎪
⎪
⎩
∑
i
xtrain
i,j
−xtest
i,j , xtest
i,j < min
i (xtrain
i,j )
xtest
i,j −
∑
i
xtrain
i,j , xtest
i,j > max
i
(xtrain
i,j )
0, others
1
ni
1
ni Eq. (1)
ED =
∑
i
(
)
1
ni
∑
j
ei,j
∑
j
ai,j ai,j =
⎧
⎪
⎪
⎪
⎪
⎪
⎪
⎨
⎪
⎪
⎪
⎪
⎪
⎪
⎩
∑
i
xtrain
i,j
−xtest
i,j , xtest
i,j < min
i (xtrain
i,j )
xtest
i,j −
∑
i
xtrain
i,j , xtest
i,j > max
i
(xtrain
i,j )
0, others
1
ni
1
ni 0, others where, i and j are the serial numbers of the independent variables and samples, respectively, and ni and nj are the number
of independent variables and samples. Adopting the EV method, the optimal models for the tested data from mathematical relationships are evaluated
(Figs. 3b, c, and d, Supplementary Information Figs. S6 and S7). Root mean squared error (RMSE) is adopted as the
main statistical parameter in this work. For distinction, the RMSEs on the test (EV) set for the developed model and the
model re-fitted by the training (EV) set are represented by RMSEtest(Model) and RMSEtest(EV). For the data from whether
linear or nonlinear relationships, the RMSEtest(EV) of models developed by methods involving tree algorithms, such as
AdaBoost, RF, and GBDT, is large. For example, RMSEtest(EV) of ML models involving tree algorithms developed on the
data obtained from linear univariate functions were all greater than 50 (Figs. 3b). Models established by such as MLP,
GPR, and SVM methods have better extrapolation ability, i.e., RMSEtest(EV) is nearly 0. Results of EV indicate that the
models developed by ML methods involving tree algorithms have poor extrapolation ability, while the models developed
by ML methods non-involving tree algorithms have good extrapolation ability, which is consistent with the results of
Section 2.1. Extrapolation Validation (EV) Method Further, since the methods involving tree algorithms have small prediction errors in model development (i.e.,
RMSEtest(Model)) but big prediction errors in the model application (i.e., RMSEtest(EV)), this creates the phenomenon of
performance degradation leading to reduced ML model trust. So, a prior evaluation of extrapolation ability using the EV
method will help in selecting a trustworthy ML model. Extrapolation Validation (EV) Method To quantitatively evaluate the extrapolation ability of a model, the extrapolation validation (EV) method is proposed. Each independent variable is serialized, and then the training and test sets are re-divided following the order in the
determined ratio. That is, the dataset is divided into training (EV) set and test (EV) set in the order of serialized
independent variables, e.g., choose the first 80% as the training (EV) set and the remaining 20% as the test (EV) set
(Fig. 3a). This provides data support for adopting performance of the test (EV) set (i.e., samples that are not exactly
within in the domain of application for the model) to evaluate extrapolation ability of a model. Considering the
contribution from all the independent variables, the serialized leverage value35 (h; Methods) is applied for dividing the
training and test sets. Both forward and backward sequences are adopted, that is, forward extrapolation validation and
backward extrapolation validation. The extrapolation performance of the serialized independent variables is evaluated
by the performance on the test (EV) set from re-fitting the model using the training (EV) set. Following this approach, all
independent variables for the developed model are evaluated. When serializing extrapolation for one independent variable, it is difficult to ensure that all independent variables in the
test (EV) set are outside the corresponding range of the training (EV) set, at which point the performance of the test (EV)
set will inevitably include the contribution from interpolation. Hence, the extrapolation degree (ED; Eq. (1), Fig. 3a) is
defined as a metric to assist in evaluating the extrapolation ability of the model. The ED quantifies the extent to which
the independent variables of the test (EV) set are outside the corresponding domain of definition of the training set
thereby digitalizing the contribution of extrapolation ability in the performance of the test (EV) set. Furthermore, for
samples far from the domain of definition of the training set, the predicted values of Ridge and SVM models developed
with linear or nonlinear multivariate functional relationship data differ significantly from their observed values, in
Section 2.1 (Figs. 1e and f). This emphasizes the fact that the reliability of the predicted values of a model for dataset
out-of-distribution samples is related to the distance between the sample independent variable and the domain of
definition of the training set. Page 4/17 Page 4/17 Eq. Application of EV method This indicates that the prediction error of this model even exceeds AE when the above mention independent variable in
the sample exists far away from the corresponding domain of definition of the training set. The MLP, MLR, LASSO, GPR, and Ridge models have RMSEtest(EV) of any I close to the corresponding RMSEtest(Model)
(Figs. 5a, b, c, and e), which indicates their good predictive ability. In addition, applying these models to predict the value
of a new sample can be considered reliable when the ED of the predicted sample is smaller than the maximum ED of the
extrapolation validation. For I10, I13, and I9 of the SVM model have low backward EDs and large RMSEtest(EV), therefore, in
applying this model, if there are I10, I13, and I9 in the prediction samples that are smaller than the minimum in the
corresponding training set, the predicted values may be unreliable, i.e., extrapolation of such independent variable is not
recommended. For the I5 of forward extrapolation validation in the SVM model, which has a high forward ED but a small
RMSEtest(EV). It means that this independent variable has little effect on the prediction reliability of the model when it
exceeds the definitional domain of the corresponding training set, therefore, the predicted value of the sample can be
considered reliable when such independent variables are extrapolated. Application of EV method An application of the EV method is demonstrated with the help of a PI-Tg model36, developed on a large dataset
containing 1321 Tg. Besides the MLR model developed in the literature (R2 = 0.8793, Q2 = 0.8718), 10 models such as
MLP, RF, and GBDT are established, all of which are fully consistent with the literature in terms of independent variables
(norm index, I), training set, and test set settings (Supplementary Information Table S10 and Fig. S8). The extrapolation
ability of 11 PI-Tg models is evaluated by the EV method (Figs. 4 and 5). In the case of EV instance for PI-Tg models, the RMSEtest(EV) for forward and backward serialized extrapolation
validations for models established by the tree-involving algorithm is always larger than that for models established by
the non-tree-involving algorithm, for every I and h (Fig. 4a). To evaluate the overall extrapolation ability of the model, the
average of RMSE for the independent variable and h extrapolation validation, i.e., the average RMSEtest(EV) and the
average RMSEtest(Model), is used as the statistical parameter. The average RMSEtest(EV) of MLP, MLR, Ridge, GPR, and
LASSO is around 20°C (Fig. 4b), which is close to experimental measurement error and acceptable37. By contrast, the Page 5/17 Page 5/17 average RMSEtest(EV) of the models developed based on RF, KNN, GBDT, XGBoost, and AdaBoost methods is larger, with
around 40°C (Fig. 4b), which suggests that these models involving tree algorithms have relatively poor extrapolation
ability. Furthermore, the standard deviation of the samples within the 95% confidence level interval (σ95) is presented as a
threshold for the evaluation of the extrapolation ability. If the RMSEtest(EV) of an independent variable is greater than σ95,
then the prediction error of the model may be greater than the difference between the actual value and the mean of the
samples within the 95% confidence level interval. The RMSEtest(EV)s of the independent variables extrapolation
validation for models established by the ML methods of involving tree algorithms are all high, with several I
extrapolation validation even near the σ95 (60.35°C; Figs. 5f, g, h, i, and j). For instance, the I10, I18, I3, and I5 forward
extrapolation validation as well as the backward extrapolation validation of I9, and I8 for the AdaBoost model (Fig. 5f),
the XGBoost model of I10 and I3 forward serialization extrapolation and I9 backward serialization extrapolation (Fig. 5g). Discussion In this contribution, the extrapolation ability of models established by multiple ML methods is explored. Further, the
extrapolation validation (EV) method is proposed to quantitatively evaluate the extrapolation ability of a model. Establishing ML models from data with deterministic functional relationships found that ML models involving tree
algorithms are fixed for predicted values out of the training set domain, confirming its extrapolation-failure phenomenon. Taking the RF model as an example reveals the intrinsic reasons for the poor extrapolation ability of the regressor
involving tree algorithms may be that the value domain constituted by the combined transformations of the potential
maximum to minimum predicted values of all the independent variables is a closed interval. The EV validation results
for the ML model with the data from defined functional relationships and with the 1321 PI-Tg data confirm that the
models developed by ML methods involving tree algorithms have poor extrapolation ability, while the models developed
by ML methods non-involving tree algorithms have good extrapolation ability. Before transitioning a model to
applications, the EV method is sufficient to evaluate the extrapolation ability of the model and help in selecting
trustworthy ML models. Meanwhile, the ED gives digital advice on the extent of reliability for the models to predict
samples. The EV method is not restricted by modeling approaches, such as advanced generative adversarial network
(GAN), convolution neural network (CNN), and recurrent neural network (RNN), and applies to any model architecture. Meanwhile, it provides the Data Science community with some insights and solutions for evaluating the reliability of out-
of-distribution sample prediction in ML models (e.g., molecular and material properties, reaction yields, etc.). Page 6/17 Page 6/17 where n is the number of samples. where n is the number of samples. Statistical parameters Statistical parameters Squared correlation coefficient (R2): R2 =
n
∑
i=1
((yi,exp−¯¯¯y exp)×(yi,cal−¯¯¯y cal))
2
n
∑
i=1
(yi,exp−¯¯¯y exp)
2×
n
∑
i=1
(yi,cal−¯¯¯y cal)
2 Squared correlation coefficient (R2): R2 =
n
∑
i=1
((yi,exp−¯¯¯y exp)×(yi,cal−¯¯¯y cal))
2
n
∑
i=1
(yi,exp−¯¯¯y exp)
2×
n
∑
i=1
(yi,cal−¯¯¯y cal)
2 Root mean squared error (RMSE):
Standard deviation (σ):
Mean absolute error (MAE):
RMSE = √
n
∑
i=1
(yi,exp −yi,cal)2
1
n
σ = √
n
∑
i=1
(y −
n
∑
i=1
y)
2
1
n
1
n
MAE =
n
∑
i=1
|yi,exp −yi,cal|
1
n Mean absolute error (MAE): MAE =
n
∑
i=1
|yi,exp −yi,cal|
1
n Three mathematical relationships for obtaining modeling data To obtain modeling data with clear functional relationships, five variables related to x, namely
,
,
,
, and
, were defined. With a tolerance of 2, arithmetic sequences in the range of [400,
1000), [488, 888), [20, 400), and [1000, 1400) were generated as the data for the training set, the test set (I), the test set
(F), and the test set (B) x, respectively. The dependent variable (y) data was calculated based on
,
, and
. This makes the dependent variable and the
x1 =
x
1
4
x2 = √x
x3 = log(x) x4 =
3√x
x5 = 1
x
y = x
y = x1 + x2 + x3 + x4 + x5
y = x1 + x2 × x3 + x4 + √x5 , and
. This makes the dependent variable and the
independent variable have linear univariate, linear multivariate, and nonlinear multivariate relationships accordingly. Complete datasets can be found in the Supplementary Data. y = x1 + x2 + x3 + x4 + x5
y = x1 + x2 × x3 + x4 + √x5 independent variable have linear univariate, linear multivariate, and nonlinear multivariate relationships accordingly. Complete datasets can be found in the Supplementary Data. Complete datasets can be found in the Supplementary Data. p
Development of mathematical relationship models Development of mathematical relationship models To establish ML models for multiple algorithms, the scikit-learn38 version 1.3.0 package for Python39 3.11 was adopted
to develop the MLR, LASSO, Ridge, GPR, MLP, AdaBoost, RF, GBDT, SVM, and KNN models, and the xgboost40 version
1.7.6 package was applied to develop the XGBoost model. The independent variable data were standardized for
modeling. The initial 33 ML models for the three datasets were established based on the default hyperparameters of a
total of 11 ML algorithms within the scikit-learn38 and xgboost40 packages. The phenomenon of extrapolation-failure was initially identified by analyzing the performance of the initial model
training set, test set (I), test set (F), and test set (B) by R2. Subsequently, the hyperparameters of 33 models were
optimized (Supplementary Information Figs. S1 ~ S3) by exhaustive enumeration to observe the influence of
hyperparameters on the performance of different algorithms. For selecting the model with the best extrapolation ability,
the model with the smallest average value of MAEtraining, MAEtest(F) and MAEtest(B) (
/ The phenomenon of extrapolation-failure was initially identified by analyzing the performance of the initial model
training set, test set (I), test set (F), and test set (B) by R2. Subsequently, the hyperparameters of 33 models were
optimized (Supplementary Information Figs. S1 ~ S3) by exhaustive enumeration to observe the influence of
hyperparameters on the performance of different algorithms. For selecting the model with the best extrapolation ability,
the model with the smallest average value of MAEtraining, MAEtest(F) and MAEtest(B) (
) statistical parameters within the tuned hyperparameters was taken as
the optimal model (Supplementary Information Tables S4, S6, and S8). The R2 of the optimal models for linear
univariate, linear multivariate, and nonlinear multivariate data relationships of 11 ML algorithms further confirms the
hypothesis that ML models involving tree algorithms may not have extrapolation ability. All models were saved and
extracted using joblib41 1.2.0 version and are available on https://github.com/https://github.com/fangyouyan. (MAEtraining + MAEtest(F) + MAEtest(B))/3 ) statistical parameters within the tuned hyperparameters was taken as
the optimal model (Supplementary Information Tables S4, S6, and S8). The R2 of the optimal models for linear
univariate, linear multivariate, and nonlinear multivariate data relationships of 11 ML algorithms further confirms the
hypothesis that ML models involving tree algorithms may not have extrapolation ability. All models were saved and
extracted using joblib41 1.2.0 version and are available on https://github.com/https://github.com/fangyouyan. Extrapolation Validation (EV) method The leverage value (h) is defined by all independent variables in the model. Within chemistry and drugs among other
related fields, h is often used to check compounds affected by structure (i.e., independent variables) in QSPR
modeling.35 Considering the contribution from all the independent variables, the serialized h is applied for dividing the
training and test sets. Both forward serialization (from small to large values) and backward serialization (from large to
small values) are adopted, i.e., forward extrapolation validation and backward extrapolation validation. The
extrapolation performance of the serialized independent variables is evaluated by the performance on the test (EV) set
from re-fitting the model using the training (EV) set. Following this approach, all independent variables for the developed
model are evaluated. Leverage value (h): h = xi(XTX)−1xT
i x i is the independent variable row-vector of the i-th compound, xi
T is the transpose of xi, X is the independent variable
matrix, and XT is the transpose of X. x i is the independent variable row-vector of the i-th compound, xi
T is the transpose of xi, X is the independent variable
matrix, and XT is the transpose of X. The performance of the re-fitted model by training (EV) set for the test (EV) set (RMSE was adopted in this work) was
used to evaluate the extrapolation ability. Of note, due to the stochastic property of ML methods such as RF, GBDT, and
GBDT, it is suggested to re-fit a model many times to obtain the average of several of the predicted values as the last
predicted value. A model was re-fitted 100 times in this effort. Considering that the extrapolation ability of the model is
related to the distance between the test samples and the training domain, the Extrapolation Degree (ED, Eq. (1)) is
defined. The ED is the measure of the extent to which the sample independent variables in the test (EV) set exceed the
range of the corresponding independent variable in the training (EV) set. Since not all sample independent variables of
the test (EV) set are outside the range of the training set, the performance of the test set (EV) includes the contribution
from interpolation ability. The ED can be an indication of the assistant evaluation of the extrapolation ability of the
model. p
Development of mathematical relationship models (MAEtraining + MAEtest(F) + MAEtest(B))/3 Page 7/17 Page 7/17 trapolation validation of mathematical relationsh Applying the EV method to evaluate the extrapolation ability of the three functional relations optimal model established
by 11 ML algorithms. Serialized extrapolation was performed on the h and independent variables of the training set, and
the training (EV) and test (EV) sets were re-divided in the ratio of 8:2, the ED was calculated and the extrapolation ability
of the model was evaluated. (Figs. 3(B), (C) and (D), Supplementary Information Fig. S6 and S7) Random Forest (RF) model visualization An RF model (Supplementary Information Fig. S4) with linear univariate relationships which has 10 DTs with a depth of
4 each (n_estimators = 10, max_depth = 4) was visualized (Supplementary Information Fig. S5) using pydotplus version
2.0.2. Extrapolation Validation (EV) method Furthermore, the standard deviation of the samples within the 95% confidence level interval (σ95) is presented as
a threshold for the evaluation of the extrapolation ability. If the RMSEtest(EV) of an independent variable is greater than
σ95, then the prediction error of the model may be greater than the difference between the actual value and the mean of
the samples within the 95% confidence level interval. By obtaining the average of all RMSEs of the independent
variables and h serialized extrapolation, i.e., the average RMSE, as a statistical parameter of the overall extrapolation
ability of the model evaluated. Development of PI- Tg models Page 8/17
The 29 norm indexes (I) required for MLR models developed in the literature were calculated. With these 29 Is as the
independent variables and the PI glass transition temperature (Tg) data provided in the literature as the dependent Page 8/17 Page 8/17 variables, 10 ML algorithmic models for LASSO, Ridge, GPR, MLP, AdaBoost, RF, GBDT, SVM, KNN, and XGBoost were
developed using scikit-learn38 version 1.3.0 and xgboost40 version 1.7.6. The I data were standardized for modeling. The
ratio of the training set to the test set in the modeling is completely consistent with the settings in the literature. Using R2
and MAE as statistical parameters to evaluate the established model, the optimal model hyperparameters were
determined with the best average performance between the training and test sets. (Supplementary Information Table
S12, Figure S8). Data availability The data for the three mathematical relationship models obtained in this work have been provided in the Supplementary
Data. The 1321 PI glass transition temperature (Tg) dataset is available from
https://pubs.acs.org/10.1021/acs.jcim.2c01389. The 33 mathematical relationship ML models developed in this paper
and the 10 PI-Tg models are available from GitHub (https://github.com/fangyouyan). Code availability Example code for the extrapolation validation (EV) method can be viewed from GitHub
(https://github.com/fangyouyan). 11 machine learning (ML) algorithm models were developed using the scikit-learn38
package version 1.3.0 in Python39 3.11 and the xgboost40 package version 1.7.6. The models were accessed via the
joblib41 package version 1.2.0. Random forest (RF) models were visualized with pydotplus package version 2.0.2. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (22222807, 22078195, and
22278319), and the Natural Science Foundation of Shanghai (20ZR1429800). Extrapolation validation of PI- Tg models Evaluation of the extrapolation ability of 11 PI-Tg models by using the EV method. Serialized extrapolation was
performed on the h and Is of the training set, and the training (EV) and test (EV) sets were re-divided in the ratio of 8:2,
the ED was calculated and the extrapolation ability of the model was evaluated. (Figs. 4 and 5) Competing interests The authors declare no competing interests. Author contributions M.X.Y. conceived the problem and carried out all detailed studies. F.Y.Y. analyzed the problem and designed the method. M.X.Y., F.Y.Y*, and Y.N.Z. co-analyzed the results. M.X.Y. wrote the manuscript, F.Y.Y* and Y.N.Z. made modifications. Q.W. provided strategic guidance. All authors contributed to useful discussions. References 1. Doudesis, D. et al. Machine learning for diagnosis of myocardial infarction using cardiac troponin concentrations. Nat Med 29, 1201-1210, doi:10.1038/s41591-023-02325-4 (2023). Page 9/17 Page 9/17 2. Fricke, E. C. et al. Collapse of terrestrial mammal food webs since the Late Pleistocene. Science 377, 1008–1011
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41. joblib. joblib, https://joblib.readthedocs.io/en/stable/ Figures Page 11/17 Figure 1
Predicted values vs. observed values (top) and R2 for the training set, test (F) set, test (I) set, and test (B) set (bottom) of
the initial hyperparametric model established for data with deterministic functional relationships i e a linear univariate Figure 1 Predicted values vs. observed values (top) and R2 for the training set, test (F) set, test (I) set, and test (B) set (bottom) of
the initial hyperparametric model established for data with deterministic functional relationships, i.e., a linear univariate,
b linear multivariate, and c nonlinear multivariate, by 11 ML methods; predicted values vs. observed values (top) and R2
for the training set, test (F) set, test (I) set and test (B) set (bottom) of the optimal hyperparametric model established for
data with deterministic functional relationships, i.e., d linear univariate, e linear multivariate, and f nonlinear multivariate,
by 11 ML methods. Page 12/17 Page 12/17 Page 12/17 Page 12/17 Figure 2
Schematic diagram of the model architecture containing 10 DTs (each with a depth of 4) for data with linear univariate
relationships via the RF method. Figure 2 Schematic diagram of the model architecture containing 10 DTs (each with a depth of 4) for data with linear univariate
relationships via the RF method. Schematic diagram of the model architecture containing 10 DTs (each with a depth of 4) for data with linear univariate
relationships via the RF method. Page 13/17 Page 13/17 Page 13/17 Figure 3
a Schematic of extrapolation degree (ED). Results of extrapolation validation (EV) for the ML model developed with data
from b linear univariate, c linear multivariate, and d nonlinear multivariate relationships. Figure 3 Figure 3 a Schematic of extrapolation degree (ED). Results of extrapolation validation (EV) for the ML model developed with data
from b linear univariate, c linear multivariate, and d nonlinear multivariate relationships. Page 14/17 Page 14/17 Figure 4
11 PI-Tg models extrapolation validation (EV) a overall result and b average RMSE statistical value. Figure 4 11 PI-Tg models extrapolation validation (EV) a overall result and b average RMSE statistical value. 11 PI-Tg models extrapolation validation (EV) a overall result and b average RMSE statistical value. Page 15/17 Figure 5
Extrapolation validation (EV) results of a MLR, b LASSO, c Ridge, d SVM, e GPR, f MLP, g AdaBoost, h XGBo
KNN and k GBDT Model. Note: The top-to-bottom order of the independent variables in the EV result plots is
with the large-to-small order of the sum of the differences between the RMSEtest(EV) and RMSEtest(Model) of t
extrapolation validation and backward extrapolation validation. Figure 5 Extrapolation validation (EV) results of a MLR, b LASSO, c Ridge, d SVM, e GPR, f MLP, g AdaBoost, h XGBoost, i RF, j
KNN and k GBDT Model. Note: The top-to-bottom order of the independent variables in the EV result plots is consistent
with the large-to-small order of the sum of the differences between the RMSEtest(EV) and RMSEtest(Model) of the forward
extrapolation validation and backward extrapolation validation. SupplementaryData.xlsx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryData.xlsx Page 16/17 SupplementaryInformation.docx Page 16/17 SupplementaryInformation.docx Page 17/17
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Contributions of EspA Filaments and Curli Fimbriae in Cellular Adherence and Biofilm Formation of Enterohemorrhagic Escherichia coli O157:H7
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PloS one
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public-domain
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RESEARCH ARTICLE Data Availability Statement: All relevant data are
within the paper. Funding: The authors have no support or funding to
report. Funding: The authors have no support or funding to
report. Contributions of EspA Filaments and Curli
Fimbriae in Cellular Adherence and Biofilm
Formation of Enterohemorrhagic Escherichia
coli O157:H7 Vijay K. Sharma1*, Indira T. Kudva1, Bradley L. Bearson2, Judith A. Stasko3 1 Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, ARS-USDA, Ames,
Iowa, United States of America, 2 Agroecosystems Management Research Unit, National Laboratory for
Agriculture and the Environment, ARS-USDA, Ames, Iowa, United States of America, 3 Microscopy Services
Laboratory, National Animal Disease Center, ARS-USDA, Ames, Iowa, United States of America * vijay.sharma@ars.usda.gov * vijay.sharma@ars.usda.gov a1111 Editor: A. Mark Ibekwe, U. S. Salinity Lab, UNITED
STATES Editor: A. Mark Ibekwe, U. S. Salinity Lab, UNITED
STATES Received: September 28, 2015
Accepted: February 4, 2016
Published: February 22, 2016 Received: September 28, 2015 Copyright: This is an open access article, free of all
copyright, and may be freely reproduced, distributed,
transmitted, modified, built upon, or otherwise used
by anyone for any lawful purpose. The work is made
available under the Creative Commons CC0 public
domain dedication. Data Availability Statement: All relevant data are
within the paper. Abstract In Escherichia coli O157:H7 (O157), the filamentous structure of the type III secretion sys-
tem is produced from the polymerization of the EspA protein. EspA filaments are essential
for O157 adherence to epithelial cells. In previous studies, we demonstrated that O157 hha
deletion mutants showed increased adherence to HEp-2 cells and produced abundant bio-
films. Transcriptional analysis revealed increased expression of espA as well as the csgA
gene, which encodes curli fimbriae that are essential for biofilm formation. In the present
study, we constructed hha espA, hha csgA, and hha csgA espA deletion mutants to deter-
mine the relative importance of EspA and CsgA in O157 adherence to HEp-2 cells and bio-
film formation. In vitro adherence assays, conducted at 37°C in a tissue culture medium
containing 0.1% glucose, showed that HEp-2 cell adherence required EspA because hha
espA and hha csgA espA mutants adhered to HEp-2 cells at higher levels only when com-
plemented with an espA-expressing plasmid. Biofilm assays performed at 28°C in a
medium lacking glucose showed dependency of biofilm formation on CsgA; however EspA
was not produced under these conditions. Despite production of detectable levels of EspA
at 37°C in media supplemented with 0.1% glucose, the biofilm formation occurred indepen-
dent of EspA. These results indicate dependency of O157 adherence to epithelial cells on
EspA filaments, while CsgA promoted biofilm formation under conditions mimicking those
found in the environment (low temperature with nutrient limitations) and in the digestive tract
of an host animal (higher temperature and low levels of glucose). OPEN ACCESS Citation: Sharma VK, Kudva IT, Bearson BL, Stasko
JA (2016) Contributions of EspA Filaments and Curli
Fimbriae in Cellular Adherence and Biofilm Formation
of Enterohemorrhagic Escherichia coli O157:H7. PLoS ONE 11(2): e0149745. doi:10.1371/journal. pone.0149745 * vijay.sharma@ars.usda.gov Introduction Enterohemorrhagic E. coli O157:H7 (O157) is an important foodborne pathogen of humans,
causing symptoms ranging from watery diarrhea to hemorrhagic colitis and hemolytic uremic Competing Interests: The authors have declared
that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 1 / 23 Cellular Adherence and Biofilm Formation syndrome [1, 2]. O157 encodes a variety of cell surface structures that directly or indirectly pro-
mote its adherence to intestinal epithelial cells as well as cultured epithelial cells in vitro [3–5]. The EspA filaments are needle-like extensions of the type-three secretion system (T3SS) [6]. The T3SS spans both inner and outer bacterial cell membranes of O157 and about 20 genes
encoded in the locus of enterocyte effacement (LEE) are required for the assembly of the T3SS
[7–10]. The T3SS secretes virulence proteins called effectors that are delivered and injected
into host epithelial cells through EspA filaments [8, 11, 12]. EspA, which is secreted by the
T3SS, polymerizes at the tip of the needle of the T3SS, located in the outer membrane, to form
hollow filaments measuring 12 nm wide and reaching a length of 260 nm or longer depending
on the availability of the secreted EspA protein [6, 8, 13, 14]. Besides serving as a conduit for
the translocation of effectors, EspA filaments also serve as adhesive elements and promote ini-
tial adherence of O157 to epithelial cells [15, 16]. LEE also encodes an effector protein called
translocated-intimin receptor (TIR) that is secreted by the T3SS and translocated through
EspA filaments into host epithelial cells [15–18]. TIR subsequently localizes in the epithelial
cell’s cytoplasmic membrane to serve as a receptor for a LEE-encoded bacterial outer mem-
brane adhesin, called intimin [17]. TIR-intimin interactions facilitate intimate adherence of
O157 bacteria to cultured mammalian cells, intestinal epithelial cells of primary reservoir ani-
mals, such as cattle, and incidental human hosts [16, 19–25]. In O157-infected cattle and other
ruminants, intimate adherence to mucosal tissues located proximal to the rectoanal junction
leads to the formation of characteristic histopathology, termed attaching and effacing lesions
[16, 19–22, 24–28]. A recent study has also demonstrated that EspA filaments are involved in
biofilm formation by enteropathogenic E. coli [29]. PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 Introduction In addition, the ability to adhere and colonize mammalian intestinal tissues by the LEE-
encoded intimate adherence mechanism initiated by EspA-epithelial cell interactions, O157
isolates have been shown to adhere to animal tissues, plants, and abiotic materials, such as plas-
tics, by employing unique sets of non-LEE encoded cell surface structures and proteins [30–
38]. For example, adherence to abiotic materials, which generally begins by the cessation of the
planktonic mode of existence, requires the expression of curli fimbriae [39–41]. Curli fimbriae
are highly aggregative bacterial functional amyloids promoting initial irreversible bacterial
adherence to abiotic surfaces as well as subsequent cell-cell interactions [42]. Curli fimbriae are
also an important constituent of the extracellular matrix of mature biofilms [39–43]. Curli fim-
briae, which range in size from 6 to 12 nm wide and 0.5 to 1 μm long, are composed primarily
of the curlin protein encoded by csgA of the csgBAC operon, which is transcribed divergently
from the csgDEFG operon [36]. The transcriptional regulator CsgD, encoded by csgD, is essen-
tial for the activation of these two operons [44, 45]. The csgDEFG operon also encodes proteins
needed for the transport of CsgA and the nucleator protein CsgB across the bacterial outer
membrane [46, 47]. Besides promoting survival of E. coli in the environment through biofilm
formation, there is increasing evidence that curli fimbriae contribute to virulence and dissemi-
nation of E. coli in animals by enhancing bacterial interactions with a variety of host matrices
and contact phase proteins [31, 36, 48]. Curli fimbriae have also been shown to promote bacte-
rial interactions with cultured epithelial cells, enhance bacterial invasion in animal models, and
contribute to severity of disease development in humans [32, 35, 49]. In previous studies, we demonstrated that hha is a negative transcriptional regulator of LEE,
including LEE-encoded espA, and the operons csgDEFG and csgBAC that encode proteins
mediating biosynthesis of curli fimbriae [33, 50]. Hha-mediated negative regulation of LEE
and the two curli operons results from direct repression of genes encoding transcriptional regu-
lators Ler and CsgD, which activate expression of genes encoded by LEE and the two curli
opreons, respectively [33, 50]. Determination of bacterial growth rates Bacterial strains were grown overnight on a shaker (200 rpm) incubator at 37°C in LB broth
containing carbenicillin (100 μg/ml). The overnight cultures were diluted 1:1000 and adjusted
to equivalent optical density readings at 600 nm (OD600) in YESCA (0.1% yeast extract and 1%
casamino acids) broth and Dulbecco’s Modified Eagle’s Medium containing 0.1% glucose
(DMEM) (Life Technologies, Grand Island, NY). Both media also contained carbenicillin at
100 μg per ml. These diluted samples (400 μl) were inoculated into the wells of 120-well Hon-
eycomb-2 plates. The plates were incubated at 37°C or 28°C in an automated growth curve
reader programmed for continuous shaking and collecting OD600 at 30 min intervals (Growth
Curves USA, Piscataway, NJ). The OD600 data were analyzed by using GraphPad Prism 6 soft-
ware (GraphPad Software, Inc., La Jolla, CA). The generation times of bacterial strains were
estimated from the exponential portions of the growth curves and equaled the time required
for the doubling of OD600. Bacterial strains, culture media, and growth conditions Bacterial strains and plasmids used in this study are listed in Table 1. All strains were derived
from a streptomycin-resistant isolate of EHEC O157:H7 strain 86–24 originally linked to a
foodborne disease outbreak in humans [52, 53]. Bacterial strains were cultivated in Luria-Ber-
tani broth (LB) or LB agar supplemented with antibiotics as needed (streptomycin 100 mg per
liter; kanamycin 50 mg per liter; and carbenicillin 100 mg per liter). Introduction Thus, a hha deletion in O157 allowed increased expression of
LEE, enhanced secretion of effector proteins including EspA, and increased adherence of the PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 2 / 23 Cellular Adherence and Biofilm Formation hha mutant strain on cultured epithelial cells [50, 51]. Deletion of hha also resulted in the pro-
duction of higher amounts of biofilm due to increased expression of csgA and other genes
required for regulation and biosynthesis of curli fimbriae [33]. In the present study, we investi-
gated whether increased expression of espA and csgA in the hha mutant strain of O157 contrib-
uted to increased adherence and biofilm formation independently or if both genes had
incremental effects on these two processes. Materials and Methods Bacterial strains, culture media, and growth conditions PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 coli O157:H7
[52, 53]
6431
Δstx2 derivative of 86–24
[50]
6491
Δhha mutant strain of 6431
[33]
6543
ΔcsgA mutant of 6491
[33]
6550
ΔespA mutant of 6491
This study
6552
ΔespA mutant of 6543
This study
TOP 10
F−mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (StrR)
endA1 nupG
Life Technologies
Plasmids
pCRXL
Cloning vector
Life Technologies
pACYC177
Low-copy cloning vector
New England
Biolabs
pBluescript II
SK
Cloning vector
Agilent
Technologies
pSM694
0.8 kb α-complementation fragment of lacZ (isolated by PCR from pBluescript) cloned at BamHI and BstEII sites
of pACYC177 for blue-white screening of bacterial colonies
This study
pAM450
Plasmid with a temperature-sensitive origin of replication
[50, 55]
pSM552
1.85 kb fragment carrying the 578 bp espA gene, 660 bp upstream, and 627 bp downstream of espA
This study
pSM556
pSM552 deleted of espA
This study
pSM601
0.86 kb fragment carrying the kanamycin resistance gene of the oBBI 92/93-neo cassette cloned at SalI site
located between upstream and downstream sequences of pSM552
This study
pSM615
2.2 kb XbaI fragment (carrying the kanamycin resistance gene flanked by the upstream sequence at the 5’ and
the downstream sequence at the 3’ end) cloned at XbaI site of pAM450
This study
pSM706
pSM694 containing the espA- complementing DNA fragment isolated by PCR from 6431
This study
pSM708
pSM694 containing the csgA- complementing DNA fragment isolated by PCR from 6431
This study
a Detailed descriptions of the construction of bacterial strains and plasmids listed are provided under material and methods. doi:10.1371/journal.pone.0149745.t001
PLOS ONE | DOI:10 1371/journal pone 0149745
February 22 2016
4 / 23 Table 1. Bacterial strains and plasmidsa. kanamycin resistance fragment (kan) generated by PCR from oBBI 92/93-neo cassette [54]
using primers VS777 and VS778 was cloned at the SalI site generated by a deletion of the espA
ORF in pSM556. The new espA-deleted plasmid (pSM601) was digested with XbaI to isolate a
2.2 kb DNA fragment containing 630 bp upstream of the espA ORF, 0.86 kb kan, and 660 bp
downstream of the espA ORF. The 2.2 kb XbaI fragment was cloned at the XbaI site of plasmid
pAM450, which encodes ampicillin resistance and a temperature-sensitive origin of replication
[50, 55]. The new recombinant plasmid (pSM615) consisting of pAM450-2.2 kb fragment was
electroporated into strains 6491 and 6543 for deleting the espA gene by using a previously
described allelic replacement method [50]. Recombinant DNA procedures The in-frame espA deletion mutants of EHEC O157:H7 strains 6491 (Δhha) and 6543 (Δhha
ΔcsgA) were constructed by using plasmid pSM552. Plasmid pSM552 was constructed by clon-
ing a 1.85 kb fragment generated by PCR using primers VS585 and VS972 (Table 2) and geno-
mic DNA of O157 strain 86–24. The 1.85 kb fragment contained 630 bp upstream of the espA
ORF (Open Reading Frame), 578 bp of the espA ORF, and 660 bp downstream of the espA
ORF. The PCR was carried out using FailSafe PCR Kit (Epicenter) according to the manufac-
turer’s instructions. The PCR fragment was purified by agarose gel electrophoresis. The DNA
fragment was recovered from an agarose gel using a Gel Extraction Kit according to manufac-
turer’s instructions (Qiagen, Valencia, CA). The gel-extracted 1.85 kb fragment was ligated
into pCRXL vector according to the manufacturer’s instructions (Invitrogen, Grand Island,
NY). The ligated DNA was electroporated into E. coli TOP10 cells as described above. The
recombinant plasmid pSM552 (pCRXL-1.85 kb fragment), thus constructed, was subjected to
PCR using primers VS257 and VS941 to delete the 578 bp espA ORF. The espA-deleted PCR
fragment of pSM552 was purified from agarose gel slices as described above and digested with
SalI to generate cohesive ends in SalI restriction sites present in primers VS257 and VS941. The SalI fragment was self-ligated and electroporated into an E. coli TOP10 bacterial host. The
resultant recombinant plasmid (pSM556) was purified from a TOP10 isolate. The 0.86 kb PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 3 / 23 doi:10.1371/journal.pone.0149745.t001 PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 Cellular Adherence and Biofilm Formation Table 1. Bacterial strains and plasmidsa. Strain or
plasmid
Genotype and description
Source or
reference
E. coli strains
86–24
stx2+ and streptomycin-resistant E. coli O157:H7
[52, 53]
6431
Δstx2 derivative of 86–24
[50]
6491
Δhha mutant strain of 6431
[33]
6543
ΔcsgA mutant of 6491
[33]
6550
ΔespA mutant of 6491
This study
6552
ΔespA mutant of 6543
This study
TOP 10
F−mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (StrR)
endA1 nupG
Life Technologies
Plasmids
pCRXL
Cloning vector
Life Technologies
pACYC177
Low-copy cloning vector
New England
Biolabs
pBluescript II
SK
Cloning vector
Agilent
Technologies
pSM694
0.8 kb α-complementation fragment of lacZ (isolated by PCR from pBluescript) cloned at BamHI and BstEII sites
of pACYC177 for blue-white screening of bacterial colonies
This study
pAM450
Plasmid with a temperature-sensitive origin of replication
[50, 55]
pSM552
1.85 kb fragment carrying the 578 bp espA gene, 660 bp upstream, and 627 bp downstream of espA
This study
pSM556
pSM552 deleted of espA
This study
pSM601
0.86 kb fragment carrying the kanamycin resistance gene of the oBBI 92/93-neo cassette cloned at SalI site
located between upstream and downstream sequences of pSM552
This study
pSM615
2.2 kb XbaI fragment (carrying the kanamycin resistance gene flanked by the upstream sequence at the 5’ and
the downstream sequence at the 3’ end) cloned at XbaI site of pAM450
This study
pSM706
pSM694 containing the espA- complementing DNA fragment isolated by PCR from 6431
This study
pSM708
pSM694 containing the csgA- complementing DNA fragment isolated by PCR from 6431
This study
a Detailed descriptions of the construction of bacterial strains and plasmids listed are provided under material and methods. Cellular Adherence and Biofilm Formation kanamycin resistance fragment (kan) generated by PCR from oBBI 92/93-neo cassette [54]
using primers VS777 and VS778 was cloned at the SalI site generated by a deletion of the espA
ORF in pSM556. The new espA-deleted plasmid (pSM601) was digested with XbaI to isolate a
2.2 kb DNA fragment containing 630 bp upstream of the espA ORF, 0.86 kb kan, and 660 bp
downstream of the espA ORF. The 2.2 kb XbaI fragment was cloned at the XbaI site of plasmid
pAM450, which encodes ampicillin resistance and a temperature-sensitive origin of replication
[50, 55]. The new recombinant plasmid (pSM615) consisting of pAM450-2.2 kb fragment was
electroporated into strains 6491 and 6543 for deleting the espA gene by using a previously
described allelic replacement method [50]. The genomic DNA from kanamycin-resistant and
ampicillin-sensitive mutants was screened by PCR using primers VS585 and VS972 (Table 2)
to confirm that the espA gene was deleted and it was substituted by the 0.86 kb kan fragment. For complementation experiments, a 4.2 kb DNA fragment containing the espA gene was iso-
lated by PCR from strain 86–24 using primers VS1085 and VS1086. Similarly, the 1.7 kb DNA
fragment containing the csgA gene was isolated from parental strain 86–24 by PCR using prim-
ers VS1087 and VS1088. These individual fragments were purified from agarose gels as
described above, digested with XbaI to produce cohesive 5’ and 3’ termini compatible for liga-
tion into the XbaI site of pSM694, a low-copy derivative of pACYC177 (New England Biolabs,
Ipswich, MA) carrying a 0.8 kb α-complementation fragment of β-galactosidase (Table 1). The
Table 1. Bacterial strains and plasmidsa. Strain or
plasmid
Genotype and description
Source or
reference
E. coli strains
86–24
stx2+ and streptomycin-resistant E. Determination of Congo red binding Binding of Congo red by bacterial cells was monitored as described previously [33, 41]. Briefly,
O157 strains were inoculated on YESCA containing 1.5% noble agar, Congo red (40 μg/ml),
coomassie brilliant blue (6.24 μg/ml) and carbenicillin (100 μg/ml). The plates were incubated
at 28°C for 24–48 h. The intensity of the red color of bacterial growth produced on this
medium was captured by photography. Cellular Adherence and Biofilm Formation Table 2. Primers used for PCR. Primer
Nucleotide sequencea
Locationb or reference
E. coli Primersc
VS257F
CAGGTCGACCTATATACCTCTTGATAATTTTTC
4662701–4662724
VS585F
GCGTCTAGACATCGACTGCCGTTTGCAGTG
4661472–4661493
VS777F
GATCGTCGACCGATAGCTGAATGAGTGACGTGC
[54]
VS778R
GATCGTCGACGCATAGAGCAGTGACGTAGTCGC
[54]
VS941R
GATCGTCGACCCGGAGATAACTATGCTTAAC
4662122–4662101
VS972R
CAGTCTAGAAGATTTATTAGGCGAAGATGATTG
4663350–4663327
VS1065F
GAAAATAGATCTCACATGTTCTTTCCTGCGTTATC
pBluescript II
VS1068R
GAAAATGGTTACCCTTAATGCGCCGCTACAGG
pBluescript II
VS1085F
GATCACTCTAGAAAAGGCACTGCCACAAAGAAAC
4659927–4659948
VS1086R
GATCACTCTAGAGTAATGGTTTATCTGCTTCATAG
4664194–4664172
VS1087F
GATCACTCTAGAACTGTCTGGTGTTTTTTGCTAG
1547911–1547932
VS1088R
GATCACTCTAGACCTCAATGATTAGTCATCCTTG
1549630–1549609
a Nucleotide sequences of primers used in this study were selected from the published genome sequence of E. coli O157:H7 strain EDL933 with the
accession number AE005174.2. b Location refers to the position of primer sequence in the genome of EDL933. c Subscripts F and R denote forward and reverse primers, respectively. Table 2. Primers used for PCR. Locationb or reference a Nucleotide sequences of primers used in this study were selected from the published genome sequence of E. coli O157:H7 strain EDL933 with the
accession number AE005174.2. b Location refers to the position of primer sequence in the genome of EDL933. b Location refers to the position of primer sequence in the genome of EDL933. c Subscripts F and R denote forward and reverse primers, respectively. doi:10.1371/journal.pone.0149745.t002 ligated DNA mixtures were separately transformed by electroporation into E. coli TOP10
according to manufacturer’s instructions. The new recombinant plasmids generated by this
ligation/electroporation were named pSM706 and pSM708. Plasmids pSM706 and pSM708
were electroporated into strains 6550 (Δhha ΔespA) and 6552 (Δhha ΔcsgA ΔespA) to comple-
ment deleted espA and csgA functions, respectively. The empty vector pSM694 was also electro-
porated into the parental and all other strains so that these strains could be used as controls in
subsequent experiments. The genomic DNA from kanamycin-resistant and
ampicillin-sensitive mutants was screened by PCR using primers VS585 and VS972 (Table 2)
to confirm that the espA gene was deleted and it was substituted by the 0.86 kb kan fragment. For complementation experiments, a 4.2 kb DNA fragment containing the espA gene was iso-
lated by PCR from strain 86–24 using primers VS1085 and VS1086. Similarly, the 1.7 kb DNA
fragment containing the csgA gene was isolated from parental strain 86–24 by PCR using prim-
ers VS1087 and VS1088. These individual fragments were purified from agarose gels as
described above, digested with XbaI to produce cohesive 5’ and 3’ termini compatible for liga-
tion into the XbaI site of pSM694, a low-copy derivative of pACYC177 (New England Biolabs,
Ipswich, MA) carrying a 0.8 kb α-complementation fragment of β-galactosidase (Table 1). The PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 4 / 23 Visualization of curli fimbriae by transmission electron microscopy For determining the presence of curli fimbriae, bacterial cells from 48 h old plates were sus-
pended in 2.5% glutaraldehyde solution, affixed to formvar grids, and stained with phospho-
tungstic acid. The stained grids were examined by transmission electron microscopy. Biofilm quantification The biofilm formation was determined by previously published procedures [33, 41]. Briefly,
bacterial cultures were grown overnight at 37°C (175 rpm) in YESCA broth containing carbe-
nicillin (100 μg/ml). The overnight cultures were diluted 1:10 in YESCA broth, YESCA broth
plus 0.1% glucose (YESCA-G), or DMEM containing carbenicillin (100 μg per ml). Two
hundred μl of diluted cultures were added to the wells of a 96-well polystyrene plate. After 48 h 5 / 23 PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 Cellular Adherence and Biofilm Formation of incubation at 28°C or 37°C, the culture liquid was aspirated, the wells were rinsed once with
PBS (phosphate-buffered saline), and the plate incubated at 80°C for 30 min. The heat-fixed
biofilms were stained with 0.1% crystal violet for 30 min and washed 3-times with distilled
water. Biofilm-bound crystal violet was eluted in 95% ethanol and absorbance of eluate was
determined at 590 nm (A590) using a spectrophotometer. Determination of bacterial adherence to HEp-2 cells Adherence assays were performed as described previously [56, 57]. Briefly, 1 x 105 bacterial
cells grown in LB broth (100 μg/ml carbenicillin) overnight at 37°C (200 rpm) were added to
HEp-2 cells (cultured in RPMI 1640 containing 10% fetal bovine serum) (Life Technologies,
Grand Island, NY) in chambered slides (for microscopic detection of adherent bacterial cells)
or in a 24-well tissue culture plate (for enumerating adherent bacterial cells) at ten bacteria to
one HEp-2 cell. After incubation for 3 h at 37°C in 5% CO2, slides and plates were processed as
follows. The slides were washed with Dulbecco’s Phosphate-Buffered Saline (DPBS) (Life tech-
nologies, Grand Island, NY) stained with toluidine blue, washed again, air dried, and examined
for adherent bacteria under a microscope equipped with a camera. The culture media was aspi-
rated from each well of the 24-well plate and saved into a sterile tube. Each well was washed
three times with one ml of DPBS and washes pooled with the aspirated culture medium corre-
sponding to that well to recover non-adherent bacterial cells. To recover adherent bacterial
cells, washed HEp-2 cells in each well were treated with 150 μl of 1% Triton-X100 for 10 min at
room temperature. The HEp-2 cell lysate was mixed with 850 μl of DPBS and homogenized by
repeated pipetting. Ten-fold serial dilutions of the pooled culture medium and HEp-2 cell
lysates were plated on LB agar (carbenicillin 100 μg/ml) plates and incubated at 37°C to deter-
mine the number of non-adherent and adherent bacterial cells, respectively. Non-adherent and
adherent bacterial counts were obtained from three independent assays with each assay per-
formed in duplicate wells. PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 Detection of EspA and CsgA Recommended volumes and dilutions of primary anti-rabbit EspA or anti-
rabbit CsgA antibodies (Pacific Immunology, Ramona, CA), iBind wash buffer, and goat-anti-
rabbit horseradish peroxidase-conjugated secondary antibody (KPL, Gaithersburg, MD) were
added in the designated slots of the iBind system. As a loading control, primary mouse anti-
Dnak antibody (AbCam, United Kingdom) was used for probing bacterial cell lysates to ensure
that the 75 kD DnaK protein was present in these lysates at similar levels. Following comple-
tion of protein transfer, the membranes were exposed to 3,3',5,5'-Tetramethylbenzidine Peroxi-
dase Substrate (KPL) to facilitate detection of blue-colored protein bands of predicted sizes. The membranes were rinsed in water and photographed using an AlphaImager (ProteinSim-
ple, Wallingford, CT). Bacterial growth rates varied in response to growth medium and
temperature The parental and the eight mutant strains showed identical growth profiles during the expo-
nential phase in DMEM or YESCA broth at 28°C and 37°C (Fig 1). For example, growth in
DMEM (Fig 1A) and YESCA broth (Fig 1B) at 28°C resulted in doubling times of about 240
min and 180 min, respectively, for all bacterial strains. Similarly, doubling times of 120 min
was observed for the parental and all mutant bacterial strains at 37°C irrespective of their
growth in DMEM (Fig 1C) or YESCA broth (Fig 1D). The longer doubling times observed
under these experimental conditions were due to the use of an automated growth reader
requiring culture volumes of less than one ml and allowing growth increases to occur in smaller
increments compared to growth curves generated by growing bacterial strains in large culture
vessels with a vigorous shaking. Overall, OD600 was markedly lower at 28°C during the station-
ary phase for bacterial strains expressing greater amounts of CsgA than the parental strain. On
the other hand, strains unable to express CsgA due to the deletion of csgA have higher OD600
compared to the parental strain during the stationary phase at 28°C in DMEM and YESCA
broth. All mutant strains had lower OD600 during the stationary phase compared to the paren-
tal strain at 37°C in DMEM or YESCA broth but the effect was less pronounced at 37°C than
that observed at 28°C. Statistical analyses A two sample, Students-t test was used to determine the significance of the differences in
adherence and biofilm formation between the mutant and the complemented mutants relative
to the parental strain. The differences were considered significant at p < 0.05. Detection of EspA and CsgA Western blotting was used for detection of EspA and CsgA produced by parental, mutant, and
complemented mutant strains. For detection of EspA, bacterial cultures were grown at 28°C or
37°C in YESCA broth, YESCA broth supplemented with 0.1% glucose (YESCA-G), or DMEM
containing 100 μg per ml of carbenicillin till the OD600 reached between 0.8–0.1(about 3.5 h). The cells were harvested by centrifugation (10, 000 x g) for 5 min and the cellular pellet was
suspended in 2X SDS-sample buffer (Bio-Rad, Hercules, CA). The detection of CsgA was
accomplished by growing bacterial strains in YESCA broth, YESCA-G broth, or DMEM con-
taining carbenicillin (100 μg per ml) for 48 h at 28°C or 37°C. An aliquot of these cultures, stan-
dardized to roughly the equivalent of OD600, was centrifuged as above and the cellular pellet
was acidified with formic acid at one-fifth of the volume of the original culture. Curli fimbriae
are highly resistant to sodium dodecyl sulfate (SDS) and other chemical treatments that are
commonly employed for preparing protein samples for analysis by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). Therefore, bacterial cells were treated with formic acid to depoly-
merize curli fimbriae to CsgA, the major constituent protein of curli fimbriae, in order to detect
this protein by SDS-PAGE. The acidified samples were kept on ice for 10 min and dried in a
miVac Centrifugal Vacuum Concentrator (Genevac, Inc., Stone Ridge, NY). The dried pellet
was dissolved in 2X SDS-sample buffer (BioRad, Hercules, CA). The 2X-SDS solubilized sam-
ples prepared for EspA and CsgA detection were heated for 10 min in a 95°C water bath,
loaded in to the wells of a 4–15% SDS-polyacrylamide gel, and subjected to electrophoresis
using a Tetra Cell electrophoresis unit according to the manufacturer’s instructions (BioRad, PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 6 / 23 Cellular Adherence and Biofilm Formation Hercules, CA). The proteins from the gels were transferred to the nitrocellulose membranes
using a mini Trans-Blot unit at 30 V overnight according to the manufacturer’s instructions
(BioRad, Hercules, CA). Following transfer, membranes were blocked in iBind solution and
placed protein-side down on an iBind Card of the iBind Western System (Thermo Scientific,
Grand Island, NY). Cellular Adherence and Biofilm Formation Fig 1. Determination of bacterial growth at 28°C and 37°C in DMEM and YESCA broth. Bacterial strains were grown overnight at 37°C in LB broth
containing carbenicillin (100 μg/ml) and diluted 1:1000 in DMEM or YESCA broth containing carbenicillin (100 μg/ml). The diluted samples were incubated at
28°C or 37°C for 24 h and optical density at 600 nm (OD600) was recorded every 30 min. Three independent cultures were tested in triplicate, and each point
on the growth curve represents an average of nine readings. The generation times were determined by computing the time required for doubling of OD600 of
the bacterial culture during the exponential phase of growth. Fig 1. Determination of bacterial growth at 28°C and 37°C in DMEM and YESCA broth. Bacterial strains were grown overnight at 37°C in LB broth
containing carbenicillin (100 μg/ml) and diluted 1:1000 in DMEM or YESCA broth containing carbenicillin (100 μg/ml). The diluted samples were incubated at
28°C or 37°C for 24 h and optical density at 600 nm (OD600) was recorded every 30 min. Three independent cultures were tested in triplicate, and each point
on the growth curve represents an average of nine readings. The generation times were determined by computing the time required for doubling of OD600 of
the bacterial culture during the exponential phase of growth. doi:10.1371/journal.pone.0149745.g001 adherence pattern resembling to that produced by the hha mutant strain (Fig 2A) and indicat-
ing the direct requirement of EspA in bacterial adherence to HEp-2 cells. On the other hand,
complementation of the hha espA csgA mutant with plasmid pSM708, encoding for CsgA,
resulted in the adherence phenotype of the complemented strain (csgA+/hha espA csgA) resem-
bling that of the uncomplemented hha espA csgA mutant strain showing very few bacterial cells
adhering to HEp2-cells (Fig 2A), indicating that CsgA is not essential for the direct adherence
of O157 to epithelial cells. Quantitative analysis of adherence (Fig 2B), which involved removing non-adherent bacte-
rial cells and recovering bacterial cells adhering to HEp-2 cells, showed 19-fold (p = 0.002)
higher recovery of adherent bacterial cells for the hha mutant compared to the parental strain. The percent adherence of the hha csgA deletion mutant was 5.5-fold higher (p = 0.026) than
the parental strain but 4-fold lower (p = 0.026) than the hha deletion mutant strain. doi:10.1371/journal.pone.0149745.g001 EspA, but not CsgA, was essential for adherence of O157 to HEp-2 cells Qualitative (microscopic) analysis of bacterial adherence to HEp-2 cells showed that the hha
mutant adhered to HEp-2 cells in large clusters consisting of several bacterial cells compared to
the parental, hha csgA, hha espA, and hha espA csgA mutant strains that adhered to HEp-2 cells
mostly as a few individual bacterial cells (Fig 2A). The complementation of hha espA and hha
espA csgA mutants with plasmid pSM706, encoding the espA gene, enabled the complemented
strains (espA+/hha espA, espA+/hha espA csgA) to adhere to HEp-2 cells as clusters, the 7 / 23 PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 Comple-
mentation with a csgA-encoding plasmid (pSM708) had no significant effect on the adherence
of the hha csgA mutant to HEp-2 cells as the percent adherence of the complemented mutant
remained 4-fold lower (p = 0.026) than the hha mutant strain. The adherence of hha espA dele-
tion mutant was not significantly (p = 0.093) different from the parental strain but was 4.5-fold 8 / 23 PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 Cellular Adherence and Biofilm Formation Fig 2. Effects of espA and csgA deletions on O157 adherence to HEp-2 cells and expression of EspA and CsgA. (A) Micrographs showing adherent
bacteria on HEp-2 cells (indicated by black arrows) of Parent (1), Δhha (2), Δhha ΔcsgA (3), csgA+/Δhha ΔcsgA (4), Δhha ΔespA (5), espA+/Δhha ΔespA (6),
Δhha ΔcsgA ΔespA (7), espA+/Δhha ΔcsgA ΔespA (8), csgA+/Δhha ΔcsgA ΔespA (9) mutant strains. (B) A bar graph showing the percent of adherent
bacteria on HEp-2 cells computed by dividing the number of adherent bacterial cells by the sum of non-adherent and adherent bacteria. Bars indicate
average of three independent assays and error bars represent standard deviation of 2. The * above the bars indicates p < 0.05 when Δhha mutant was
compared to the parent and the double and triple mutants were compared to the Δhha mutant strains. (C) Western blot analysis of cell lysates (prepared by
growing bacterial strains in DMEM at 37°C to an OD600 of 0.8 to 1.0) using anti-DnaK (top blot), anti-EspA (middle blot), and anti-CsgA (bottom blot) Fig 2. Effects of espA and csgA deletions on O157 adherence to HEp-2 cells and expression of EspA and CsgA. (A) Micrographs showing adherent
bacteria on HEp-2 cells (indicated by black arrows) of Parent (1), Δhha (2), Δhha ΔcsgA (3), csgA+/Δhha ΔcsgA (4), Δhha ΔespA (5), espA+/Δhha ΔespA (6),
Δhha ΔcsgA ΔespA (7), espA+/Δhha ΔcsgA ΔespA (8), csgA+/Δhha ΔcsgA ΔespA (9) mutant strains. (B) A bar graph showing the percent of adherent
bacteria on HEp-2 cells computed by dividing the number of adherent bacterial cells by the sum of non-adherent and adherent bacteria. Bars indicate
average of three independent assays and error bars represent standard deviation of 2. The * above the bars indicates p < 0.05 when Δhha mutant was
compared to the parent and the double and triple mutants were compared to the Δhha mutant strains. Cellular Adherence and Biofilm Formation antibodies. Molecular weights of protein standards (Lane M) are indicated in kD on right side of each blot. The locations of predicted or identified DnaK, EspA,
and CsgA proteina are indicated on the left. Lanes numbered 1 through 9 contains cell lysates of strains listed in the same order as in Fig 2A. antibodies. Molecular weights of protein standards (Lane M) are indicated in kD on right side of each blot. The locations of predicted or identified DnaK, EspA,
and CsgA proteina are indicated on the left. Lanes numbered 1 through 9 contains cell lysates of strains listed in the same order as in Fig 2A. antibodies. Molecular weights of protein standards (Lane M) are indicated in kD on right side of each blot. The locations of predicted or identified DnaK, EspA,
and CsgA proteina are indicated on the left. Lanes numbered 1 through 9 contains cell lysates of strains listed in the same order as in Fig 2A. antibodies. Molecular weights of protein standards (Lane M) are indicated in kD on right side of each blot. The locations of predicted or identified DnaK, EspA,
and CsgA proteina are indicated on the left. Lanes numbered 1 through 9 contains cell lysates of strains listed in the same order as in Fig 2A. doi:10.1371/journal.pone.0149745.g002 doi:10.1371/journal.pone.0149745.g002 lower (p = 0.0087) than the hha mutant strain. However, complementation of the hha espA
mutant with the espA-encoding plasmid (pSM706) enhanced its adherence by 16.5-fold
(p = 0.002) compared to the parental strain, the level of adherence that was not significantly
different (p = 0.305) from that of the hha mutant strain. The percent adherence of the hha
mutant deleted of both espA and csgA (hha espA csgA) was not significantly different
(p = 0.305) from that of the parental strain. Although complementation of hha espA csgA
mutant with the espA-encoding plasmid (pSM706) enhanced its adherence by about 7-fold
(p = 0.002) relative to the parental strain and by about 3-fold (p = 0.032) compared to the
uncomplemented hha espA csgA mutant strain, the adherence of the complemented (espA+/
hha espA csgA) mutant was still 3-fold lower (p = 0.02) than the hha mutant strain. On the
other hand, complementation with the csgA-encoding plasmid (pSM708) kept the percent
adherence of the complemented (csgA+/hha espA csgA) mutant at a level not significantly
(p = 0.53) different from the uncomplemented hha espA csgA mutant strain, the level of adher-
ence that was still 6-fold lower than the hha mutant strain. Western blot analysis of cell lysates (prepared from bacterial cultures grown to an OD600 of
0.8 to 1.0 at 37°C in DMEM-carbenicillin) using anti-EspA and anti-CsgA antibodies showed
the presence of 25 kD EspA and 17 kD CsgA bands in lysates of bacterial strains carrying either
a chromosomal copy of these two genes or were complemented for the deleted chromosomal
copies of these genes with a plasmid-encoded espA (pSM706) or csgA (pSM708) (Fig 2C). The
intensities of the bands corresponding to these two proteins were lowest for the parental strain
(Fig 2C) that also showed lowest adherence to HEp-2 cells (Fig 2A and 2B). On the other hand,
both EspA and CsgA bands were detected at much higher intensities in the highly adherent
hha mutant and hha espA and hha espA csgA mutants that were complemented with plasmid
pSM706 (Fig 2C). However, the hha espA and hha espA csgA mutants, which were unable to
produce EspA due to the espA deletion (Fig 2C), complemented with plasmid pS708 showed
highly reduced adherence compared to the hha mutant indicating that EspA is essential for epi-
thelial cell adherence (Fig 2A and 2B). Since 75 kD DnaK protein, used as a loading control,
was detected in the whole-cell lysates of all strains at identical levels (Fig 2C), the detection of
higher amounts of EspA and CsgA in the hha mutant strains harboring either the chromo-
somal or plasmid-encoded copies of these genes were likely due to the differential expression of
these two proteins in these strains. (C) Western blot analysis of cell lysates (prepared by
growing bacterial strains in DMEM at 37°C to an OD600 of 0.8 to 1.0) using anti-DnaK (top blot), anti-EspA (middle blot), and anti-CsgA (bottom blot) PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 9 / 23 PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 Biofilm formation was CsgA-dependent and did not require EspA Biofilm formation was initially examined after growth of each of the nine bacterial strains in
YESCA broth lacking glucose and incubation for 48 h at 28°C. Under these growth conditions,
which are normally considered conducive for biofilm formation, the hha and hha espA mutants
produced the highest biofilm biomass. This was indicated by the development of a dark blue
color (shown as black color) when the biofilms produced by these two mutants were stained
with a crystal violet solution (Fig 3A). On the other hand, the hha csgA and hha csgA espA
mutants were unable to produce detectable amounts of biofilms as the wells of the microtiter
plate corresponding to these strains did not stain with crystal violet and appeared similar in
coloration to the growth medium control (Fig 3A). When the hha csgA and hha csgA espA
mutants were complemented with csgA-encoding plasmid (pSM708) or with the espA-encod-
ing plasmid (pSM706), only pSM708 restored biofilm formation to these mutants as indicated
by the development of dark blue color with crystal violet staining of the wells inoculated with PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 10 / 23 Cellular Adherence and Biofilm Formation Fig 3. Qualitative and quantitative analysis of biofilm formation in YESCA broth at 28°C. (A) Single row of a microtiter plate showing the relative
intensity of crystal violet-stained biofilms produced in the wells inoculated with the Parent (1), Δhha (2), Δhha ΔcsgA (3), csgA+/Δhha ΔcsgA (4), Δhha ΔespA Fig 3. Qualitative and quantitative analysis of biofilm formation in YESCA broth at 28°C. (A) Single row of a microtiter plate showing the relative
intensity of crystal violet-stained biofilms produced in the wells inoculated with the Parent (1), Δhha (2), Δhha ΔcsgA (3), csgA+/Δhha ΔcsgA (4), Δhha ΔespA
(5), espA+/Δhha ΔespA (6), Δhha ΔcsgA ΔespA (7), espA+/Δhha ΔcsgA ΔespA (8), csgA+/Δhha ΔcsgA ΔespA (9) mutant strains. The last well in the row
represents the uninoculated growth medium. (B) Bar graph of the amount of biofilms produced by each of the nine strains listed on the X-axis. Bars represent
the means of three independent assays and error bars represent standard deviation of 2. The * above the bars indicates p < 0.05 when biofilm formation in
the mutant strains was compared to the parental strain. (C) Color photograph of the bacterial growth produced after 48 h of growth at 28°C on YESCA agar
containing Congo red. Biofilm formation was CsgA-dependent and did not require EspA The numbers 1–9 correspond to strains listed in the same order as in Fig 3A. doi:10.1371/journal.pone.0149745.g003 Fig 3. Qualitative and quantitative analysis of biofilm formation in YESCA broth at 28°C. (A) Single row of a microtiter plate showing the relative
intensity of crystal violet-stained biofilms produced in the wells inoculated with the Parent (1), Δhha (2), Δhha ΔcsgA (3), csgA+/Δhha ΔcsgA (4), Δhha ΔespA
(5), espA+/Δhha ΔespA (6), Δhha ΔcsgA ΔespA (7), espA+/Δhha ΔcsgA ΔespA (8), csgA+/Δhha ΔcsgA ΔespA (9) mutant strains. The last well in the row
represents the uninoculated growth medium. (B) Bar graph of the amount of biofilms produced by each of the nine strains listed on the X-axis. Bars represen
the means of three independent assays and error bars represent standard deviation of 2. The * above the bars indicates p < 0.05 when biofilm formation in
the mutant strains was compared to the parental strain. (C) Color photograph of the bacterial growth produced after 48 h of growth at 28°C on YESCA agar
containing Congo red. The numbers 1–9 correspond to strains listed in the same order as in Fig 3A. Fig 3. Qualitative and quantitative analysis of biofilm formation in YESCA broth at 28°C. (A) Single row of a microtiter plate showing the relative
intensity of crystal violet-stained biofilms produced in the wells inoculated with the Parent (1), Δhha (2), Δhha ΔcsgA (3), csgA+/Δhha ΔcsgA (4), Δhha ΔespA
(5), espA+/Δhha ΔespA (6), Δhha ΔcsgA ΔespA (7), espA+/Δhha ΔcsgA ΔespA (8), csgA+/Δhha ΔcsgA ΔespA (9) mutant strains. The last well in the row
represents the uninoculated growth medium. (B) Bar graph of the amount of biofilms produced by each of the nine strains listed on the X-axis. Bars represent
the means of three independent assays and error bars represent standard deviation of 2. The * above the bars indicates p < 0.05 when biofilm formation in
the mutant strains was compared to the parental strain. (C) Color photograph of the bacterial growth produced after 48 h of growth at 28°C on YESCA agar
containing Congo red. The numbers 1–9 correspond to strains listed in the same order as in Fig 3A. doi:10.1371/journal.pone.0149745.g003 PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 11 / 23 Cellular Adherence and Biofilm Formation the complemented (csgA+/hha csgA and csgA+/hha csgA espA) mutant strains (Fig 3A). Biofilm formation was CsgA-dependent and did not require EspA Quanti-
fication (A590) of the intensity of the blue color showed that there was a 3-fold (p < 0.0001)
increase in the production of biofilm by the hha and hha espA deletion mutants compared to
the parental strain (Fig 3B). On the other hand, biofilm production declined by 4.6-fold for the
hha csgA (p < 0.0001) and 5.25-fold for hha csgA espA mutants compared to the parental strain
(Fig 3B). When compared to the quantity of biofilm biomass produced by the hha or hha espA
mutant, there were15 to 17-fold lower amounts of biofilm produced by hha csgA and hha csgA
espA mutants (Fig 3B). Only plasmid pSM708 but not pSM706 restored biofilm production to
the hha csgA and hha cgsA espA mutants at levels similar to that produced by the hha and hha
espA mutants (Fig 3B). Since relative ability of O157 isolates to produce biofilms is also correlated with their ability
to bind the Congo red dye, we examined the Congo red binding phenotypes of bacterial strains
on media containing Congo red. The parental strain that produced very low amounts of bio-
film produced lighter red-colored colonies on Congo red medium (Fig 3C). The hha and hha
espA mutants that produced higher amounts of biofilms produced darker red colonies. The
hha csgA and hha csgA espA mutants produced white colonies but upon complementation with
pSM708, these strains were able to produce darker red colonies similar to those of hha and hha
espA mutants (Fig 3C). Western blot analysis of formic acid-solubilized bacterial cells (grown in YESCA broth at
28°C for 48 h) using anti-CsgA antibody showed the presence of a ~ 17 kD protein band in the
parental, hha, and hha espA mutant strains that were wild-type for csgA (Fig 4A). Similarly, the
hha csgA and hha csgA espA mutant strains that were complemented for csgA deletion with
plasmid pSM708 also produced a 17 kD band (Fig 4A). However, probing of whole-cell lysates
with anti-EspA antibody showed no detectable 25 kD protein bands corresponding to EspA in
all nine strains grown at 28°C in YESCA broth at 28°C (Fig 4B). A 75 kD DnaK band was
detected in the whole-cell lysates of all strains at similar levels indicating efficient and equiva-
lent extraction of proteins in these lysates (Fig 4C). Biofilm formation was CsgA-dependent and did not require EspA In O157, the presence of curli fimbriae at the bacterial cell surface is dependent on the pro-
duction of CsgA, which is a major structural protein of curli fimbriae. Phenotypic expression
of curli fimbriae on bacterial cell surfaces could be determined by negative staining of glutaral-
dehyde-fixed bacterial cells using a transmission electron microscope [58]. As shown in Fig
4D, with the exception of hha csgA, hha csgA espA, and espA+/hha csgA espA, all other strains
possessed curli fimbriae as determined by electron microscopy of glutaraldehyde-fixed cells. The production of curli fimbriae was restored on hha csgA and hha csgA espA mutants via com-
plementation with pSM708 (Fig 4D). PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 Growth at 28°C in the presence of 0.1% glucose did not induce EspA
production but allowed biofilm formation Since EspA could not be detected in whole-cell extracts prepared from bacterial strains grown
under conditions that maximize biofilm formation (growth at 28°C for 48 h in YESCA broth
lacking glucose) (Fig 4B), the role of EspA in biofilm formation could not be discounted
unequivocally under these growth conditions. Growth of O157 at 37°C in a minimal essential
medium supplemented with glucose has been shown to enhance the production of EspA and
other LEE-encoded effector proteins [59]. We were also able to detect EspA in whole-cell
lysates of strains harboring a wild-type copy of the espA gene after growth at 37°C in DMEM
containing 0.1% glucose (Fig 2C). Therefore, we first examined whether growth of bacterial
strains at 28°C in DMEM and YESCA broth containing 0.1% glucose (YESCA-G) would
induce EspA production and help infer the role of EspA in biofilm formation. The amount of PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 12 / 23 Cellular Adherence and Biofilm Formation Fig 4. Western blot analysis of cell lysates for detection of EspA and CsgA under growth conditions conducive for biofilm formation. (A) Western
blot of acid-solubilized cell lysates (prepared from bacterial cells grown in YESCA broth at 28°C for 48 h) probed with anti-CsgA antibody. A predicted protein
of 17 kD detected by anti-CsgA antibody in strains carrying the wild-type csgA gene is labeled on the left side of the blot. (B) Western blot prepared from
bacterial lysates and probed with anti-EspA antibody. The location of a predicted EspA protein of 25 kD, not detected in any of the nine strains, is indicated on
the left side of the blot. (C) Western blot of the loading control protein DnaK generated by probing of bacterial cell lysates with anti-DnaK antibodies. Predicted location of a 75 kD DnaK band detected with anti-DnaK antibody is labeled on the left side of the blot. Molecular weights of protein standards (Lane
M) used in the blot are indicated in kD on the right side of each blot. (D) Transmission electron micrographs of glutaraldehyde-fixed bacterial cells of each of
the nine strains listed on the top of Fig 4A. Dark-stained structures represent bacterial cells and hair like structures present around them are curli fimbriae
(indicated by an arrow). Fig 4. Western blot analysis of cell lysates for detection of EspA and CsgA under growth conditions conducive for biofilm formation. doi:10.1371/journal.pone.0149745.g004 Growth at 28°C in the presence of 0.1% glucose did not induce EspA
production but allowed biofilm formation (A) Western
blot of acid-solubilized cell lysates (prepared from bacterial cells grown in YESCA broth at 28°C for 48 h) probed with anti-CsgA antibody. A predicted protein
of 17 kD detected by anti-CsgA antibody in strains carrying the wild-type csgA gene is labeled on the left side of the blot. (B) Western blot prepared from
bacterial lysates and probed with anti-EspA antibody. The location of a predicted EspA protein of 25 kD, not detected in any of the nine strains, is indicated on
the left side of the blot. (C) Western blot of the loading control protein DnaK generated by probing of bacterial cell lysates with anti-DnaK antibodies. Predicted location of a 75 kD DnaK band detected with anti-DnaK antibody is labeled on the left side of the blot. Molecular weights of protein standards (Lane
M) used in the blot are indicated in kD on the right side of each blot. (D) Transmission electron micrographs of glutaraldehyde-fixed bacterial cells of each of
the nine strains listed on the top of Fig 4A. Dark-stained structures represent bacterial cells and hair like structures present around them are curli fimbriae
(indicated by an arrow). doi:10.1371/journal.pone.0149745.g004 biofilm produced by the hha mutant was about 2-fold (p < 0.0001) and 1.5 (p < 0.0001) higher
than the parental strain in DMEM (Fig 5A) and YESCA-G broth (Fig 5B), respectively. Although the magnitude of biofilm biomass produced by the hha csgA mutant in DMEM and
YESCA-G broth was > 2-fold (p < 0.0001) lower than the hha mutant strain, complementa-
tion of hha csgA mutant with csgA-encoding plasmid pSM708 increased biofilm formation in
the complemented (csgA+/hha csgA) mutant by > 2-fold (p < 0.0001), levels similar to those
produced by the hha mutant strain in DMEM (Fig 5A) and YESCA-G broth (Fig 5B). These PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 13 / 23 Cellular Adherence and Biofilm Formation Fig 5. Effect of glucose on EspA and CsgA production and biofilm formation. Bar graphs of biofilms produced in the wells of a microtiter plate
of growth at 28°C in DMEM (A) and YESCA-glucose broth (B). Bars represent means of three independent assays and error bars represent standa
deviation of 2. Growth at 37°C in media containing glucose induced EspA production
but EspA was not required for biofilm formation When bacterial strains were grown for 48 h in DMEM and YESCA-G at 37°C, with the excep-
tion of the hha csgA, hha csgA espA, and pSM706-complemented hha csgA espA mutant strains,
all other strains produced significantly (p < 0.05) higher biofilm biomass than the parental
strain (Fig 6A and 6B). For example, the magnitude of biofilm produced by the hha mutant
was about 2-fold (p < 0.0001) higher than the parental strain, but biofilm formation in the hha
csgA mutant was > 2-fold less than the hha mutant strain in DMEM (Fig 6A) and YESCA-G
broth (Fig 6B). However, complementation of the hha csgA mutant with plasmid pSM708
increased biofilm formation in the complemented (csgA+/hha csgA) mutant by > 2-fold
(p < 0.0001), levels similar to those produced by the hha mutant strain in DMEM (Fig 6A) and
YESCA-G broth (Fig 6B). The biofilm production in the hha espA mutant and hha espA
mutant complemented with plasmid pSM706 remained at levels 2-fold (p < 0.0001) higher
than the parental strain but similar to those produced by the hha mutant strain (Fig 6A) and
(Fig 6B), indicating that csgA is indispensable for biofilm formation. The absolute requirement
of csgA in biofilm formation was affirmed by results showing 2-fold (p < 0.0001) reduction
in biofilm formation in hha csgA espA mutant compared to the hha and hha espA mutants, and
restoration of biofilm formation to levels (> 2-fold higher than the parental strain; p < 0.0001)
similar to those produced by the hha mutant by complementing the hha csgA espA mutant
with plasmid pSM708 but not with pSM706 (Fig 6A and 6B). Western blot analysis using anti-
CsgA antibody of formic acid-solubilized bacterial cell pellets, prepared from cultures grown at
37°C in DMEM (Fig 6C) or YESCA-G (Fig 6D), showed the presence of a predicted 17 kD pro-
tein band in all strains except for the hha csgA, hha csgA espA, and pSM706-complemented
hha csgA espA mutant strains. Similarly, with the exception of the hha espA, hha csgA espA,
and pSM708-complemented hha csgA espA mutant strains, all other strains showed the pres-
ence of a 25 kD EspA in Western blots of cell lysates prepared from bacterial cultures grown to
an OD600 of 0.8 to 1.0 at 37°C in DMEM (Fig 6C) or YESCA-G (Fig 6D). Growth at 28°C in the presence of 0.1% glucose did not induce EspA
production but allowed biofilm formation The
importance of csgA in biofilm formation was further evidenced by the results showing 2 to PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 14 / 23 Cellular Adherence and Biofilm Formation 3-fold (p < 0.0001) reduction in biofilm formation in hha csgA espA mutant compared to the
hha and hha espA mutants, and restoration of biofilm formation to levels (> 2-fold higher than
the parental strain; p < 0.0001) produced by the hha and hha espA mutants by complementing
hha csgA espA mutant with plasmid pSM708 but not with pSM706 (Fig 5A and 5B). The maxi-
mal amounts of biofilm produced in DMEM and YESCA-G at 28°C were 6- (Fig 5A) and
2-fold (Fig 5B) lower than produced in glucose-free YESCA at 28°C (Fig 3B) indicating that
glucose is inhibitory to biofilm formation. Western blotting and probing of formic acid-solubi-
lized bacterial cell pellets, which were collected by centrifugation of cultures grown at 28°C in
DMEM (Fig 5C) or YESCA-G (Fig 5D) for 48 h, with anti-CsgA antibody showed the presence
of a predicted 17 kD protein band in all strains except the hha csgA, hha csgA espA, and
pSM706-complemented hha csgA espA mutant strain. However, no predicted protein band of
25 kD was detected when whole-cell lysates prepared from cultures grown to OD600 of 0.8 to
1.0 at 28°C in DMEM (Fig 5C) or YESCA-G (Fig 5D) were probed with anti-EspA antibody
indicating that EspA is not required for biofilm formation at 28°C. Growth at 28°C in the presence of 0.1% glucose did not induce EspA
production but allowed biofilm formation The * above the bars indicates p < 0.05 when biofilm formation in the mutant strains was compared to the parental or the hha mutant
Western blot analysis of cell lysates prepared from bacterial strains grown in DMEM (C) and YESCA-glucose (D) and probed using anti-DnaK (top b
EspA (middle blot), and anti-CsgA (bottom blot) antibodies. Molecular weights of protein standards are indicated in kD on the right side of each blot. locations of predicted or identified DnaK, EspA, and CsgA proteins are indicated on the left. Lanes numbered 1 through 9 contain cell lysates of stra
in the same order as in Fig 5A and 5B. doi:10.1371/journal.pone.0149745.g005 Fig 5. Effect of glucose on EspA and CsgA production and biofilm formation. Bar graphs of biofilms produced in the wells of a microtiter plate a Fig 5. Effect of glucose on EspA and CsgA production and biofilm formation. Bar graphs of biofilms produced in the wells of a microtiter plate after 48 h
of growth at 28°C in DMEM (A) and YESCA-glucose broth (B). Bars represent means of three independent assays and error bars represent standard
deviation of 2. The * above the bars indicates p < 0.05 when biofilm formation in the mutant strains was compared to the parental or the hha mutant strain. Western blot analysis of cell lysates prepared from bacterial strains grown in DMEM (C) and YESCA-glucose (D) and probed using anti-DnaK (top blot), anti-
EspA (middle blot), and anti-CsgA (bottom blot) antibodies. Molecular weights of protein standards are indicated in kD on the right side of each blot. The
locations of predicted or identified DnaK, EspA, and CsgA proteins are indicated on the left. Lanes numbered 1 through 9 contain cell lysates of strains listed
in the same order as in Fig 5A and 5B. doi:10.1371/journal.pone.0149745.g005 results indicated that biofilm formation is dependent on csgA. The biofilm production in the
hha espA and hha espA mutant complemented with espA-encoding plasmid pSM706 remained
at levels (> 2-fold; p < 0.0001) as high as in the hha mutant strain both in DMEM (Fig 5A)
and YESCA-G broth (Fig 5B), indicating that espA is not required for biofilm formation. PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 Growth at 37°C in media containing glucose induced EspA production
but EspA was not required for biofilm formation These results indi-
cated that the addition of glucose and an incubation temperature of 37°C are critical to induce
espA expression. Despite production of EspA in DMEM and YESCA-G at 37°C, EspA was dis-
pensable for biofilm formation because complementation of the hha espA csgA mutant with
plasmid pSM706 failed to restore biofilm formation on this mutant to the levels produced by
the hha mutant strain. PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 15 / 23 Cellular Adherence and Biofilm Formation Fig 6. Effect of temperature and glucose on EspA and CsgA production and biofilm formation. Bar graphs of biofilms produced in the wells of a
microtiter plate after 48 h of growth at 37°C in DMEM (A) and YESCA-glucose broth (B). Bars represent means of three independent assays and error bars
represent standard deviation of 2. The * above the bars indicates p < 0.05 when biofilm formation in the mutant strains was compared to the parental or the
hha mutant strain. Western blot analysis of cell lysates prepared from bacterial strains grown in DMEM (C) and YESCA-glucose (D) and probed using anti-
DnaK (top blot), anti-EspA (middle blot), and anti-CsgA (bottom blot) antibodies. Molecular weights of protein standards are indicated in kD on the right side
of each blot. The locations of predicted or identified DnaK, EspA, and CsgA proteins are indicated on the left. Lanes numbered 1 through 9 contain cell
lysates of strains listed in the same order as in Fig 6A and 6B. Fig 6. Effect of temperature and glucose on EspA and CsgA production and biofilm formation. Bar graphs of biofilms produced in the wells of a
microtiter plate after 48 h of growth at 37°C in DMEM (A) and YESCA-glucose broth (B). Bars represent means of three independent assays and error bars
represent standard deviation of 2. The * above the bars indicates p < 0.05 when biofilm formation in the mutant strains was compared to the parental or the
hha mutant strain. Western blot analysis of cell lysates prepared from bacterial strains grown in DMEM (C) and YESCA-glucose (D) and probed using anti-
DnaK (top blot), anti-EspA (middle blot), and anti-CsgA (bottom blot) antibodies. Molecular weights of protein standards are indicated in kD on the right side
of each blot. The locations of predicted or identified DnaK, EspA, and CsgA proteins are indicated on the left. Growth at 37°C in media containing glucose induced EspA production
but EspA was not required for biofilm formation Lanes numbered 1 through 9 contain cell
lysates of strains listed in the same order as in Fig 6A and 6B. doi:10.1371/journal.pone.0149745.g006 PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 Discussion In the present study, we show the relative importance of two outer membrane filamentous
adhesins in adherence of O157 to cultured epithelial cells and abiotic surfaces. The EspA fila-
ments along with other virulence proteins encoded by the LEE pathogenicity island are needed
for initial adherence to and subsequent translocation of effector proteins into epithelial cells
[15, 16]. Curli fimbriae belong to the amyloid class of proteins and are responsible for bacterial
interactions with both biotic and abiotic surfaces and subsequent interbacterial interactions
considered important during biofilm formation [42]. We have shown previously that hha is a
negative transcriptional regulator of LEE-encoded pathogenicity island and operons csgDEFG
and csgBAC encoding genes for biosynthesis of curli fimbriae that are essential for biofilm for-
mation [33, 50]. Thus the hha deletion mutant, in which expression of genes encoded by LEE
and the two curli operons is highly upregulated, provided an important model system to exam-
ine the specific contributions of EspA filaments and curli fimbriae in adherence of O157 to 16 / 23 PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 Cellular Adherence and Biofilm Formation epithelial cells and to polystyrene surfaces during biofilm formation. By deleting espA and
csgA, or both espA and csgA genes in the hha mutant strain, we showed that espA and csgA,
which encodes for EspA filaments and curli fimbriae, respectively, function independently of
each other with each having a dedicated function. epithelial cells and to polystyrene surfaces during biofilm formation. By deleting espA and
csgA, or both espA and csgA genes in the hha mutant strain, we showed that espA and csgA,
which encodes for EspA filaments and curli fimbriae, respectively, function independently of
each other with each having a dedicated function. It has been shown that EspA filaments are produced by only a fraction of O157 bacterial
population through a posttranscriptional mechanism controlled by regulators whose expres-
sion are controlled by specific environmental signals and phase-variable events [60]. In agree-
ment with these findings, Western blot analysis of whole-cell lysates with anti-EspA antibody
showed very low-levels of EspA production in the parent compared to the isogenic hha dele-
tion mutant strain. We also observed a direct correlation between magnitudes of adherence to
HEp-2 cells with the levels of EspA production. The espA deletion mutant of hha showed
highly reduced adherence compared to the parental strain whose adherence was several-fold
lower than the hha mutant strain. Discussion Moreover, presence of high levels of curli fimbriae alone was
not able to compensate for the loss of adherence of the hha espA deletion mutants. These find-
ings clearly demonstrated that increased production of EspA not only enhanced the magnitude
of adherence, but it was also a critical element for the adherence of O157 to epithelial cells. Curli fimbriae are encoded by the csgA gene and are highly diverse in their functional activi-
ties [3]. Curli fimbriae, because of their amyloid-like properties, are adhesive cell surface struc-
tures and have been shown to interact with a variety of animal tissues and abiotic surfaces [31,
32, 43, 61]. One of the best studied functions of curli fimbriae is in biofilm formation but there
are only a few studies describing the importance of curli fimbriae in direct interactions with
animal epithelial cells [3, 32, 35, 61]. In the current study employing the hha mutant producing
higher levels of curli fimbriae, we were able to show that contrary to the EspA-dependency of
adherence of O157 to epithelial cells, curli fimbriae were dispensable for direct adherence to
HEp-2 cells. This inference is supported by the results showing that complementing hha csgA
and hha espA csgA mutants with a csgA encoding plasmid (pSM708) did not enhance adher-
ence of the complemented mutants relative to the non-complemented mutant strains. p
p
However, curli might indirectly contribute to the adherence of O157 to epithelial cells by
promoting interbacterial interactions as indicated by the reduction in the adherence of the hha
csgA mutants to HEp-2 cells compared to the hha mutant strain. For example, microscopic
observations revealed that the hha mutant produced an adherent phenotype characterized by
the presence of clusters of adherent cells. This would suggests that once a hha mutant bacterial
cell adheres to an HEp-2 cell using EspA and other LEE-encoded adherence factors, curli fim-
briae present on the adherent mutant bacterial cells might interact with nearby non-adherent
cells to produce clusters consisting of many cells. These clusters were not produced by the low-
level curli-producing parental and the curli-lacking hha csgA mutants indicating that increased
recovery of hha mutant adherent cells compared to the hha csgA mutant might be due to the
production of clustered adherence phenotype by the hha mutant. PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 Discussion Interestingly, the csgA-
encoding plasmid (pSM708) was able to restore both the Congo red binding and biofilm pro-
duction to the hha csgA mutant after 48 h of incubations, but it failed to restore adherence of
this mutant to HEp-2 cells at the levels shown by the hha mutant. These results suggest that the
length of adherence assays that lasted only three hours might not be long enough for this plas-
mid to produce substantial levels of curli fimbriae needed to promote clustered adherence phe-
notype. It is also possible that temporal differences in the expression of csgA from the csgA-
encoding plasmid relative to the chromosomal copy of csgA in the hha mutant might be a limit-
ing factor for the csgA-complemented hha csgA mutant to achieve high levels of adherence that
is typical of the hha mutant strain. The regulation of the expression of csgA and other genes required for biogenesis of curli fim-
briae is highly complex and triggered by factors, such as low temperature, low osmolarity, and PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 17 / 23 Cellular Adherence and Biofilm Formation nutrient limitation [3, 40, 62]. We first examined the abilities of hha and the hha mutant
deleted of espA or csgA or both espA and csgA to produce biofilms at 28°C in a medium (low
osmolarity and lacking glucose as a carbon source) conducive for biogenesis of curli fimbriae
[31]. Under these growth conditions, only the hha csgA and hha csgA espA mutants were
unable to produce detectable amounts of biofilms unless complemented with csgA-expressing
plasmid, confirming that CsgA production is essential for O157 to produce biofilms. Interest-
ingly, CsgA was detectable in cell-lysates of all mutants as long as they harbored the wild-type
csgA. However, EspA was not detected in lysates of all nine strains tested in this study. The expression of espA and LEE is also very complex as numerous transcriptional regulators
and environmental conditions tend to modulate their expression [50, 63–70]. For example, the
expression of LEE, which encodes the espA gene, is induced under conditions mimicking the
host animal intestinal environment [59]. It has been demonstrated that optimal expression of
LEE can be induced in minimal essential medium supplemented with glucose and by utilizing
a growth temperature of 37°C. Discussion Thus, in order to understand the role of EspA in biofilm forma-
tion, we used DMEM and the YESCA broth supplemented with glucose to induce espA expres-
sion at 28°C and 37°C. None of the strains produced EspA in either of the two media at 28°C
but incubation at 37°C allowed EspA production in strains carrying a wild-type espA. Although
substantial amounts of biofilms were produced at 37°C by the hha mutant and the hha mutants
harboring the wild-type copy of the csgA gene, biofilm formation occurred independent of the
production of EspA. While low temperature promotes biofilm formation, glucose at a concentration of 0.1% or
higher could potential inhibits initial stages or lower the amount of biofilm formation in E. coli
and other enterobacteriacea [71]. The inhibitory effects of glucose on biofilm formation have
in part been linked to cAMP and cAMP receptor protein-mediated catabolite repression affect-
ing expression of genes mediating biofilm formation. We did observe about 60% to 75% reduc-
tion in biofilm production in DMEM and YESCA containing 0.1% glucose at 28°C and 37°C
despite the detection of roughly the similar levels of CsgA under these conditions. The forma-
tion of lower amounts of biofilms observed in the presence of glucose could not be attributed
to the differences in bacterial growth rates (120 min and 240 min of doubling times at 37°C
and 28°C, respectively) because roughly the similar amounts of biofilms were produced in glu-
cose-containing DMEM and YESCA at 28°C or 37°C. Since highest amounts of biofilms were
produced at 28°C in YESCA broth lacking glucose (Fig 3), both the absence of a rapidly metab-
olizable energy source and growth at low temperatures are necessary for the expression of fac-
tors conducive for optimal levels of biofilm formation. Nonetheless, the ability to produce even
lower amounts of biofilms at 37°C in the presence of glucose could potentially be advantageous
to survival and persistence of O157 in the animal host, considering that low levels of glucose
are present in the intestinal environment [71–73]. Acknowledgments We thank Lindsay Andersen and Bryan Wheeler for technical assistance in this study. We also
thank Shawn Bearson for the critical review of the manuscript. Mention of trade names or commercial products in this article is solely for the purpose of
providing specific information and does not imply recommendation or endorsement by the U. S. Department of Agriculture. USDA is an equal opportunity provider and employer. Conclusions In this study, we demonstrated that EspA and CsgA contribute independently to tissue adher-
ence and biofilm formation, respectively. We confirm previous findings that EspA production
requires bacterial growth at 37°C in the presence of glucose, conditions that attenuate biofilm
formation but still allow substantial expression of this adherence phenotype. Although we
determined that curli fimbriae have no direct role in O157 adherence to HEp-2 cells, curli fim-
briae might indirectly enhance adherence of O157 by promoting interactions between adherent
(mediated through EspA) bacterial cells with the non-adherent cells on epithelial cell surfaces. Since curli fimbriae are also able to interact with a variety of host molecules, curli fimbriae
could exploit other matrices or cell surface molecules in the animal intestinal environment to PLOS ONE | DOI:10.1371/journal.pone.0149745
February 22, 2016 18 / 23 Cellular Adherence and Biofilm Formation promote bacterial adherence and biofilm formation. A high proportion of clinical isolates of
uropathogenic E. coli and sorbitol fermenting Shiga toxigenic E. coli isolated from patients
express curli fimbriae at 37°C and have been shown to interact with cells of the urinary tract
system and colonic cells, respectively [49, 61, 74]. Author Contributions Conceived and designed the experiments: VKS. Performed the experiments: VKS JAS. Ana-
lyzed the data: VKS. Contributed reagents/materials/analysis tools: ITK BLB. Wrote the paper:
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Investigating the inequalities in route to diagnosis amongst patients with diffuse large B-cell or follicular lymphoma in England
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British journal of cancer
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Investigating the inequalities in route to diagnosis amongst
patients with diffuse large B-cell or follicular lymphoma Matthew J. Smith
1✉, Miguel Angel Luque Fernandez
1,2, Aurélien Belot
1, Matteo Quartagno3, Audrey Bonaventure4,
Sara Benitez Majano1, Bernard Rachet1 and Edmund Njeru Njagi1 Matthew J. Smith
1✉, Miguel Angel Luque Fernandez
1,2, Aurélien Belot
1, Matteo Quartagno3, Audrey Bonaventure4,
Sara Benitez Majano1, Bernard Rachet1 and Edmund Njeru Njagi1 © The Author(s) 2021 © The Author(s) 2021 INTRODUCTION: Diagnostic delay is associated with lower chances of cancer survival. Underlying comorbidities are known to
affect the timely diagnosis of cancer. Diffuse large B-cell (DLBCL) and follicular lymphomas (FL) are primarily diagnosed amongst
older patients, who are more likely to have comorbidities. Characteristics of clinical commissioning groups (CCG) are also known to
impact diagnostic delay. We assess the association between comorbidities and diagnostic delay amongst patients with DLBCL or FL
in England during 2005–2013. g
g
METHODS: Multivariable generalised linear mixed-effect models were used to assess the main association. Empirical Bayes
estimates of the random effects were used to explore between-cluster variation. The latent normal joint modelling multiple
imputation approach was used to account for partially observed variables. RESULTS: We included 30,078 and 15,551 patients diagnosed with DLBCL or FL, respectively. Amongst patients from the same CCG,
having multimorbidity was strongly associated with the emergency route to diagnosis (DLBCL: odds ratio 1.56, CI 1.40–1.73; FL:
odds ratio 1.80, CI 1.45–2.23). Amongst DLBCL patients, the diagnostic delay was possibly correlated with CCGs that had higher
population densities. p p
CONCLUSIONS: Underlying comorbidity is associated with diagnostic delay amongst patients with DLBCL or FL. Results suggest a
possible correlation between CCGs with higher population densities and diagnostic delay of aggressive lymphomas. British Journal of Cancer; https://doi.org/10.1038/s41416-021-01523-6 In the UK, the cancer diagnostic route is defined as the first of
eight possible points of contact between the patient and the
healthcare system [11]. Emergency diagnosis is defined as a
diagnosis of cancer following presentation to an accident and
emergency unit, or following an emergency pathway for in/out-
patients: it is used as an indicator of diagnostic delay for cancer
patients [12]. Underlying comorbidities are known to affect the
timely diagnosis of other cancers [13–15]. Comorbidity expressing
symptoms similar to cancer may delay the diagnosis: dissimilar
symptoms may hasten the cancer diagnosis. Investigating the inequalities in route to diagnosis amongst
patients with diffuse large B-cell or follicular lymphoma For example,
some symptoms are present in both lymphomas and other
chronic diseases, such as swollen abdomen and fatigue in
diabetes [16], chest pain in congestive heart failure [17] and
shortness of breath in chronic obstructive pulmonary disease [18]. Furthermore, all three of these diseases are prevalent amongst
patients with lymphoma, which could explain misdiagnosis and
diagnostic delay [19, 20]. 1Inequalities in Cancer Outcomes Network, Department of Non-Communicable Disease Epidemiology, London School of Hygiene and Tropical Medicine, London, UK.
2Noncommunicable Disease and Cancer Epidemiology Group, Instituto de Investigación Biosanitaria de Granada, Ibs.GRANADA, Andalusian School of Public Health, Granada,
Spain. 3MRC Clinical Trials Unit, Institute of Clinical Trials and Methodology, University College London, London, UK. 4CRESS, Université de Paris, INSERM, UMR 1153, Epidemiology
of Childhood and Adolescent Cancers Team, Villejuif, France. ✉email: matthew.smith1@lshtm.ac.uk Received: 23 November 2020 Revised: 23 June 2021 Accepted: 3 August 2021 British Journal of Cancer British Journal of Cancer British Journal of Cancer www.nature.com/bjc METHODS
St d
d
i Study design, participants, data and setting
We developed a population-based cross-sectional study comprising all
patients, aged 18 to 99 years, diagnosed with non-Hodgkin lymphoma
(NHL) between January 1, 2005 and December 31, 2013. NHL was coded
(C82.0-C85.9) according to the 10th revision of the International Statistical
Classification of Diseases and Related Problems (ICD) [26]. Morphology (cell
type) and topography (tumour site) were defined using renewed updates
of the ICD for Oncology (ICD-O); ICD-O-3 [27] was used for diagnoses up to
2010, and ICD-O-3.1 [28] for diagnoses after 2011. Patients diagnosed with
either DLBCL or FL were included in the study and are hereby referred to as
subtype (Supplementary Table S1) [26]. logit πij
¼ β0 þ bi þ β1Aij þ β2Gij þ β3Eij þ
X
5
k¼2
β4k Dijk þ
X
3
k¼2
β5k Cijk where bi Nð0; σ2
bÞ. The patient, and tumour, characteristics were age (A),
gender (G), ethnicity (E), deprivation (D) and comorbidity score (C). The model was estimated using maximum likelihood. Likelihood-ratio
tests were used to compare between models with and without each
covariate and for linear trend. Note that these and subsequent estimates
are for any given CCG as results from logistic mixed-effects models have
cluster-specific interpretation [42–45]. Empirical Bayes estimates of the
random effect bbi were used to explore the between-CCG variability in the
odds of the emergency route to diagnosis. The random-effect variance
parameter was tested for using a mixture of Chi-squares with 0 and 1
degrees of freedom [42, 43]. The mixture of the Chi-square test is a
likelihood-ratio-type test, where an appropriate reference distribution is
used to account for the fact that the null hypothesis in this case is at the
boundary of the parameter space [42, 46]. Combining likelihood-ratio tests
after multiple imputation requires derivation of a particularly modified
likelihood-ratio test statistic, which is compared with a particularly derived
reference distribution. For tests of fixed-effect parameters, the relevant
methodology exists [47]. We are not aware of the existing corresponding
methodology for combining after multiple imputation likelihood-ratio-type
tests for random-effect variance parameters. Information on patients’ cancer diagnosis was collected by the national
cancer registry and analysis service (NCRAS) [29]. The NCRAS contains England
national cancer registry data and Hospital Episode Statistics [30] (HES)
datasets that are accessed via the Cancer Analysis System [31] (CAS). INTRODUCTION Non-Hodgkin lymphoma is a heterogeneous disease comprising
over 60 morphological entities with diverse histological patterns
[1]. The most common of which are diffuse large B-cell (DLBCL)
and follicular lymphomas (FL), exhibiting an annual rate of 8.2 and
3.3 cases (respectively) per 100,000 people in the UK. These
subtypes
are
relatively
common
in
adults,
with
incidence
increasing amongst older ages [2]. Each of these subtypes has
markedly differing treatments and health outcomes [1]. Survival of DLBCL or FL patients in England has steadily
increased over the past decades [3, 4], yet the proportion of
patients surviving trails that of other European countries [5]. Evidence has highlighted that diagnostic delay (compared to an
earlier diagnosis) is associated with a less intensive treatment plan,
which then impacts on the chances of survival [6]. Public health
policies have aimed to increase awareness, encourage more
patient and healthcare system interactions and set targets for
earlier cancer diagnosis [7–10]. Published on Behalf of CRUK M.J. Smith et al. 2 A universal healthcare system (UHS), such as the National
Health Service (NHS) in England, aims to provide all residents with
access to healthcare [21]. However, variability in health outcomes
amongst patients with the same lymphoma still occurs [22, 23]. Clinical Commissioning Groups (CCGs) commission the hospital
and community NHS services, and decide on local priorities
(informed by general practices), for their respective geographical
areas; however, CCGs have shown variability in health outcomes
since their inception [24, 25], which may partly explain differences
in diagnostic delay. the Index of Multiple Deprivation [40] (IMD), the income domain was
classified into one of five quintiles based on the national distribution of
ranked deprivation scores in the 32,844 LSOAs. Each patient was linked
with one of the 209 Clinical Commissioning Groups (CCG) where their
LSOA resides [41]. Lastly, ethnicity (HES dataset) was recorded as either
white or other. Statistical analysis We described the study population, tabulated the patient characteristics
with diagnostic delay markers (route to diagnosis), and calculated
unadjusted odds ratios (and 95% confidence intervals [CI]) with Wald test
P values. We aim to assess the association between pre-diagnosed
comorbidities and diagnostic delay (i.e., route to diagnosis)
amongst patients with DLBCL or FL, accounting for patient
sociodemographic characteristics. We conducted analysis for DLBCL and FL separately. Univariable
independent logistic regression models were used to explore the crude
association between the route to diagnosis and each of the patient
characteristics. Then, multivariable generalised linear mixed-effect models
(GLMM) were used to account for the dependency between patients j = 1,
…,ni from CCG i = 1,…,209. The GLMM model for the route to diagnosis
was defined as Variables Route to diagnosis, obtained from NCRAS, was originally recorded as one of
eight routes to diagnosis [11]. Patients with a ‘death certificate only’ route
to diagnosis were excluded to remove bias. There is no nationally
recognised screening programme for NHL, and no patients were
diagnosed via a ‘screen-detected’ route. An ‘unknown’ route to diagnosis
was recoded as a missing record. The remaining routes were dichotomised
into a binary variable indicating whether the patient was diagnosed
following an emergency or elective presentation: elective presentation
consisted of patients diagnosed through 2-week-wait, general practitioner
referral, inpatient elective and other outpatient. METHODS
St d
d
i Cancer
registry (CAS dataset) contained information on subtype (morphology), age at
diagnosis, ethnicity, gender and date of diagnosis. This was linked to HES,
which contained information on patient’s previous hospital admissions,
accident and emergency presentations, outpatient appointments. Missing data analysis Variables with missing data were the outcome (route to diagnosis [DLBCL:
1.9%, FL: 2.1%]), and ethnicity [DLBCL: 22.8%, FL: 24.9%]. Using logistic
regression models, we explored the missing data mechanism for each
partially observed variable. The imputation model included all fully- and
partially observed covariates and the cluster variable indicator. To reduce
potential
bias
[47],
the
auxiliary
variables
(patient’s
vital
status,
Nelson–Aalen estimate of the cumulative mortality hazard, and stage at
diagnosis) were included as, per the missing data indicator model, they
were predictive of the chance of missing values and, as per subject matter
knowledge, associated with the underlying values themselves [48]. We
used the latent normal joint modelling multiple imputation approach,
under a missing at random assumption, and generated ten imputed
datasets. The multilevel logistic regression models for each outcome were
fitted to each of these datasets and results combined using Rubin’s rules
[49, 50]. Comorbidity status, based on the Charlson comorbidity index [32] (CCI),
was defined as “the existence of disorders, in addition to a primary disease
of interest, which are causally unrelated to the primary disease” [33, 34]. Comorbidities were coded within HES according to the International
Classification of Diseases, 10th revision (Supplementary Table S2). Previous
records of comorbidity were obtained from HES data. Patients with any
previous malignancy were removed. For each patient, we defined a time
window of 6–24 months prior to cancer diagnosis for a comorbidity to be
recorded. A patient’s CCI was determined using an algorithm developed by
Maringe et al. [35]. CCI was classified according to the Royal College of
Surgeons (RCS) Charlson Score [36], which was categorised into three
groups: 0 for no previous comorbidity, 1 for single comorbidity and 2 or
more for multimorbidity. We tabulated the prevalence of comorbidity for
DLBCL and FL (Supplementary Table S3). We used R software for all analysis; the glmer function of the lme4
package was used for generalised linear mixed-effects models, and the
jomo [51] package for multiple imputation, which allows imputation of
clustered data. Stage at diagnosis is based on the Ann Arbor classification system (CAS
dataset) [37]. A lower tumour stage is predictive of a higher survival
outcome compared to a higher tumour burden. For NHL subtypes, stages
I/II is a criterion for treatment of low tumour stage; stages III/IV is a criterion
for treatment of high tumour stage [38]. Therefore, early-stage was
dichotomised as I/II, and late-stage as III/IV. Summary statistics y
In this study, we included 45,629 patients diagnosed with DLBCL
(30,078; 65.9%) or FL (15,551; 34.1%) between January 1, 2005 and
December 31, 2013 (Table 1A, B). The prevalence of emergency Deprivation level (HES dataset) is based on the Lower Super Output Area
[39] (LSOA) of residence of the patient at the date of cancer diagnosis. An
LSOA is a geographical location with a median of 1500 inhabitants. From British Journal of Cancer British Journal of Cancer M.J. Smith et al. 3 Table 1. (A) Summary statistics of the emergency route to diagnosis amongst patients diagnosed with diffuse large B-cell lymphoma (n = 30,078) in
England during 2005–2013; (B) summary statistics of the emergency route to diagnosis amongst patients diagnosed with follicular lymphoma (n =
15,551) in England during 2005–2013. Table 1. (A) Summary statistics of the emergency route to diagnosis amongst patients diagnosed with diffuse large B-cell lymphoma (n = 30,078) in
England during 2005–2013; (B) summary statistics of the emergency route to diagnosis amongst patients diagnosed with follicular lymphoma (n =
15,551) in England during 2005–2013. Table 1. (A) Summary statistics of the emergency route to diagnosis amongst patients diagnosed with diffuse large B-cell lymphoma (n = 30,078) in
England during 2005–2013; (B) summary statistics of the emergency route to diagnosis amongst patients diagnosed with follicular lymphoma (n =
15,551) in England during 2005–2013. 15,551) in England during 2005–2013. Crude odds ratios for emergency vs elective.
cIncrease in odds of the emergency route for each 10-year increase in age.
d Summary statistics diagnostic routes amongst those diagnosed with DLBCL or FL was
9683 (34.1%) and 1879 (12.3%), respectively, there was no
evidence of a yearly trend. Amongst these patients, the average
age at diagnosis was 68.2 and 66.3 years, respectively. We found that comorbidity status was significantly associated
with the emergency route to diagnosis, after adjusting for age,
gender, ethnicity and deprivation and accounting for clustering
due to CCG did not explain the relative difference. The more severe
the comorbidity score, and those living in more deprived areas,
increased the odds of the emergency route to diagnosis. Our
results are consistent with previous findings of an increase in the
probability of emergency route to diagnosis [6, 52], and, in other
countries and for other cancers, comorbidities were associated
with diagnostic delay [53]. Similar results were found amongst
studies investigating colon cancer [54, 55]. Since the proportion of
patients with emergency route remains stable over calendar time,
this phenomenon is not thought to be time-dependent. g
g
y
p
y
The prevalence of emergency diagnostic routes (compared to
elective) was higher amongst FL males, ethnic minorities in DLBCL,
and those living in the most deprived areas (both DLBCL and FL). The emergency route, compared to elective, was more common
amongst those with multimorbidity: DLBCL (7.2% vs 4.6%,
respectively) and FL (6.2% vs 3.1%, respectively). Similarly, for
both DLBCL and FL, an increase in the crude odds of the
emergency route to diagnosis was strongly associated with an
increase in age and living in most deprived areas, while for an
ethnic minority it was observed in DLBCL only. There was an
increase in the odds of the emergency route to diagnosis with
each increase in deprivation level. Deprivation level was a strong independent predictor of the
route to diagnosis after adjusting for comorbidity and other
factors (Table 2A, B); however, accounting for clustering increased
the strength of the association for patients living in more deprived
areas. This suggests that the difference in diagnostic delays
between deprivation groups is partly explained by unobserved,
and possibly unmeasured, characteristics of CCGs. A characteristic
of CCGs, not explored in this study but for other cancers, could be
accessed to the healthcare system (e.g., accessibility to a GP
appointment) [56]. Multivariable mixed-effect logistic regression models Table 2A, B shows the results from the multivariable GLMM for odds
of the emergency route to the diagnosis of DLBCL and FL,
respectively. For both DLBCL and FL, under complete case analysis,
we found that for any given CCG, the presence of comorbidity was
associated with the emergency route to diagnosis: the association
was largest amongst those with a comorbidity status of two or more
(Table 2A, B). Living in more deprived areas was strongly associated
with the emergency route to diagnosis. g
y
g
After multiple imputation (Table 2A, B), there were similar
conclusions to the complete case analysis. Amongst patients from
the same CCG, having a comorbidity score of 2 or more, compared
to no comorbidity, was strongly associated with an emergency
route to diagnosis (DLBCL: OR 1.56, CI 1.40–1.73; FL: OR 1.80, CI
1.45–2.23). There was weak evidence of a trend for deprivation
and comorbidity index amongst DLBCL (P = 0.054 and P = 0.060,
respectively); however, there was no evidence of a trend amongst
FL (P = 0.206 and P = 0.113, respectively). Using a mixture of Chi-square tests with 0 and 1 degree of
freedom (i.e. half the P value from a Chi-square with 1 degree of
freedom), we found strong evidence of between-CCG variability in
the odds of the emergency route to diagnosis (DLBCL: P < 0.005;
FL: P < 0.001). The variance of the CCG random effects of the
models for DLBCL and FL indicated some heterogeneity between
CCGs in routes to diagnosis. Contrary to the assurances of a universal healthcare system,
such as the NHS, our results suggest inequitable access to
healthcare services between CCGs (i.e., more densely populated
CCGs appear to have patients with a greater chance of diagnostic
delay compared to less densely populated CCGs). Patients
diagnosed through the emergency route are patients that either
could not access a GP appointment or the GP appointment was
inconclusive: during this waiting time, cancer can progress and
the patient admitted themselves to the emergency department. Inequalities may be due to a combination of competing demands
and a lack of clinical guidance regarding symptoms. However, lack
of clinical guidance would be a non-differential misclassification
and this would not explain the inequalities in the emergency route
amongst patient characteristics. Summary statistics (A)
Route to diagnosisa
cORb
95% CI
P value
Elective
N = 19,833
(65.9%)
Emergency
N = 9683
(34.1%)
Age (mean, s.d.)
67.2 (14.8)
68.2 (15.5)
1.04c
1.03–1.06
<0.001
Gender
Male
10,658 (53.7)
5292 (54.7)
Ref
Ref
Ref
Female
9175 (46.3)
4391 (45.4)
0.96
0.92–1.01
0.139
Ethnicity
White
14,583 (94.8)
6,898 (92.6)
Ref
Ref
Ref
Minorities
802 (5.2)
549 (7.4)
1.44
1.29–1.62
<0.001
Missingd
4448 (22.4)
2236 (23.1)
–
–
–
Deprivation
Least deprived
4410 (22.2)
1823 (18.8)
Ref
Ref
Ref
2
4455 (22.5)
2105 (21.7)
1.14
1.06–1.23
<0.001
3
4145 (20.9)
2031 (21.0)
1.19
1.10–1.28
<0.001
4
3806 (19.2)
1993 (20.6)
1.27
1.17–1.37
<0.001
Most deprived
3017 (15.2)
1731 (17.9)
1.39
1.28–1.50
<0.001
Comorbidity
None
17,957 (90.5)
8396 (86.7)
Ref
Ref
Ref
One
970 (4.9)
590 (6.1)
1.30
1.17–1.45
<0.001
Multimorbidity
906 (4.6)
697 (7.2)
1.65
1.49–1.82
<0.001
(B)
Route to diagnosise
cORb
95% CI
P value
Elective
N = 13,353
(87.7%)
Emergency
N = 1879
(12.3%)
Age (mean, s.d.)
63.5 (13.5)
66.3 (14.2)
1.17c
1.13–1.21
<0.001
Gender
Male
6209 (46.5)
962 (51.2)
Ref
Ref
Ref
Female
7144 (53.5)
917 (48.8)
0.83
0.75–0.91
<0.001
Ethnicity
White
9459 (94.9)
1399 (94.8)
Ref
Ref
Ref
Minorities
510 (5.1)
77 (5.2)
1.02
0.80–1.31
0.870
Missingd
3384 (25.3)
403 (21.5)
–
–
–
Deprivation
Least deprived
3100 (23.2)
375 (20.0)
Ref
Ref
Ref
2
3040 (22.8)
405 (21.6)
1.10
0.95–1.28
0.205
3
2857 (21.4)
375 (20.0)
1.09
0.93–1.26
0.292
4
2462 (18.4)
412 (21.9)
1.38
1.19–1.61
<0.001
Most deprived
1894 (14.2)
312 (16.6)
1.36
1.16–1.60
<0.001
Comorbidity
None
12,410 (92.9)
1667 (88.7)
Ref
Ref
Ref
One
536 (4.0)
95 (5.1)
1.32
1.05–1.65
0.015
Multimorbidity
407 (3.1)
117 (6.2)
2.14
1.73–2.65
<0.001
cOR crude odds ratio, CI confidence interval. aIn all, 562 (1.9%) missing route to diagnosis records. b
d
dd
f
l cOR crude odds ratio, CI confidence interval. cOR crude odds ratio, CI confidence interval. aIn all, 562 (1.9%) missing route to diagnosis records. b aIn all, 562 (1.9%) missing route to diagnosis records. b g
y
cIncrease in odds of the emergency route for each 10-year increase in age. cIncrease in odds of the emergency route for each 10-year increase in age. d d Proportions of missing records amongst all ethnicity records (including observed records). British Journal of Cancer M.J. Smith et al. 4 patients diagnosed with non-Hodgkin lymphoma, adjusting for
patient and healthcare pathway characteristics. Multivariable mixed-effect logistic regression models We graphically illustrate, from our analysis accounting for both
clustering and missing data, the Empirical Bayes (EB) estimates of
the CCG random effects for odds of the emergency route to
diagnosis (Figs. 1 and 2). These are used to explore the between-
CCG variability. A positive EB estimate indicated a higher
probability of emergency route to diagnosis for a patient from
that CCG in comparison to a patient who has similar observed
characteristics but from a CCG with either a less positive, or a
negative EB estimate. For DLBCL, there are possibly a few outlying
CCGs with the lowest probabilities, and possibly an outlying one
with the highest probability. For FL, there are possibly a few
outlying ones with the highest probabilities. To explore possible
patterns, the size of the markers were weighted by the population
density for the respective CCG and have a lighter shade for a
higher proportion of missing records of the route to diagnosis. g
p
Our results challenge previous research that did not find
evidence of a difference in diagnostic delay between deprivation
levels using unadjusted analyses; although, previous studies were
based on a smaller sample size that were potentially under-
powered in comparison to our study [6]. We highlight that
deprivation is predictive of the diagnostic route if analyses do not
account for CCGs that widely differ, among other dimensions, in
healthcare provision [64]. Furthermore, the late lymphoma stage
at diagnosis seems associated with poorer survival. Evidence is
limited due to the extended use of the FL and DLBCL International
Prognostic Indices (FLIPI and IPI, respectively) and for lymphoma
prognosis and survival outcomes. The indices, in addition to the
lymphoma stage, integrate other prognostic factors such as serum
lactate dehydrogenase, the number of nodal site involvement,
patient ages, and haemoglobin. Evidence shows that a higher
index score, and thus a higher stage, is associated with poorer
health outcomes and survival: highlighting the necessity of
prompt management among patients at an advanced stage [65]. For DLBCL (Fig. 1), the results show a slight pattern such that
there were more CCGs with a larger population density (larger-
sized markers) that had a higher probability for their patients
being diagnosed through an emergency route to diagnosis
(markers with EB estimates above 0). There was no apparent
pattern for patients with FL (Fig. 2). Summary statistics Previous studies [57] have found delays in
diagnosis since first symptoms and suggested introducing rapid
access to lymph node diagnostic clinics [58] and providing: less
variability in the number of GP appointments attended before a
diagnosis [59, 60], clearer definitions of symptoms [61], and
appropriate patient-oriented information when previous investi-
gations rule out cancer [15]. These unmeasured characteristics of
CCGs could explain the large between-CCG variation in outcomes. In the United States, and for other malignancies, physician supply
is associated with early detection of breast cancer [62], and higher
primary care physician density is associated with a lower incidence
of late-stage colorectal cancer [63]. British Journal of Cancer DISCUSSION
d We aimed to assess the association between comorbidity status
and a marker of diagnostic delay (route to diagnosis), amongst British Journal of Cancer M.J. Smith et al. 5 Table 2. (A) Multivariable GLMM for the odds of the emergency route to diagnosis in (a) complete case analysis, (b) multiple imputation amongst
patients (n = 30,078) diagnosed with diffuse large B-cell lymphoma in England during 2005–2013; (B) multivariable GLMM for the odds of the
emergency route to diagnosis in (a) complete case analysis, (b) multiple imputation amongst patients (n = 15,551) diagnosed with follicular
lymphoma in England during 2005–2013. Table 2. (A) Multivariable GLMM for the odds of the emergency route to diagnosis in (a) complete case analysis, (b) multiple imputation amongst
patients (n = 30,078) diagnosed with diffuse large B-cell lymphoma in England during 2005–2013; (B) multivariable GLMM for the odds of the
emergency route to diagnosis in (a) complete case analysis, (b) multiple imputation amongst patients (n = 15,551) diagnosed with follicular
lymphoma in England during 2005–2013. Table 2. (A) Multivariable GLMM for the odds of the emergency route to diagnosis in (a) complete case analysis, (b) multiple imputation amongst
patients (n = 30,078) diagnosed with diffuse large B-cell lymphoma in England during 2005–2013; (B) multivariable GLMM for the odds of the
emergency route to diagnosis in (a) complete case analysis, (b) multiple imputation amongst patients (n = 15,551) diagnosed with follicular
lymphoma in England during 2005–2013. lymphoma in England during 2005–2013. DISCUSSION
d (A)
(a) Complete case analysis (n = 22,832)
(b) After multiple imputation (n = 30,078)
OR
95% CI
P value
OR
95% CI
P value
Agea
1.03
1.02–1.04
0.002
1.05
1.04–1.06
<0.001
Gender
Male
Ref
Ref
Ref
Ref
Female
0.95
0.90–1.01
0.082
0.95
0.91–1.00
0.061
Ethnicity
White
Ref
Ref
Ref
Ref
Minority
1.44
1.28–1.62
<0.001
1.42
1.26–1.60
<0.001
Deprivation
Least deprived
Ref
Ref
Ref
Ref
2
1.14
1.04–1.24
0.003
1.13
1.05–1.22
0.001
3
1.18
1.08–1.29
<0.001
1.17
1.08–1.27
<0.001
4
1.23
1.12–1.34
<0.001
1.23
1.14–1.34
<0.001
Most deprived
1.24
1.13–1.36
<0.001
1.32
1.21–1.43
<0.001
Comorbidity
None
Ref
Ref
Ref
Ref
One
1.26
1.12–1.41
<0.001
1.27
1.14–1.41
<0.001
Multimorbidity
1.58
1.41–1.78
<0.001
1.56
1.40–1.73
<0.001
Variance of RE (s.d.)
0.007
(0.09)
–
–
0.008
(0.09)
–
–
(B)
(a) Complete case analysis (n = 11,445)
(b) After multiple imputation (n = 15,551)
OR
95% CI
P value
OR
95% CI
P value
Agea
1.15
1.12–1.17
<0.001
1.17
1.15–1.19
<0.001
Gender
Male
Ref
Ref
Ref
Ref
Female
0.76
0.68–0.85
<0.001
0.80
0.73–0.89
<0.001
Ethnicity
White
Ref
Ref
Ref
Ref
Minority
1.03
0.80–1.32
0.835
1.03
0.81–1.29
0.833
Deprivation
Least deprived
Ref
Ref
Ref
Ref
2
1.16
0.98–1.38
0.084
1.11
0.95–1.29
0.190
3
1.09
0.92–1.30
0.312
1.07
0.92–1.24
0.396
4
1.42
1.20–1.69
<0.001
1.38
1.18–1.61
<0.001
Most deprived
1.38
1.14–1.66
<0.001
1.39
1.18–1.64
<0.001
Comorbidity
None
Ref
Ref
Ref
Ref
One
1.18
0.92–1.51
0.190
1.19
0.94–1.49
0.143
Multimorbidity
1.78
1.40–2.26
<0.001
1.80
1.45–2.23
<0.001
Variance of RE (s.d.)
0.016
(0.128)
–
–
0.017
(0.130)
–
–
OR odds ratio, CI confidence interval. aIncrease in odds of emergency route to diagnosis for each 10-year increase in age at diagnosis. (b) After multiple imputation (n = 15,551) OR odds ratio, CI confidence interval. aIncrease in odds of emergency route to diagnosis for each 10-year increase in age at diagnosis. CCGs with higher population densities have a higher probability of
emergency route to diagnosis. On one hand, deprivation tends to
be correlated with high population density in England [66], and is
also associated with higher use of emergency services [67]. On the
other hand, population density is independently associated with We graphically illustrated that patients living in CCGs with more
dense populations have a higher probability of emergency route
to diagnosis. To our knowledge, there is yet no research into the
relationship between population density and diagnostic delay of
cancer in England. British Journal of Cancer DISCUSSION
d 1
Variance of diagnostic delay of diffuse large B-cell lymphoma amongst clinical commissioning groups. Empirical Bayes estimates of
the random effects from the model for the route to diagnosis, by each Clinical Commissioning Group, amongst patients (n = 30,078)
diagnosed with diffuse large B-cell lymphoma in England during 2005–2013. (B) Follicular lymphoma (B) Follicular lymphoma −1.0
−0.5
0.0
0.5
1.0
CCG ID
EB estimates of the random effects
Population density (persons per square−Km)
500
1000
0
4
8
12
16
% missing records
Fig. 2
Variance of diagnostic delay of follicular lymphomas amongst clinincal commissioning groups. Empirical Bayes estimates of the
random effects from the model for the route to diagnosis, by each Clinical Commissioning Group amongst patients (n = 15,551) diagnosed
with follicular lymphoma in England during 2005–2013. Fig. 2
Variance of diagnostic delay of follicular lymphomas amongst clinincal commissioning groups. Empirical Bayes estimates of the
random effects from the model for the route to diagnosis, by each Clinical Commissioning Group amongst patients (n = 15,551) diagnosed
with follicular lymphoma in England during 2005–2013. high emergency calls [68]. This could be because highly dense
areas accumulate high demands that are not completely covered
by available healthcare resources; accordingly, this demand could
be exacerbated by the association between deprivation and the
prevalence of comorbidities. This association has not been well
explored, but it is likely that cancers other than NHL are affected
by the association between the prevalence of emergency route to
diagnosis and population density. Further research should be
conducted to determine the need for greater availability of
healthcare services in more populated areas. provide a full interpretation of these results. Densely populated areas
may be associated with populations from less favourable back-
grounds and potentially higher pressure on the healthcare system. CCGs were established from the Health and Social Care Act 2012 and
replaced Primary Care Trusts (PCTs). However, CCGs and PCTs were
constructed based on administrative boundaries, and the population
size of CCGs are similar to the PCTs they replaced. Since 2013, the
number of CCGs have reduced due to mergers [69], and the
proportion of late-staged lymphomas has increased [70], possibly
indicating competition for healthcare services. Our study is strengthened by the large population-based
sample capturing all patients with a diagnosis of DLBCL and FL
between 2005 and 2013. To date, this is the largest study of
diagnostic delay amongst patients with NHL. DISCUSSION
d This study shows that NHL patients living in British Journal of Cancer British Journal of Cancer M.J. Smith et al. −1.0
−0.5
0.0
0.5
1.0
CCG ID
EB estimates of the random effects
Population density (persons per square−Km)
500
1000
0
4
8
12
16
% missing records
(B) Follicular lymphoma
Fig. 2
Variance of diagnostic delay of follicular lymphomas amongst clinincal commissioning groups. Empirical Bayes estimates of the
random effects from the model for the route to diagnosis, by each Clinical Commissioning Group amongst patients (n = 15,551) diagnosed
with follicular lymphoma in England during 2005–2013. −1.0
−0.5
0.0
0.5
1.0
CCG ID
EB estimates of the random effects
Population density (persons per square−Km)
500
1000
0
4
8
12
% missing records
(A) DLBCL
Fig. 1
Variance of diagnostic delay of diffuse large B-cell lymphoma amongst clinical commissioning groups. Empirical Bayes estimates of
the random effects from the model for the route to diagnosis, by each Clinical Commissioning Group, amongst patients (n = 30,078)
diagnosed with diffuse large B-cell lymphoma in England during 2005–2013. M.J. Smith et al. 6 (B) Follicular lymphoma
−1.0
−0.5
0.0
0.5
1.0
CCG ID
EB estimates of the random effects
Population density (persons per square−Km)
500
1000
0
4
8
12
% missing records
(A) DLBCL
Fig. 1
Variance of diagnostic delay of diffuse large B-cell lymphoma amongst clinical commissioning groups. Empirical Bayes estimates of
the random effects from the model for the route to diagnosis, by each Clinical Commissioning Group, amongst patients (n = 30,078)
diagnosed with diffuse large B-cell lymphoma in England during 2005–2013. (B) Follicular lymphoma
Fig. 1
Variance of diagnostic delay of diffuse large B-cell lymphoma amongst clinical commissioning groups. Empirical Bayes estimates of
the random effects from the model for the route to diagnosis, by each Clinical Commissioning Group, amongst patients (n = 30,078)
diagnosed with diffuse large B-cell lymphoma in England during 2005–2013. −1.0
−0.5
0.0
0.5
1.0
CCG ID
EB estimates of the random effects
Population density (persons per square−Km)
500
1000
0
4
8
12
16
% missing records
(B) Follicular lymphoma
Fig. 2
Variance of diagnostic delay of follicular lymphomas amongst clinincal commissioning groups. Empirical Bayes estimates of the
random effects from the model for the route to diagnosis, by each Clinical Commissioning Group amongst patients (n = 15,551) diagnosed
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from
each
other
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Surg. 2010;97:772–81. 66. Venerandi A, Quattrone G, Capra L. AUTHOR CONTRIBUTIONS Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41416-021-01523-6. MS, ENN and BR contributed to the conception of the study and designed the study. ENN, BR, ABe, MALF and MQ provided advice on statistical methods. MS conducted
the analyses of the data and prepared the draft of the manuscript, tables and figures. ENN and BR supervised the study and provided comments on the manuscript draft. ENN, BR, MALF, MQ, SBM, ABe and ABo provided comments on the final draft of the
manuscript. All authors read and approved the final manuscript. Correspondence and requests for materials should be addressed to M.J.S. Reprints and permission information is available at http://www.nature.com/
reprints Reprints and permission information is available at http://www.nature.com/
reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations. ACKNOWLEDGEMENTS We would like to thank Adrian Turculet, Data Manager of the LSHTM Inequalities in
Cancer Outcomes Network, for his support and assistance with the data linkage. The authors declare no competing interests. CONCLUSION Multilevel and longitudinal modelling using stata,
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the joint latent normal model. Biom J. 2019;61:1003–19. 46. Verbeke G, Molenberghs G. Linear mixed models for longitudinal data. New York:
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Sons, Ltd; New Jersey. 2013. 48. Haematological Malignancy Research Network. Survival of non-Hodgkin lymphoma. 2016. Date accessed: 2020-09-01. https://www.hmrn.org/statistics/survival. 78. Verbeke G, Molenberghs G. Arbitrariness of models for augmented and coarse
data, with emphasis on incomplete data and random effects models. Stat
Modelling 2010;10:391–419. 49. Little RJ, Rubin DB. Statistical analysis with missing data. John Wiley & Sons, Inc.;
New Jersey. 1987. 79. Molenberghs G, Njagi E, Kenward M, Verbeke G. Enriched-data problems and
essential non-identifiability. Int J Stat Med Res. 2012;1:16–44. 50. Rubin DB. Multiple imputation for nonresponse in surveys. Wiley; New York. 1987. 80. Swann R, Lyratzopoulos G, Rubin G, Pickworth E, McPhail S. The frequency, nature
and impact of GP-assessed avoidable delays in a population-based cohort of
cancer patients. Cancer Epidemiol. 2020;64:101617. 51. Quartagno M, Carpenter J R. jomo: a package for multilevel joint modeling
multiple imputation. 2016. Date accessed: 2020-01-01. http://cran.r-project.org/
package=jomo. British Journal of Cancer British Journal of Cancer M.J. Smith et al. 9 FUNDING This research was funded by Cancer Research UK grant number C7923/A18525. The
authors declare no support from any organisations for the submitted work. The design
of the study, the analyses and the writing of the manuscript were solely the
responsibility of the authors. The findings and conclusions in this manuscript are those
of the authors and do not necessarily represent the views of Cancer Research UK. Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
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Commons license, and indicate if changes were made. The images or other third party
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indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. ETHICS APPROVAL AND CONSENT TO PARTICIPATE We
obtained
the
statutory
approvals
required
for
this
research
from
the
Confidentiality Advisory Group (CAG) of the Health Research Authority (HRA): PIAG
1–05(c) 2007. Ethical approval was obtained from the Research Ethics Committee
(REC) of the Health Research Authority (HRA): 07/MRE01/52. This work uses the data
provided by patients and collected by the National Health Service as part of their care
and support. We used anonymised National Cancer Registry and Hospital Episode
Statistics data. No consent to participate was sought from patients. © The Author(s) 2021 CONSENT TO PUBLISH
Not applicable. Not applicable. British Journal of Cancer British Journal of Cancer
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Workplace flourishing: Measurement, antecedents and outcomes
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Page 1 of 11
Original Research Page 1 of 11
Original Research Original Research Page 1 of 11 Dates: Practical/managerial implications: Organisations should continually modify their
strategic frameworks to maintain a healthy balance between individual and environmental
characteristics. This will lay the foundation for a favourable work environment. When such an
environment is institutionalised, talent retention and performance should follow. How to cite this article:
Redelinghuys, K.,
Rothmann, S., & Botha, E. (2019). Workplace
flourishing: Measurement,
antecedents and outcomes. SA Journal of Industrial
Psychology/SA Tydskrif vir
Bedryfsielkunde, 45(0),
a1549. https://doi.org/
10.4102/sajip.v45i0.1549 How to cite this article:
Redelinghuys, K.,
Rothmann, S., & Botha, E. (2019). Workplace
flourishing: Measurement,
antecedents and outcomes. SA Journal of Industrial
Psychology/SA Tydskrif vir
Bedryfsielkunde, 45(0),
a1549. https://doi.org/
10.4102/sajip.v45i0.1549 Contribution/value-add: The study results should provide new insight into the relationship
between the person–environment fit and WF, as well as the effect it may have on ITL and
performance. Note: This article is partially based on the author’s thesis for the degree Doctor of Philosophy in Industrial Psychology at the North-West
University (Vaal Triangle Campus), South Africa, with promoter Prof. S. Rothmann and co-promoter Dr E. Botha, November 2016,
available here: http://dspace.nwu.ac.za/bitstream/handle/10394/24959/Redelinghuys_JJ.pdf?isAllowed=y&sequence=1 Workplace flourishing: Measurement,
antecedents and outcomes Orientation: The continuous growth of employee attrition, especially within the highly skilled
talent pool, is becoming increasingly problematic. Therefore, one should continually explore
the different factors that impact employee retention and performance. This casts the attention
to the person–environment fit and workplace flourishing (WF). Affiliations:
1Optentia Research Focus
Area, North-West University,
South Africa Research purpose: This study explored relationships among person–environment fit, WF,
intention to leave (ITL), in-role performance and organisational citizenship behaviour. Motivation for the study: Disease-driven research heavily outweighs health promotion
research. Therefore, more research is needed regarding positive employee behaviours such
as strengths, optimal functioning and flourishing. 2School of Industrial
Psychology and Human
Resource Management,
Optentia Research Focus
Area, North-West University,
South Africa Research approach/design and method: A cross-sectional survey design was used with 258
secondary school teachers from two Gauteng districts. The Perceived Fit, Flourishing-at-Work,
Turnover Intention, In-Role Behaviour and Organisational Citizenship Behaviour scales were
administered. Structural equation modelling and mediational analyses were performed. Main findings: Results confirmed WF’s three-factor structure. Person–environment fit
positively associated with WF. Workplace flourishing negatively related to ITL, while positively
relating to in-role performance and organisational citizenship behaviour. Person–environment
fit indirectly affected in-role performance and organisational citizenship behaviour via WF. Introduction The continuous growth and manifestation of employee attrition, especially within the highly
skilled talent pool, is becoming increasingly problematic. When organisations establish policies
that retain productive personnel, remove unproductive personnel and select the most suitable
new candidates, they should have a favourable composition of qualified and talented employees
(Adnot, Dee, Katz, & Wyckoff, 2017). Thus, two factors are emphasised: employee retention and
employee performance. To address this persisting issue, researchers need to establish different
factors that impact employee retention and employee performance, factors in addition to well-
established disease-driven aspects such as stress, burnout and depression (e.g. Steinhardt, Smith
Jaggars, Faulk, & Gloria, 2011). Although the preceding aspects are undeniably significant in their
own right, they tend to ignore or fail in furthering our understanding of the role flourishing plays
in employee well-being (Youssef-Morgan & Luthans, 2014). SA Journal of Industrial Psychology
ISSN: (Online) 2071-0763, (Print) 0258-5200 Flourishing at work Fishbein and Ajzen (1975) contend that beliefs precede
attitudes, intentions and ultimately behaviours. Meta-
analytic studies (Hoffman & Woehr, 2006; Kristof-Brown,
Zimmerman, & Johnson, 2005; Oh et al., 2014; Verquer,
Beehr, & Wagner, 2003) suggest that person–organisation and
person–job fit generally has a strong association with
attitudes (e.g. job satisfaction), while having a weaker
association with intentions (e.g. ITL) and behaviours (e.g. in-role, extra-role). Therefore, although PEF strongly relates
to important job attitudes, intervening variables may be
required to amplify its association with intention and
behaviour. A feasible intervening variable in the relationship
between PEF and the outcomes contained in this study
(ITL, in-role performance, and organisational citizenship
behaviour [OCB]), could be WF. WF contains various
components that directly link to PEF and the study outcome,
as well as numerous components that could mediate fit–
outcome relationships. Emotional
well-being
incorporates
three
employee
judgements: job satisfaction, positive emotions and negative
emotions. Job satisfaction reflects the amount of congruence
between employees’ perceptions and standards (Weiss &
Cropanzano, 1996). Positive affect stands key towards an
individual’s capacity to flourish. It refers to pleasant reactions
at work (e.g. joy, interest, and gratitude), while negative
affect encompasses unpleasant reactions (e.g. sadness,
boredom and anxiety). Although both job satisfaction and
affect tap into employee emotions, the focus of the constructs
differs slightly, making both constructs valuable flourishing
components. Job satisfaction reveals employees’ perceptions
that their wants are attended to, while affect relates to
employees’ perceptions that their needs are attended to
(Rojas & Veenhoven, 2013). Psychological well-being at work includes dimensions
(autonomy, personal growth, mastery, meaning, purpose and
positive relations) in Ryff and Singer’s (1998) model, as well
as engagement (Seligman, 2011). Three dimensions in the
model of Ryff and Singer (1998) relate to three psychological
needs (Deci & Ryan, 1985), namely autonomy (i.e. autonomy
satisfaction – experiencing independence and choice
in executing work-related tasks), mastery (i.e. competence
satisfaction – feeling effective in work environment
interaction) and positive relations (i.e. relatedness
satisfaction – experiencing a sense of connectedness to others
in the workplace). Meaning reflects the perceived significance
of employees’ work experiences, while purpose refers to
possessing a sense of the preferred outcomes connected to
one’s work-related behaviour (Barrick, Mount, & Li, 2013). Learning (similar to personal growth in the model of Ryff &
Singer, 1998) is defined as the acquisition and application
of knowledge and expertise to one’s job (Spreitzer, Lam, &
Fritz, 2010). Page 2 of 11 Original Research Page 2 of 11 Using the Flourishing-at-Work Scale (FAWS; Rautenbach,
2015), Janse van Rensburg, Rothmann, and Diedericks (2017)
found that person–environment fit relates (PEF) positively
with flourishing and negatively with intention to leave (ITL)
via flourishing. However, the effects of WF (in addition to
PEF) on other organisational outcomes have not been studied. Moreover, the social well-being scale of the FAWS as
developed by Rautenbach (2015) and used by Janse van
Rensburg et al. (2017) included only one item per social
well-being dimension. Therefore, it is vital to investigate
additional factors associated with WF to strengthen the
FAWS’s psychometric properties. Social well-being includes five dimensions: social acceptance
(the acceptance of the diversity of colleagues), actualisation
(the belief in one’s organisation, team and colleagues’
potential), coherence (the belief that one’s organisation
and social relations at work are both meaningful and
comprehensible), contribution (the belief that one’s daily
work tasks add value to one’s team, department, and
organisation) and integration (the belief that one experiences
a sense of communal connectedness and belongingness) and
the mental health continuum (MHC) (Keyes, 1998, 2005). The
social element of work plays a pivotal part in flourishing
(Grant, 2008), as employees are embedded within social
organisational structures, facing endless social tasks and
challenges (Keyes, 1998). The study aim was to examine relationships among PEF,
WF, ITL, in-role performance and organisational citizenship
behaviour (OCB). Copyright: Copyright:
© 2019. The Authors. Licensee: AOSIS. This work
is licensed under the
Creative Commons
Attribution License. Read online:
Scan this QR
code with your
smart phone or
mobile device
to read online. Read online:
Scan this QR
code with your
smart phone or
mobile device
to read online. The concept of flourishing in life (Keyes, 2002) has positioned itself as the most prominent
multidimensional well-being model (Keyes, 2002; Seligman, 2011). Building on the study of Keyes
(2002), Rothmann (2013) studied the multidimensionality of flourishing, comprising emotional
well-being, psychological well-being and social well-being in work and organisational contexts. Workplace flourishing (WF) has been defined as an employee’s perception that he or she is feeling
and functioning well in the workplace (Rautenbach, 2015). Read online:
Scan this QR
code with your
smart phone or
mobile device
to read online. Read online:
Scan this QR
code with your
smart phone or
mobile device
to read online. Open Access http://www.sajip.co.za Original Research what is expected in the basic job requirements. Four
dimensions constitute OCB: helping, loyalty, advocacy, as
well as functional participation and obedience (Coyle-
Shapiro, 2002; Van Dyne, Graham, & Dienesch, 1994). Helping reflects the extent to which employees offer
assistance to others. Loyalty relates to the identification
with or loyalty towards the organisation, which involves
cooperation and serving the organisational interests. Advocacy refers to behaviour aimed at others in the
organisation, which includes the maintenance of high
standards, the challenging of others and the proposition of
change. Functional participation assumes a more personal
stance, while simultaneously contributing to organisational
efficiency (Coyle-Shapiro, 2002; Van Dyne et al., 1994). (TWA; Dawis & Lofquist, 1984) and job embeddedness
theory (Mitchell, Holtom, Lee, Sablynski, & Erez, 2001)
suggest that when congruence is achieved between
employees and their work environment and when employees
establish meaningful relationships in the workplace,
they will experience increased satisfaction, which will
subsequently affect their turnover decisions. For instance,
job satisfaction (an emotional well-being element) plays a
central part in various employee turnover models (Hom,
Lee, Shaw, & Hausknecht, 2017), suggesting that dissatisfied
employees will start to explore other possibilities through a
range of evaluation processes. Similarly, the attraction–
selection–attrition (ASA) model (Schneider, 1987) suggests
that employees are attracted to and remain within
organisations with which they share similar preferences, as
this allows them to achieve their goals. Regarding social
well-being, the social identity theory (Tajfel & Turner, 1986)
proposes that employees who experience fit with their
organisation’s values become part of a psychological group,
defined as the ‘collection of people who share the same
social identification or define themselves in terms of the
same social category membership’ (Turner, 1984, p. 530). Therefore, when employees experience a favourable balance
between their personal (values, abilities and needs) and
environmental
characteristics
(values,
demands
and
supplies), it lays the foundation for a positive work
environment. Within this environment, employees may
experience a sense of acceptance, enjoyment, integration,
meaningfulness and relatedness, among others (Janse van
Rensburg et al., 2017). Subsequently, employees should
experience little to no urge to leave their work setting (Janse
van Rensburg et al., 2017). Intention to leave is defined as an
employee’s cognisant and intentional frame of mind to part
ways with his or her organisation (Tett & Meyer, 1993). Original Research Although the association between WF, in-role performance
and OCB has been studied before (Redelinghuys et al.,
2018), the indirect effect of PEF on performance (in-role
and extra-role) via WF is yet to be investigated. Social
exchange theory (Blau, 1964) may offer a valuable framework
for these relationships. The latter theory posits that employees
and organisations enter into social exchanges with one
another when they perceive the other party to be a worthy
contributor to the relationship. Therefore, when one party
(the organisation) positively impacts another (the employee),
the latter should return the favour to honour their part of the
exchange. Thus, when organisations positively impact its
employees by providing an environment that sufficiently
attends to their needs, demands and the things they value
and subsequently increase their probability of experiencing
positive
work-related
well-being,
employees
should
upwardly adjust their performance and helping behaviours
to express their gratitude. In addition to the preceding framework, numerous studies
suggest that WF elements link to performance (in-role
and extra-role) to varying degrees. For example, job
satisfaction (Judge, Thoresen, Bono, & Patton, 2001; LePine,
Erez, & Johnson, 2002), positive affect (Ziegler, Schlett,
Casel,
&
Diehl,
2012),
psychological
well-being
(Cropanzano & Wright, 2001) and work engagement
(Babcock-Roberson & Strickland, 2010) have been linked
to performance. Furthermore, studies suggest that PEF may
indirectly affect in-role performance via psychological need
satisfaction (Greguras & Diefendorff, 2009) and OCB via job
satisfaction (Van Dyne et al., 1994). Apart from the preceding frameworks, various studies
suggest that PEF links to WF elements, including job
satisfaction (Cable & DeRue, 2002), positive affect (Gabriel,
Diefendorff, Chandler, Moran, & Greguras, 2014), meaningful
work (Duffy, Autin, & Bott, 2015), psychological need
satisfaction (Greguras & Diefendorff, 2009) and work
engagement (Chen, Yen, & Tsai, 2014). Subsequently, studies
also link various WF elements to ITL, including affect
(Thoresen, Kaplan, Barsky, Warren, & De Chermont, 2003),
work engagement (Alarcon & Edwards, 2011), job satisfaction
(Redelinghuys & Botha, 2016) and meaningful work (Janik &
Rothmann, 2015); while PEF has indirectly affected ITL via
job satisfaction (Wheeler, Gallagher, Brouer, & Sablynski,
2007), work engagement (Peng, Lee, & Tseng, 2014) and more
recently WF (Janse van Rensburg et al., 2017). Flourishing at work Finally, work engagement comprises three
components, namely a physical component (involvement
and the exhibition of vitality), a cognitive component
(absorption and involvement) and an affective component
(connectedness to one’s job and dedication) (Kahn &
Heaphy, 2014). Cable and DeRue (2002) adopted a three-factor person–
environment
fit
conceptualisation
(person–organisation,
needs–supplies, and demands–abilities). Person–organisation
fit reflects the degree of perceived similarity in terms of
values between employees and organisations (Cable &
DeRue, 2002). Employees feel attached to the organisation’s
broader mission when they believe that their values match
that of their organisation and colleagues. Employees form
separate cognitions regarding organisational and job-related
fit. Needs–supplies fit reflects the perceived similarity
between job rewards (what the organisation offers) and
employee needs (Cable & DeRue, 2002). When employees
perceive a match between what they expect from their job
and what they receive, job satisfaction materialises (Dawis &
Lofquist, 1984; Locke, 1976). Demands–abilities fit reflects
the perceived similarity between job demands and an
employee’s inhabited knowledge, skills and capabilities
(Cable & DeRue, 2002). Traditionally, fit literature posits that if congruence is
reached between the employee and the work environment,
positive outcomes ensue. The work adjustment theory http://www.sajip.co.za Open Access Page 3 of 11 Page 3 of 11 Original Research The following hypotheses stemmed from the discussion: The following hypotheses stemmed from the discussion: H1: Person–environment fit positively associates with WF. H2: WF negatively associates with ITL. H2: WF negatively associates with ITL. Apart from ITL, in-role performance and OCB have also
been associated with WF (Redelinghuys, Rothmann, & Botha,
2018). In-role performance refers to the undertakings an
employee is expected to fulfil as stipulated in his or her
formal job requirements (Borman & Motowidlo, 1997;
Williams & Anderson, 1991). In contrast, Lambert (2006)
defines OCB as employee behaviour that contributes beyond H3: WF positively associates with in-role performance. H4: WF positively associates with OCB. H5: Person–environment fit indirectly affects ITL via WF. H6: Person–environment fit indirectly affects in-role performance
via WF. H7: Person–environment fit indirectly affects OCB via WF. H7: Person–environment fit indirectly affects OCB via WF. http://www.sajip.co.za Open Access Page 4 of 11 Original Research (three items), relatedness satisfaction (three items), learning
(two items), meaningful work (three items) and engagement
(seven items). A sample item for this dimension includes:
‘How often did you become enthusiastic about your job?’
Social well-being comprises five dimensions (three items per
dimension): social acceptance, actualisation, coherence,
contribution and integration. A sample item for this
dimension includes: ‘How often did you feel included at
your school?’ Rautenbach (2015) confirmed the FAWS’s
three-factor structure, with rho coefficients ranging from 0.77
to 0.95. Research design
Research participants (three items), relatedness satisfaction (three items), learning
(two items), meaningful work (three items) and engagement
(seven items). A sample item for this dimension includes:
‘How often did you become enthusiastic about your job?’
Social well-being comprises five dimensions (three items per
dimension): social acceptance, actualisation, coherence,
contribution and integration. A sample item for this
dimension includes: ‘How often did you feel included at
your school?’ Rautenbach (2015) confirmed the FAWS’s
three-factor structure, with rho coefficients ranging from 0.77
to 0.95. A survey was conducted among personnel fulfilling a
teaching role in the Sedibeng East and West districts in
Gauteng. The teaching profession is a good framework for
studies on flourishing, as research that assumes a disease-
driven or dysfunctional behaviour stance (e.g. O’Brennan,
Pas, & Bradshaw, 2017), heavily outweighs a health promotion
or positive functioning stance (e.g. Li, Wang, Gao, & You,
2017). Therefore, more research is needed regarding positive
employee behaviours, which include aspects such as
strengths, optimal functioning and flourishing (Youssef-
Morgan & Luthans, 2014). This is especially important when
considering the demands teachers face (e.g. student ill-
discipline, fellow educator absenteeism, overinvolvement or
lack of parental involvement) and the ever-increasing list of
role-players (e.g. department of education, school-governing
body, management, parents and learners) they need to satisfy. Approximately 800 surveys were circulated, while 258 were
completed adequately (32% response rate). Table 1 provides
the sample characteristics. The Perceived Fit Scale (PFS; Cable & DeRue, 2002) measured
PEF. It contains nine items recorded on a 7-point scale, which
ranges from 1 (strongly disagree) to 7 (strongly agree). The
PFS comprises three dimensions (three items each): person–
organisation fit: ‘My personal values match my school’s
values and culture’, need–supplies fit: ‘The attributes that I
look for in a job are fulfilled very well by my present job’, and
demands–abilities fit: ‘My abilities and training are a good fit
with the requirements of my job’. Redelinghuys and Botha
(2016) confirmed the PFS’s three-factor structure, with rho
coefficients ranging from 0.85 to 0.88. Measuring instruments The Turnover Intention Scale (Sjöberg & Sverke, 2000)
measured ITL. It contains three items recorded on a 5-point
scale, which ranges from 1 (strongly disagree) to 5 (strongly
agree). Covering a solitary dimension, a sample item
includes: ‘I feel that I could leave this job’. Janse van Rensburg
et al. (2017) yielded a rho coefficient of 0.71. The Flourishing-at-Work Scale (FAWS; Rautenbach, 2015)
measured WF. It comprises 46 items recorded on a 6-point
scale ranging from 1 (never) to 6 (every day). Participants were
required to respond to questions concerning the regularity
with which they experienced particular symptoms at work
during the preceding month. The FAWS encompasses three
dimensions: emotional well-being, psychological well-being
and social well-being. Emotional well-being comprises three
dimensions (three items per dimension): positive affect,
negative affect and job satisfaction. A sample item for this
dimension includes: ‘How often did you feel grateful?’. Psychological
well-being
comprises
six
dimensions:
autonomy satisfaction (three items), competence satisfaction The In-role Behaviour Scale (Williams & Anderson, 1991)
measured in-role performance. It entails seven items scored
on a 7-point scale ranging from 1 (strongly disagree) to 7
(strongly agree). Covering a solitary dimension, a sample
item includes: ‘I adequately complete assigned duties’. Participants were required to rate their own performance, as
external evaluation was prohibited. Redelinghuys et al. (2018) yielded a rho coefficient of 0.73. TABLE 1: Characteristics of the participants (N = 258). Item
Frequency
%
Gender
Male
62
24.0
Female
90
73.7
Missing values
6
2.3
Age
21–35 years
106
41.1
Older than 35 years
129
50.0
Missing values
23
8.9
Race
African people
48
18.6
Mixed Race people
2
0.8
Indian people
9
3.5
White people
192
74.4
Other people
1
0.4
Missing values
6
2.3
Years of service
0–10 years
104
40.3
More than 10 years
134
51.9
Missing values
20
7.8 The Organisational Citizenship Behaviour Scale (OCBS;
Rothmann, 2010) measured OCB. It entails six items recorded
on a 7-point scale, which ranges from 1 (strongly disagree) to
7 (strongly agree). The OCBS comprises two dimensions
(three items each): assistance to co-workers: ‘I assist others
with their duties’ and assistance to the organisation: ‘I defend
the school when other employees criticise it’. Diedericks and
Rothmann (2014) confirmed the OCBS’s two-factor structure,
with adequate reliability coefficients (> 0.70). Statistical analysis Confirmatory factor analyses were carried out with the
scales through Mplus 7.41 (Muthén & Muthén, 1998–2016). A
hypothesised measurement model (Model 1) was specified
and tested against opposing models (Models 2–5) to establish
which model fitted the data best. Mplus 7.41 (Muthén & Muthén, 1998–2016) was applied to
analyse the data. The weighted least-squares with mean and
variance adjustment (WLSMV) estimator was utilised as it
does not assume normally distributed variables, providing
the most suitable selection for categorical data modelling. To evaluate the reliability of the measuring battery, rho
coefficients (Raykov, 2009) were utilised. The practical
significance of results was determined by effect sizes (Cohen,
1988). The confidence interval (CI) level was set at a value of
95% (p ≤ 0.05) for statistical significance. A measurement
model was specified and tested against numerous goodness-
of-fit indices. Descriptive statistics were computed with
SPSS23 (IBM Corp, 2016). The model comprised one third-order factor (WF), two
second-order factors (PEF and OCB) and two first-order
factors (ITL: measured by three observed variables, and in-
role performance: measured by seven observed variables). WF comprised three second-order factors: emotional well-
being, psychological well-being, and social well-being. Emotional well-being comprised three first-order factors
(each measured by three observed variables): positive affect,
negative affect and job satisfaction. Psychological well-being
comprised six first-order factors: autonomy, competence,
relatedness (each measured by three observed variables),
learning (measured by two observed variables), meaningful
work (measured by three observed variables) and engagement
(measured by seven observed variables). Social well-being
comprised five first-order factors (each measured by
three observed variables): social contribution, integration,
actualisation, acceptance and coherence. Person–environment
fit comprised three first-order factors (each measured by
three observed variables): person–organisation fit, needs–
supplies fit and demands–abilities fit. Organisational
citizenship behaviour comprised two first-order factors (each
measured by three observed variables): citizenship behaviour
towards co-workers and citizenship behaviour towards the
organisation. All latent variables were correlated. Four opposing measurement models were specified and
tested to make model comparison possible as suggested by
Wang and Wang (2012). The best fitting model (Model 1) was
respecified as a structural model (Model 6) and compared to
opposing structural models. The chi-square statistic, root
mean square error of approximation (RMSEA), Tucker–Lewis
index (TLI), comparative fit index (CFI) and the weighted
root mean square residual (WRMR) were utilised. Comparative fit index and TLI values of ≥ 0.90 were
considered satisfactory. Statistical analysis Root mean square error of
approximation values of < 0.08 indicated close model fit. Discriminant validity of the constructs was assessed using a
method proposed by Farrell (2010). To establish discriminant
validity, three values are important. Firstly, the correlations
between constructs (see the values below the diagonal in
Table 3), as this is used to compute squared correlations. Secondly, the average variance extracted (AVE) (see the
values on the diagonal in Table 3), which is calculated for
each construct by adding the R-square values of each
construct item, and then dividing it by the number of items
the construct has. For example, person–organisation fit has
three items; therefore, its AVE is calculated as follows: 0.838 +
0.966 + 0.838/3 = 0.88. Lastly, squared correlation values
(see the values above the diagonal in Table 3) are horizontally
and vertically compared to AVE values. Average variance
extracted values greater than the squared correlation values,
indicate sufficient discriminant validity. A c2 of 4668.46 (df = 2313) was achieved for the original
measurement model. Acceptable CFI and TLI fit indices
(both 0.93) were achieved. The RMSEA (0.06) and c2/df (2.02)
were acceptable, while the WRMR (1.62) value was lower
compared to the alternative models. Following the same blueprint as Model 1, Models 2–5 were
similarly specified and tested, with minor changes to the
models. In Model 2, PEF consisted of one first-order variable:
fit (measured by nine observed variables). In Model 3, WF
consisted of 3 second-order variables: emotional well-being
(measured by 9 observed variables), psychological well-
being (measured by 21 observed variables) and social well-
being (measured by 15 observed variables). In Model 4, WF
consisted of two second-order variables: hedonic well-being
(measured by nine observed variables) and eudaimonic well-
being (measured by 36 observed variables). In Model 5, PEF
and WF jointly consisted of one first-order variable: well-
being (measured by 54 observed variables). Indirect effects were assessed in Mplus 7.41. Bootstrapping
with 10 000 samples was applied to construct two-sided bias-
corrected 95% CIs (Hayes, 2018). Lower and upper CIs were
conveyed. Research procedure Once ethical permissions had been obtained from the
necessary authorities, the researchers communicated with
the secondary school principals in the selected districts. The
researchers arranged dates and times with probable research
participants at their respective schools to discuss the study
purpose and to obtain informed consent. Paper questionnaires, Open Access Page 5 of 11 Original Research West District offices, as well as ethical clearance from the
North-West University’s Ethics Committee. West District offices, as well as ethical clearance from the
North-West University’s Ethics Committee. with English as instructional language, were distributed to
consenting participants; granting them a 2-week period to
complete the questionnaires. Arrangements were made for
participants to securely return their questionnaires. Ethical consideration Testing indirect effects
To establish whether PEF indirectly aff
performance and OCB, the authors us
guidelines Descriptive statistics, reliabilities, correlation
coefficients and discriminant validity Model 6b: χ² = 4691.298; df = 2316; p < 0.001; TLI = 0.93;
CFI = 0.93; RMSEA = 0.06 (90% CI: 0.060, 0.066); WRMR =
1.64. The WLSMV estimator was used in these calculations. The chi-square values for WLSMV cannot be used for
chi-square difference testing (Satorra & Bentler, 2010). Therefore, the Difftest Mplus function was utilised. Table 4
illustrates the difference testing of the opposing structural
models and designates Model 6b as the best fitting opposing
model. Model 6b: χ² = 4691.298; df = 2316; p < 0.001; TLI = 0.93;
CFI = 0.93; RMSEA = 0.06 (90% CI: 0.060, 0.066); WRMR =
1.64. The WLSMV estimator was used in these calculations. The chi-square values for WLSMV cannot be used for
chi-square difference testing (Satorra & Bentler, 2010). Therefore, the Difftest Mplus function was utilised. Table 4
illustrates the difference testing of the opposing structural
models and designates Model 6b as the best fitting opposing
model. Table 3 reports the descriptive statistics, reliabilities,
correlation coefficients and discriminant validity of the
constructs. The reliabilities of the measuring instruments were
acceptable, ranging from 0.75 to 0.94 (Nunnally & Bernstein,
1994). All the PEF dimensions were practically and
statistically significantly related to the three emotional well-
being dimensions, six psychological well-being dimensions
and five social well-being dimensions, ranging from
medium to large effects. Person–environment fit dimensions
were practically and statistically significantly related to job
satisfaction (emotional well-being), autonomy satisfaction
(psychological well-being) and social actualisation (social
well-being) with a large effect. Figure 1 illustrates the standardised path coefficients
estimated by Mplus 7.41 (Muthén & Muthén, 1998–2016) for
Model 6. Figure 1 illustrates the standard path coefficients found with
PEF as independent variable and WF, ITL, in-role performance
and OCB as dependent variables and also WF as an
independent variable with ITL, in-role performance and OCB
as dependent variables. Most of the flourishing dimensions were practically and
statistically significantly related to ITL (large effects), except
for negative affect (0.46), competence (−0.43), meaning
(−0.48) and learning (−0.48). Most flourishing dimensions
were practically and statistically significantly related to in-
role performance with a medium effect, except for negative
affect (−0.29) and competence (0.28). Most flourishing
dimensions were practically and statistically significantly
related to OCB (to co-workers) with a medium effect, as well
as OCB (to the organisation), ranging from medium to large
effects. Structural model testing A structural model (Model 6) was specified and tested
based on Model 1 (the best fitting measurement model). No difference in the chi-square values (4668.462) of the
best fitting measurement and structural models, indicated
acceptable model specification. Model 6 yielded the following
fit statistics: χ² = 4668.462; df = 2313; p < 0.001; TLI = 0.93;
CFI = 0.93; RMSEA = 0.06 (90% CI: 0.060, 0.065); WRMR =
1.62. As a result of the cross-sectional nature of the study, two
opposing models (6a and 6b) were tested for comparable fit
purposes, as suggested by Hancock and Mueller (2010). Model 6a: χ² = 10761.737; df = 2314; p < 0.001; TLI = 0.73; CFI =
0.75; RMSEA = 0.12 (90% CI: 0.117, 0.121); WRMR = 3.31. Regarding effect sizes (Cohen, 1988), the model accounted
for the following percentages of variance explained (R2):
WF = 67% (large effect), ITL = 62% (large effect), in-role
performance = 21% (medium effect) and OCB = 40% (large
effect). Descriptive statistics, reliabilities, correlation
coefficients and discriminant validity For the model portion predicting WF, PEF’s path coefficient
(b = 0.82; p ≤ 0.01) was statistically significant and displayed
the anticipated sign. Therefore, Hypothesis 1 is accepted. For the model portion predicting ITL, the path coefficients
of PEF (b = –0.60; p ≤ 0.01) and WF (b = −0.22; p ≤ 0.05) were
statistically significant and displayed the anticipated sign. Therefore, Hypothesis 2 is accepted. For the model portion predicting in-role performance, WF’s
path coefficient (b = 0.34; p ≤ 0.01) was statistically significant
and displayed the anticipated sign. Therefore, Hypothesis 3
is accepted. Discriminant validity is supported. Table 3 shows that the
AVE per construct (on the diagonal) was greater than the
squared correlation values (above the diagonal). For the model portion predicting OCB, WF’s path coefficient
(b = 0.54; p ≤ 0.01) was statistically significant and displayed
the anticipated sign. Therefore, Hypothesis 4 is accepted. Ethical consideration Authorisation for the study was acquired from the
Gauteng Department of Education, the Sedibeng East and Table 2 presents the goodness-of-fit statistics for the five
competing measurement models described above. http://www.sajip.co.za Open Access Page 6 of 11
Original Research
TABLE 2: Goodness-of-fit statistics of competing measurement models. Model
b2
df
TLI
CFI
RMSEA
WRMR
Estimate
90% CI
1
4668.462*
2313
0.93
0.93
0.06
0.060, 0.065
1.62
2
5028.588*
2316
0.92
0.92
0.07
0.065, 0.070
1.74
3
6133.391*
2327
0.88
0.89
0.08
0.077, 0.082
2.03
4
7080.640*
2328
0.85
0.86
0.09
0.087, 0.091
2.27
5
8894.941*
2337
0.80
0.80
0.10
0.102, 0.107
2.68
χ2, chi-square statistic; df, degrees of freedom; TLI, Tucker–Lewis index; CI, confidence interval; CFI, comparative fit index; RMSEA, root mean square error of approximation; WRMR, weighted root
mean square residual. ties,
the
were
ein,
and
well-
ions
rom
ions
job
tion
ocial
and
cept
ning
ions
in-
tive
hing
ntly
well
arge
the
the
sted
del). the
ated
wing
0.93;
R =
two
e fit
10). FI =
3 31
Model 6b: χ² = 4691.298; df = 2316; p <
CFI = 0.93; RMSEA = 0.06 (90% CI: 0.060
1.64. The WLSMV estimator was used in
The chi-square values for WLSMV ca
chi-square difference testing (Satorra
Therefore, the Difftest Mplus function w
illustrates the difference testing of the o
models and designates Model 6b as the be
model. Figure 1 illustrates the standardised
estimated by Mplus 7.41 (Muthén & Muth
Model 6. Figure 1 illustrates the standard path coef
PEF as independent variable and WF, ITL, i
and OCB as dependent variables and
independent variable with ITL, in-role per
as dependent variables. For the model portion predicting WF, PE
(b = 0.82; p ≤ 0.01) was statistically signif
the anticipated sign. Therefore, Hypothes
For the model portion predicting ITL, th
of PEF (b = –0.60; p ≤ 0.01) and WF (b = −
statistically significant and displayed th
Therefore, Hypothesis 2 is accepted. For the model portion predicting in-role
path coefficient (b = 0.34; p ≤ 0.01) was stat
and displayed the anticipated sign. There
is accepted. For the model portion predicting OCB, W
(b = 0.54; p ≤ 0.01) was statistically signif
the anticipated sign. Therefore, Hypothes
Regarding effect sizes (Cohen, 1988), the
for the following percentages of varian
WF = 67% (large effect), ITL = 62% (la
performance = 21% (medium effect) and
effect). Page 7 of 11
Original Research Original Research TABLE 3: Descriptive statistics, reliabilities, correlation coefficients and discriminant validity. Variable
M
SD
q
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
1. Person–organisation fit
5.14
1.48
0.94
0.88
0.40
0.27
0.22
0.14
0.25
0.25
0.13
0.18
0.15
0.24
0.16
0.18
0.24
0.25
0.18
0.22
0.09
0.07
0.15
0.31
2. Needs–supplies fit
4.89
1.42
0.92
0.63
0.84
0.41
0.34
0.22
0.38
0.37
0.19
0.27
0.23
0.36
0.24
0.27
0.36
0.37
0.27
0.34
0.14
0.11
0.23
0.46
3. Demands–abilities fit
5.85
.95
0.81
0.52
0.64
0.72
0.22
0.14
0.26
0.25
0.13
0.18
0.16
0.24
0.16
0.18
0.24
0.26
0.18
0.23
0.09
0.07
0.15
0.31
4. Positive affect
4.40
1.05
0.83
0.47
0.58
0.47
0.65
0.36
0.64
0.46
0.24
0.33
0.29
0.45
0.29
0.33
0.44
0.46
0.33
0.42
0.13
0.12
0.24
0.32
5. Negative affect
2.90
1.16
0.75
-0.38
-0.47
-0.38
-0.60
0.54
0.42
0.30
0.15
0.22
0.18
0.29
0.19
0.22
0.29
0.30
0.22
0.27
0.08
0.08
0.16
0.21
6. Job satisfaction
4.01
1.25
0.90
0.50
0.62
0.51
0.80
-0.65
0.84
0.52
0.27
0.38
0.32
0.50
0.34
0.38
0.50
0.53
0.38
0.48
0.15
0.14
0.28
0.37
7. Autonomy satisfaction
4.29
1.26
0.80
0.50
0.61
0.50
0.68
-0.55
0.72
0.65
0.30
0.44
0.37
0.56
0.37
0.38
0.49
0.52
0.37
0.47
0.14
0.13
0.27
0.36
8. Competence satisfaction
4.83
0.92
0.88
0.36
0.44
0.36
0.49
-0.39
0.52
0.55
0.76
0.22
0.19
0.29
0.19
0.19
0.26
0.27
0.19
0.24
0.08
0.07
0.14
0.18
9. Relatedness satisfaction
4.09
1.15
0.85
0.43
0.52
0.43
0.58
-0.47
0.62
0.66
0.47
0.70
0.27
0.42
0.28
0.28
0.36
0.38
0.28
0.35
0.11
0.10
0.20
0.27
10. Meaning
4.59
1.21
0.91
0.39
0.48
0.40
0.54
-0.43
0.57
0.61
0.44
0.52
0.86
0.36
0.24
0.24
0.31
0.32
0.24
0.29
0.09
0.08
0.17
0.23
11. Engagement
4.75
0.97
0.89
0.49
0.60
0.49
0.67
-0.54
0.71
0.75
0.54
0.65
0.60
0.71
0.36
0.37
0.48
0.50
0.36
0.46
0.14
0.13
0.26
0.35
12. Learning
4.61
0.96
0.81
0.40
0.49
0.40
0.54
-0.44
0.58
0.61
0.44
0.53
0.49
0.60
0.69
0.24
0.31
0.34
0.24
0.33
0.10
0.08
0.18
0.23
13. Social contribution
4.51
1.16
0.84
0.43
0.52
0.43
0.58
-0.47
0.62
0.62
0.44
0.53
0.49
0.61
0.49
0.72
0.56
0.58
0.42
0.53
0.11
0.10
0.20
0.27
14. Testing indirect effects To establish whether PEF indirectly affected ITL, in-role
performance and OCB, the authors used Hayes’s (2018)
guidelines. http://www.sajip.co.za Open Access Page 7 of 11
Original Research Page 7 of 11
Original Research *, p < 0.05; **, p < 0.01. FIGURE 1: The structural model – Standardised solution with standard errors in
parentheses. TABLE 4: Difference testing for competing structural models. Model
∆χ2
∆df
p
Model 6a
277.888
1
<0.0001*
Model 6b
23.568
3
<0.0001*
*, p < 0.01. TABLE 4: Difference testing for competing structural models. Model
∆χ2
∆df
p
Model 6a
277.888
1
<0.0001*
Model 6b
23.568
3
<0.0001*
*, p < 0.01. satisfaction, meaning, engagement and learning) and social
well-being (social contribution, integration, actualisation,
acceptance and coherence). Similar to previous studies (Janse
van Rensburg et al., 2017; Rautenbach, 2015), all the FAWS
dimensions
yielded
acceptable
reliability
coefficients,
varying from 0.75 to 0.92. Discriminant validity was also
supported as the AVE per construct was greater than the
associated squared correlation values. PEF
OCB
R2 = 0.40
IRP
R2 = 0.21
ITL
R2 = 0.62
WF
R2 = 0.67
SWB
PWB
EWB
β = 0.93**
S.E. = 0.02
β = 0.60**
S.E. = 0.10
β = 0.97**
S.E. = 0.01
β = 0.90**
S.E. = 0.02
β = 0.22**
S.E. = 0.01
β = 0.34**
S.E. = 0.13
β = 0.54**
S.E. = 0.11
β = 0.82**
S.E. = 0.03
β = 0.72**
S.E. = 0.04
β = 0.88**
S.E. = 0.03
β = 0.71**
S.E. = 0.04
DAF
NSF
POF
POF, person–organisation fit; NSF, needs–supplies fit; DAF, demands–abilities fit; PEF,
person environment fit WF workplace flourishing EWB emotional well being PWB PEF
β = 0.72**
S.E. = 0.04
β = 0.88**
S.E. = 0.03
β = 0.71**
S.E. = 0.04
DAF
NSF
POF The results indicated that PEF positively associated with
WF. Therefore, when employees perceive high similarity
between their own values and the values of their organisation,
between the compensation they receive in response to the
work they deliver, and similarity between their job demands
and their capabilities, they should experience elevated
emotional well-being, psychological well-being and social
well-being levels at work. The results are consistent with the
TWA (Dawis & Lofquist, 1984), the ASA theory (Schneider,
1987) and other PEF theories, indicating that person–
environment congruence equates to positive outcomes. Page 7 of 11
Original Research The results also support the notion of cognitive appraisal
theories of affect (Roseman, Spindel, & Jose, 1990; Scherer,
1999) that cognitive circumstantial evaluations yield affective
responses and the social identity theory (Tajfel & Turner,
1986) which proposes that workers who experience fit with
their organisation’s values, become part of a ‘psychological
group’. This also concurs with prior findings (Janse van
Rensburg et al., 2017). POF POF POF, person–organisation fit; NSF, needs–supplies fit; DAF, demands–abilities fit; PEF,
person–environment fit; WF, workplace flourishing; EWB, emotional well-being; PWB,
psychological well-being; SWB, social well-being; ITL, intention to leave; IRP, in-role
performance; OCB, organisational citizenship behaviour; SE, standard error; R2, variance
explained; ß, regression coefficients. *, p < 0.05; **, p < 0.01. FIGURE 1: The structural model – Standardised solution with standard errors in
parentheses. FIGURE 1: The structural model – Standardised solution with standard errors in
parentheses. TABLE 5: Indirect effect of person–environment fit on intention to leave, in-role
performance and organisational citizenship behaviour via workplace flourishing. TABLE 5: Indirect effect of person–environment fit on intention to leave, in-role
performance and organisational citizenship behaviour via workplace flourishing. Variable
Estimate
SE
p
95% BC CI
Indirect effect on intention to leave
0.18
0.12
0.12
0.33, 0.23
Indirect effect on in-role performance
0.28
0.12
0.02
0.05, 0.55
Indirect effect on organisational
citizenship behaviour
0.44
0.13
0.00
0.21, 0.68
SE, standard error; BC CI, bias-corrected confidence interval. WF negatively associated with ITL. Numerous theories
suggest that WF elements relate to ITL. Lee and Mitchell’s
(1994) unfolding model of voluntary turnover, as well as
Mobley’s (1977) turnover model, indicate that dissatisfied
employees will start to explore other possibilities through a
range of evaluation processes. Consistent with previous
findings (Diedericks & Rothmann, 2014; Janse van Rensburg
et al., 2017; Rothmann, 2013), employees will be less inclined
to consider vacating their job when they flourish in the
workplace. SE, standard error; BC CI, bias-corrected confidence interval. Table 5 shows the indirect effect of PEF on ITL, in-role
performance and OCB. Person–environment fit had a
statistically significant indirect effect (p ≤ 0.05) on in-role
performance and OCB (p ≤ 0.01) via WF. Person–environment
fit did not have a statistically significant indirect effect
(p > 0.05) on ITL via WF. Therefore, Hypothesis 6 and 7 are
accepted. Hypothesis 5 is rejected. WF positively associated with in-role performance and
OCB. Numerous theories can explain the flourishing–
performance relationship. Page 7 of 11
Original Research The happy and/or productive
worker thesis suggests that happy (predominantly measured
by job satisfaction) employees are productive employees. Quantitative and qualitative reviews of the job satisfaction–
job performance relationship have also shown that job
satisfaction positively associates with job performance (Judge
et al., 2001). Studies have also shown that psychological well-
being predicts job performance (Cropanzano & Wright, 2001). Furthermore, the social exchange theory (Blau, 1964) proposes
that when one party (the organisation) positively impacts
another (the employee), the latter should return the favour to
honour their part of the exchange. Therefore, employees who
perceive that their needs, demands and the things they value
are sufficiently attended to are more likely to experience a
positive work environment, which should propel them
towards better performance (in-role and extra-role). Page 7 of 11
Original Research WF negatively associate
suggest that WF element
(1994) unfolding model
Mobley’s (1977) turnove
l
ill t
t t
PEF
OCB
R2 = 0.40
IRP
R2 = 0.21
ITL
R2 = 0.62
WF
R2 = 0.67
SWB
PWB
EWB
β = 0.93**
S.E. = 0.02
β = 0.60**
S.E. = 0.10
β = 0.97**
S.E. = 0.01
β = 0.90**
S.E. = 0.02
β = 0.22**
S.E. = 0.01
β = 0.34**
S.E. = 0.13
β = 0.54**
S.E. = 0.11
β = 0.82**
S.E. = 0.03
β = 0.72**
S.E. = 0.04
β = 0.88**
S.E. = 0.03
β = 0.71**
S.E. = 0.04
DAF
NSF
POF
POF, person–organisation fit; NSF, needs–supplies fit; DAF, demands–abilities fit; PEF,
person–environment fit; WF, workplace flourishing; EWB, emotional well-being; PWB,
psychological well-being; SWB, social well-being; ITL, intention to leave; IRP, in-role
performance; OCB, organisational citizenship behaviour; SE, standard error; R2, variance
explained; ß, regression coefficients. *, p < 0.05; **, p < 0.01. FIGURE 1: The structural model – Standardised solution with standard errors in
parentheses. TABLE 5: Indirect effect of person–environment fit on intention to leave, in-role
performance and organisational citizenship behaviour via workplace flourishing. Variable
Estimate
SE
p
95% BC CI
Indirect effect on intention to leave
0.18
0.12
0.12
0.33, 0.23
Indirect effect on in-role performance
0.28
0.12
0.02
0.05, 0.55
Indirect effect on organisational
citizenship behaviour
0.44
0.13
0.00
0.21, 0.68i
TABLE 4: Difference testing for competing structural models. Model
∆χ2
∆df
p
Model 6a
277.888
1
<0.0001*
Model 6b
23.568
3
<0.0001*
*, p < 0.01. Page 8 of 11 Original Research Page 8 of 11
PEF
OCB
R2 = 0.40
IRP
R2 = 0.21
ITL
R2 = 0.62
WF
R2 = 0.67
SWB
PWB
EWB
β = 0.93**
S.E. = 0.02
β = 0.60**
S.E. = 0.10
β = 0.97**
S.E. = 0.01
β = 0.90**
S.E. = 0.02
β = 0.22**
S.E. = 0.01
β = 0.34**
S.E. = 0.13
β = 0.54**
S.E. = 0.11
β = 0.82**
S.E. = 0.03
β = 0.72**
S.E. = 0.04
β = 0.88**
S.E. = 0.03
β = 0.71**
S.E. = 0.04
DAF
NSF
POF
POF, person–organisation fit; NSF, needs–supplies fit; DAF, demands–abilities fit; PEF,
person–environment fit; WF, workplace flourishing; EWB, emotional well-being; PWB,
psychological well-being; SWB, social well-being; ITL, intention to leave; IRP, in-role
performance; OCB, organisational citizenship behaviour; SE, standard error; R2, variance
explained; ß, regression coefficients. Page 7 of 11
Original Research Social integration
4.30
1.29
0.91
0.49
0.60
0.49
0.66
-0.54
0.71
0.70
0.51
0.60
0.56
0.69
0.56
0.75
0.82
0.76
0.55
0.69
0.14
0.13
0.51
0.35
15. Social actualisation
3.69
1.34
0.89
0.50
0.61
0.51
0.68
-0.55
0.73
0.72
0.52
0.62
0.57
0.71
0.58
0.76
0.87
0.78
0.58
0.72
0.15
0.13
0.28
0.37
16. Social acceptance
4.18
1.23
0.91
0.42
0.52
0.43
0.58
-0.47
0.62
0.61
0.44
0.53
0.49
0.60
0.49
0.65
0.74
0.76
0.82
0.53
0.11
0.10
0.20
0.27
17. Social coherence
4.06
1.29
0.92
0.47
0.58
0.48
0.65
-0.52
0.69
0.69
0.49
0.59
0.54
0.68
0.55
0.73
0.83
0.85
0.73
0.88
0.14
0.12
0.25
0.34
18. In-role performance
6.21
0.73
0.81
0.30
0.37
0.30
0.36
-0.29
0.39
0.38
0.28
0.33
0.30
0.38
0.31
0.33
0.38
0.39
0.33
0.37
0.70
0.12
0.26
0.04
19. OCB co-workers
5.29
1.14
0.80
0.27
0.33
0.27
0.34
-0.28
0.37
0.36
0.26
0.31
0.29
0.36
0.29
0.31
0.36
0.36
0.31
0.35
0.35
0.68
0.46
0.05
20. OCB organisation
5.42
1.16
0.85
0.39
0.48
0.39
0.49
-0.40
0.53
0.52
0.38
0.45
0.41
0.51
0.42
0.45
0.51
0.53
0.45
0.50
0.51
0.68
0.72
0.10
21. Intention to leave
2.81
1.29
0.91
-0.56
-0.68
-0.56
-0.57
0.46
-0.61
-0.60
-0.43
-0.52
-0.48
-0.59
-0.48
-0.52
-0.59
-0.61
-0.52
-0.58
-0.20
-0.22
-0.32
0.81
AVE, average variance extracted estimates on the diagonal, and squared correlations above the diagonal; OCB, organisational citizenship behaviour; SD, standard deviation; M, mean. Note: All correlations are statistically significant (p < 0.01). Correlations are presented below the diagonal. Bold values indicate that the average variance extracted per construct and create a line of separation between the correlations (below the diagonal) and the
d
l ti
( b
th di
l) Page 8 of 11
Original Research
satisfaction, meaning, en
well-being (social contr
acceptance and coherence
van Rensburg et al., 2017
dimensions
yielded
a
varying from 0.75 to 0.9
supported as the AVE p
associated squared correl
The results indicated th
WF. Therefore, when em
between their own values
between the compensati
work they deliver, and sim
and their capabilities,
emotional well-being, ps
well-being levels at work
TWA (Dawis & Lofquist,
1987) and other PEF t
environment congruenc
The results also support
theories of affect (Rosem
1999) that cognitive circum
responses and the socia
1986) which proposes tha
their organisation’s value
group’. This also concu
Rensburg et al., 2017). Authors’ contribution K.R. coordinated the data collection process. S.R. assisted
with data analysis and interpretation. K.R., S.R. and E.B. K.R. coordinated the data collection process. S.R. assisted
with data analysis and interpretation. K.R., S.R. and E.B. made contributions regarding the conceptualisation, as well
as writing and editing, of the article. made contributions regarding the conceptualisation, as well
as writing and editing, of the article. Acknowledgements
Competing interests With regard to the indirect effect of PEF on in-role performance
and OCB via WF, respectively, results confirmed this effect. Hence, PEF’s association with in-role performance and
OCB is an indirect one, suggesting that PEF increases
participant performance (in-role and extra-role), as long as
participant flourishing levels remain sufficiently high. Therefore, PEF should first elevate employee flourishing
levels to subsequently increase in-role performance and
OCB. Although the preceding associations have not been
studied before, they seem consistent with Fishbein and
Ajzen’s (1975) framework which contends that beliefs
precede attitudes, intentions and ultimately behaviours. The authors declare that they have no financial or personal
relationships that may have inappropriately influenced them
in writing this article. The views expressed in this article
are our own and do not reflect the official position of the
institution or funder. References Several study limitations are prominent. Firstly, the cross-
sectional research approach impedes the assessment of
causality among the variables under scrutiny. Secondly, the
study did not assess interpersonal PEF aspects such as
person–group and person–supervisor fit. Thirdly, because
of certain restrictions (e.g. occupation and geographical
location), the generalisation of findings to other settings
should proceed with caution. Lastly, a specific modelling
strategy was used to assess the constructs, another strategy
(e.g. bifactor modelling) may possibly yield different results. Adnot, M., Dee, T., Katz, V., & Wyckoff, J. (2017). Teacher turnover, teacher quality, and
student achievement in DCPS. Educational Evaluation and Policy Analysis, 39(1),
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charismatic leadership, work engagement, and organizational citizenship
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10, 99–109. https://doi.org/10.1207/s15327043hup1002_3 Discussion The study aim was to examine relationships among PEF, WF,
ITL, in-role performance and OCB. When employees fit, feel
well and function well both psychologically and socially,
positive outcomes ensue (i.e. lower ITL, higher in-role
performance and OCB). Results confirmed WF’s three-factor structure, endorsing its
construct validity beyond the fast-moving consumer goods
industry (Rautenbach, 2015) and tertiary education sector
(Janse van Rensburg et al., 2017). Flourishing at work
consisted of emotional well-being (job satisfaction, positive
affect and low negative affect), psychological well-being
(autonomy satisfaction, competence satisfaction, relatedness Open Access http://www.sajip.co.za Original Research Page 9 of 11 Page 9 of 11 Results showed a direct association between PEF and ITL,
proposing that PEF reduces participant intent to leave,
regardless of their flourishing levels. The ASA model
(Schneider, 1987) suggests that employees who experience
work environment incongruence should be more inclined
to leave. Similarly, WF significantly associated with ITL,
regardless of employee PEF levels. Thus, PEF and WF, both
in their own right and independently, significantly associated
with individuals’ thoughts of leaving their organisation. As individuals experience fit with their school and job
aspects, and experience links within the organisation (e.g. social integration), they have much more to sacrifice when
leaving the school, resulting in lower ITL. This is inconsistent
with previous findings (Janse van Rensburg et al., 2017). A possible explanation for this inconsistency could be
ascribed to increased model complexity. is maintained between individual (values, abilities and
needs) and environmental (values, demands, and supplies)
characteristics. This will lay the foundation for a favourable
work environment, an environment where employees can
experience a sense of acceptance, enjoyment, integration,
meaningfulness and relatedness (among others). When such
an environment is institutionalised, talent retention and
performance should follow. Future studies should aim to explore additional outcomes
and antecedents related to WF. Research should also aim to
assess the causality between the constructs, as none of the
constructs is static in nature. A bifactor modelling strategy
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Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles
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PLOS pathogens
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cc-by
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Abstract Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of
hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic a-helices in the
apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB)
and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight
reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the
formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious
HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast
to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3
in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued
the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the
formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic a-helices
in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral
particles. These results suggest that amphipathic a-helices in the exchangeable apolipoproteins play crucial roles in the
infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of
novel anti-HCV therapeutics targeting for viral assembly. Citation: Fukuhara T, Wada M, Nakamura S, Ono C, Shiokawa M, et al. (2014) Amphipathic a-Helices in Apolipoproteins Are Crucial to the Formation of Infectious
Hepatitis C Virus Particles. PLoS Pathog 10(12): e1004534. doi:10.1371/journal.ppat.1004534 Editor: Timothy L. Tellinghuisen, The Scripps Research Institute, United States of America Editor: Timothy L. Tellinghuisen, The Scripps Research Institute, United States of America Received August 3, 2014; Accepted October 21, 2014; Published December 11, 2014 Copyright: 2014 Fukuhara 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: The authors confirm that all data underlying the findings are fully available without restriction. Abstract Generally, ApoA, ApoB, ApoC and ApoE bind
the surface of lipoprotein through the interaction between
amphipathic a-helices and ER-derived membrane [13,14]. This Takasuke Fukuhara1., Masami Wada1., Shota Nakamura2, Chikako Ono1, Mai Shiokawa1,
Satomi Yamamoto1, Takashi Motomura1, Toru Okamoto1, Daisuke Okuzaki3, Masahiro Yamamoto4,
Izumu Saito5, Takaji Wakita6, Kazuhiko Koike7, Yoshiharu Matsuura1* 1 Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan, 2 Department of Infection Metagenomics, Research Institute
for Microbial Diseases, Osaka University, Osaka, Japan, 3 DNA-Chip Developmental Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka
University, Osaka, Japan, 4 Department of Immunoparasitology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan, 5 Laboratory of Molecular
Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan, 6 Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan, 7 Department
of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Abstract All relevant data are within the paper and its
Supporting Information files except for the cDNA array data of GSE32886 which is available from GEO (Gene Expression Omnibus) under the accession number
GSE32886. Funding: This work was supported in part by grants-in-aid from the Japanese Ministry of Health, Labor, and Welfare (Research on Hepatitis), the Japanese
Ministry of Education, Culture, Sports, Science, and Technology, the Naito Foundation, and the Takeda Science Foundation. 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. s: The authors have declared that no competing interests exist. * Email: matsuura@biken.osaka-u.ac.jp * Email: matsuura@biken.osaka-u.ac.jp . These authors contributed equally to this work. RNA replication have been identified, respectively [5,6]. In
addition, development of a robust in vitro propagation system of
HCV based on the genotype 2a JFH1 strain (HCVcc) has
gradually clarified the mechanism of assembly of HCV particles
[7,8]. It has been shown that the interaction of NS2 protein with
structural and non-structural proteins facilitates assembly of the
viral capsid and formation of infectious particles at the connection
site between the ER membrane and the surface of lipid droplets
(LD) [9]. On the other hand, very low density lipoprotein (VLDL)
associated proteins, including apolipoprotein B (ApoB), ApoE, and
microsomal triglyceride transfer protein (MTTP), have been
shown to play crucial roles in the formation of infectious HCV
particles [10–12]. Generally, ApoA, ApoB, ApoC and ApoE bind
the surface of lipoprotein through the interaction between
amphipathic a-helices and ER-derived membrane [13,14]. This RNA replication have been identified, respectively [5,6]. In
addition, development of a robust in vitro propagation system of
HCV based on the genotype 2a JFH1 strain (HCVcc) has
gradually clarified the mechanism of assembly of HCV particles
[7,8]. It has been shown that the interaction of NS2 protein with
structural and non-structural proteins facilitates assembly of the
viral capsid and formation of infectious particles at the connection
site between the ER membrane and the surface of lipid droplets
(LD) [9]. On the other hand, very low density lipoprotein (VLDL)
associated proteins, including apolipoprotein B (ApoB), ApoE, and
microsomal triglyceride transfer protein (MTTP), have been
shown to play crucial roles in the formation of infectious HCV
particles [10–12]. PLOS Pathogens | www.plospathogens.org Amphipathic a-Helices in Apolipoproteins Are Crucial to
the Formation of Infectious Hepatitis C Virus Particles Takasuke Fukuhara1., Masami Wada1., Shota Nakamura2, Chikako Ono1, Mai Shiokawa1,
Satomi Yamamoto1, Takashi Motomura1, Toru Okamoto1, Daisuke Okuzaki3, Masahiro Yamamoto4,
Izumu Saito5, Takaji Wakita6, Kazuhiko Koike7, Yoshiharu Matsuura1* ApoB and ApoE have a redundant role in HCV particle
formation To obtain more convincing data on the involvement of
apolipoproteins in the production of infectious HCV particles,
we established knockout (KO) Huh7 cells deficient in either ApoB
(B-KO1 and B-KO2) or ApoE (E-KO1 and E-KO2) by using
ZFN (Figure S1). Deficiencies of ApoB or ApoE expression in
these cell lines were confirmed by ELISA and immunoblotting
analyses (Figure S1). First, we examined the roles of ApoB and
ApoE on the entry and RNA replication of HCV by using HCV
pseudotype particles (HCVpp) and subgenomic replicon (SGR) of
the JFH1 strain, respectively. The B-KO and E-KO cell lines
exhibited no significant effect on the infectivity of HCVpp and the
colony formation of SGR (Figure S2A and Figure S2B), suggesting
that ApoB and ApoE are not involved in the entry and replication
processes of HCV. To examine the role of ApoB and ApoE in the
propagation of HCV, HCVcc was inoculated into parental, B-KO
and E-KO cell lines at an MOI of 1, and intracellular viral RNA
and infectious titers in the supernatants were determined (Figure
S2C and Figure S2D). Although RNA replication and infectious
particle formation in B-KO cells upon infection with HCV were
comparable with those in parental Huh7 cells, E-KO cells
exhibited slight reduction of particle formation, and the expression
of ApoE in E-KO cells rescued infectious particle formation
(Figure S2C, Figure S2D, Figure S2E). Next, to examine the
redundant role of ApoB, the effect of knockdown of ApoB on
HCV assembly was determined in parental and E-KO Huh7 cell
lines (Fig. 2A). Knockdown of ApoB in E-KO cells resulted in a
more efficient reduction of infectious particle production than that
in parental Huh7 cells, suggesting that ApoB and ApoE have a
redundant role in the formation of infectious HCV particles. Recently, several novel genome editing techniques have been
developed, including methods using zinc finger nucleases (ZFN),
transcription
activator
like-effector
nucleases
(TALEN)
and
CRISPR/Cas9 systems [17–19]. DNA double strand breaks
(DSBs) induced by these artificial nucleases can be repaired by
error-prone non-homologous end joining (NHEJ), resulting in
mutant mice or cell lines carrying deletions, insertions, or
substitutions at the cut site. To clarify the detailed function of
gene family with redundant functions, the generation of animals or
cell lines carrying multiple mutated genes may be essential. Results To further confirm the redundant role of ApoB and ApoE in the
HCV life cycle, especially in the particle formation, 2 clones of
ApoB and ApoE double-knockout (BE-KO1 and BE-KO2) Huh7
cells were established by ZFNs (Figure S3A and Figure S3B). The
lack of ApoB and ApoE expressions was confirmed by immuno-
blotting and ELISA analyses (Figure S3C, Figure S3D, Figure Author Summary However, the evidence of the involvement of apolipoproteins
in HCV particle formation is dependent on knockdown data and
exogenous expression of the apolipoproteins, and thus the precise
mechanisms of participation of the apolipoproteins in HCV
assembly have not been elucidated [10,11,16]. ApoB and ApoE have a redundant role in HCV particle
formation In this study, Huh7 cell lines deficient in both ApoB and ApoE
genes were established by using ZFNs and revealed that ApoB and
ApoE redundantly participate in the formation of infectious HCV
particles. Interestingly, the expression of other exchangeable apolipo-
proteins, i.e., ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3, facilitated
HCV assembly in ApoB and ApoE double-knockout cells. In addition,
the expression of amphipathic a-helices in the exchangeable
apolipoproteins restored the production of infectious particles in the
double-knockout cells through an interaction with viral particles. Author Summary In vitro systems have been developed for the study of
hepatitis C virus (HCV) infection and have revealed many
details of the life cycle of HCV. Apolipoprotein B (ApoB)
and ApoE have been shown to play crucial roles in the
particle formation of HCV, based on data obtained by
siRNA-mediated gene knockdown and overexpression of
the proteins. However, precise roles of the apolipoproteins
in HCV assembly have not been elucidated yet. In this
study, we show that infectious particle formation of HCV in
Huh7 cells was severely impaired by the knockout of both
ApoB and ApoE genes by artificial nucleases, and this
reduction was cancelled by the expression of not only
ApoE,
but
also
other
exchangeable
apolipoproteins,
including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3. In
addition, expression of amphipathic a-helices in the
exchangeable
apolipoproteins
restored
the
infectious
particle formation in the double-knockout cells through
an interaction with viral particles. These results provide
clues to the understanding of life cycle of HCV and the
development of novel antivirals to HCV. binding of apolipoproteins enhances the stability and hydrophi-
licity of lipoprotein. However, the specific roles played by the
apolipoproteins in HCV particle formation are controversial. Gastaminza et al. demonstrated that ApoB and MTTP are cellular
factors essential for an efficient assembly of infectious HCV
particles [10]. However, studies by other groups demonstrated that
ApoE is a major determinant of the infectivity and particle
formation of HCV, and the ApoE fraction is highly enriched with
infectious particles [11]. In addition, Mancone et al. showed that
ApoA1 is required for production of infectious particles of HCV
[15]. However, the evidence of the involvement of apolipoproteins
in HCV particle formation is dependent on knockdown data and
exogenous expression of the apolipoproteins, and thus the precise
mechanisms of participation of the apolipoproteins in HCV
assembly have not been elucidated [10,11,16]. binding of apolipoproteins enhances the stability and hydrophi-
licity of lipoprotein. However, the specific roles played by the
apolipoproteins in HCV particle formation are controversial. Gastaminza et al. demonstrated that ApoB and MTTP are cellular
factors essential for an efficient assembly of infectious HCV
particles [10]. However, studies by other groups demonstrated that
ApoE is a major determinant of the infectivity and particle
formation of HCV, and the ApoE fraction is highly enriched with
infectious particles [11]. In addition, Mancone et al. showed that
ApoA1 is required for production of infectious particles of HCV
[15]. Exchangeable Apolipoproteins in HCV Assembly This inhibitory effect is well consistent with the high
level of expression of these apolipoproteins in the hepatic cancer
cell lines, suggesting that the apolipoproteins involved in HCV
assembly are dependent on the expression pattern in hepatic
cancer cell lines, including Huh7 cells [20]. Therefore, we
examined the effects of exogenous expression of the apolipopro-
teins highly expressed in the liver tissues on the infection of HCV
in the stable ApoE-knockdown Huh7 cells (Fig. 1D). In contrast to
the control-knockdown cells, expression of not only ApoE but also
ApoA1, ApoA2, and ApoC1 rescued the infectious particle
formation in the ApoE-knockdown cells (Fig. 1E), suggesting that
various exchangeable apolipoproteins participate in the efficient
production of infectious HCV particles. Exchangeable Apolipoproteins in HCV Assembly HepG2 cells (Fig. 1A and B). The web-based search engine
NextBio (NextBio, Santa Clara, CA) revealed that ApoB, ApoH
and the exchangeable apolipoproteins ApoA1, ApoA2, ApoC1,
ApoC2, ApoC3, and ApoE are highly expressed in human liver
tissues (Fig. 1A). On the other hand, the expressions of ApoA1,
ApoC1, ApoC2, ApoC3 and ApoH in hepatic cancer cell lines
were suppressed compared to those in hepatocytes (Fig. 1B). To
examine the roles of apolipoproteins in the formation of infectious
HCV particles, the effects of knockdown of ApoA2, ApoB and
ApoE on the infectious particle production in the supernatants
were determined in Huh7 cells by focus forming assay (Fig. 1C). The transfection of siRNAs targeting to ApoA2, ApoB and ApoE
significantly
suppressed
the
production
of
infectious
HCV
particles. This inhibitory effect is well consistent with the high
level of expression of these apolipoproteins in the hepatic cancer
cell lines, suggesting that the apolipoproteins involved in HCV
assembly are dependent on the expression pattern in hepatic
cancer cell lines, including Huh7 cells [20]. Therefore, we
examined the effects of exogenous expression of the apolipopro-
teins highly expressed in the liver tissues on the infection of HCV
in the stable ApoE-knockdown Huh7 cells (Fig. 1D). In contrast to
the control-knockdown cells, expression of not only ApoE but also
ApoA1, ApoA2, and ApoC1 rescued the infectious particle
formation in the ApoE-knockdown cells (Fig. 1E), suggesting that
various exchangeable apolipoproteins participate in the efficient
production of infectious HCV particles. HepG2 cells (Fig. 1A and B). The web-based search engine
NextBio (NextBio, Santa Clara, CA) revealed that ApoB, ApoH
and the exchangeable apolipoproteins ApoA1, ApoA2, ApoC1,
ApoC2, ApoC3, and ApoE are highly expressed in human liver
tissues (Fig. 1A). On the other hand, the expressions of ApoA1,
ApoC1, ApoC2, ApoC3 and ApoH in hepatic cancer cell lines
were suppressed compared to those in hepatocytes (Fig. 1B). To
examine the roles of apolipoproteins in the formation of infectious
HCV particles, the effects of knockdown of ApoA2, ApoB and
ApoE on the infectious particle production in the supernatants
were determined in Huh7 cells by focus forming assay (Fig. 1C). The transfection of siRNAs targeting to ApoA2, ApoB and ApoE
significantly
suppressed
the
production
of
infectious
HCV
particles. Introduction More than 160 million individuals worldwide are infected with
hepatitis
C virus
(HCV), and
cirrhosis
and hepatocellular
carcinoma induced by HCV infection are life-threatening diseases
[1]. Current standard therapy combining peg-interferon (IFN),
ribavirin (RBV) and a protease inhibitor has achieved a sustained
virological response (SVR) in over 80% of individuals infected with
HCV genotype 1 [2]. In addition, many antiviral agents targeting
non-structural proteins and host factors involved in HCV
replication have been applied in clinical trials [3,4]. In vitro systems have been developed for the study of HCV
infection and have revealed many details of the life cycle of HCV. By using pseudotype particles bearing HCV envelope proteins and
RNA replicon systems, many host factors required for entry and December 2014 | Volume 10 | Issue 12 | e1004534 1 Exchangeable Apolipoproteins in HCV Assembly PLOS Pathogens | www.plospathogens.org Several apolipoproteins participate in the production of
infectious viral particles (C) Huh7 cells infected with HCVcc at an MOI of 1 at 6 h post-
transfection with siRNAs targeting ApoA2 (A2), ApoB (B), ApoE (E) and control (Cont), and expression levels of apolipoproteins (upper panel) and
infectious titers in the culture supernatants (lower panel) were determined by immunoblotting and a focus-forming assay at 72 h post-infection,
respectively. (D) ApoA1, ApoA2, ApoC1, ApoE and ApoH were exogenously expressed in control and ApoE-knockdown Huh7 cells by lentiviral
vectors. Expressions of the apolipoproteins were determined by immunoblotting analysis. (E) Infectious titers in the culture supernatants of control
and ApoE-knockdown Huh7 cells expressing the apolipoproteins were determined by focus-forming assay at 72 h post-infection. In all cases, asterisks
indicate significant differences (*, P,0.05; **, P,0.01) versus the results for control cells. doi:10.1371/journal.ppat.1004534.g001 further clarify the roles of ApoB in the life cycle of HCV, we
established knockout Huh7 cell lines deficient in MTTP (M-KO1
and M-KO2) and in both ApoE and MTTP (EM-KO1 and EM-
KO2) by using the ZFN and CRISPR/Cas9 system (Figure S4A
and Figure S4E). The lack of MTTP, ApoB and ApoE expressions
was confirmed by immunoblotting and ELISA analyses (Figure
Figure S4B, Figure S4C, Figure S4D, Figure S4F, Figure S4G,
Figure S4H). As previously reported, the secretion of ApoB was
completely abrogated in M-KO and EM-KO cells, while the
mRNA levels of ApoB were comparable among Huh7, M-KO
and EM-KO cells (Figure S4I). To examine the roles of MTTP in
the assembly of HCV through the secretion of ApoB, HCVcc was
inoculated into the Huh7, B-KO, M-KO, E-KO, BE-KO and
EM-KO cell lines at an MOI of 1, and intracellular HCV
genomes and infectious titers in the supernatants were determined
(Fig. 3A–C). Although intracellular RNA replication in M-KO
and EM-KO cells was comparable with that in Huh7, B-KO, E-
KO and BE-KO cells (Fig. 3B), infectious titers in the superna-
tants of EM-KO cells were severely impaired as seen in BE-KO
cells, while those of M-KO cells were comparable to those of
parental Huh7cells (Fig. 3C), suggesting that MTTP participates S3E). The BE-KO cell lines also exhibited no significant effect on
the infectivity of HCVpp (Fig. 2B) and the colony formation of
SGR (Fig. 2C). Next, we examined the redundant role of ApoB
and ApoE on the propagation of HCVcc. Several apolipoproteins participate in the production of
infectious viral particles Upon infection with
HCVcc at an MOI of 1, infectious titers in the supernatants of BE-
KO1 and BE-KO2 cells were 50 to 100 times lower than those of
parental Huh7 cells at 72 h post-infection, while the level of
intracellular RNA replication was comparable (Fig. 2D and E). In
addition, exogenous expression of ApoE in BE-KO (ApoE-res)
cells rescued the production of infectious viral particles to levels
comparable to those in parental Huh7 cells (Fig. 2F and G),
suggesting that ApoB and ApoE redundantly participate in the
particle formation of HCV. Several apolipoproteins participate in the production of
infectious viral particles First, we compared expression levels of apolipoproteins between
hepatocyte and hepatic cancer cell lines including Huh7 and PLOS Pathogens | www.plospathogens.org December 2014 | Volume 10 | Issue 12 | e1004534 2 Exchangeable Apolipoproteins in HCV Assembly Figure 1. Several apolipoproteins participate in HCV propagation. (A) Relative mRNA expression of the apolipoproteins in the liver tissues
(left columns) was determined using the NextBio Body Atlas application. The median expression (right columns) was calculated across all 128 human
tissues from 1,068 arrays using the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. mRNA expression for each gene was log10
transformed. (B) Log10 transformed, normalized signal intensity of the apolipoproteins in Huh7 (left columns) and HepG2 (right columns) cells were
extracted from previously published expression microarray dataset GSE32886. (C) Huh7 cells infected with HCVcc at an MOI of 1 at 6 h post-
transfection with siRNAs targeting ApoA2 (A2), ApoB (B), ApoE (E) and control (Cont), and expression levels of apolipoproteins (upper panel) and
infectious titers in the culture supernatants (lower panel) were determined by immunoblotting and a focus-forming assay at 72 h post-infection,
respectively. (D) ApoA1, ApoA2, ApoC1, ApoE and ApoH were exogenously expressed in control and ApoE-knockdown Huh7 cells by lentiviral
vectors. Expressions of the apolipoproteins were determined by immunoblotting analysis. (E) Infectious titers in the culture supernatants of control
and ApoE-knockdown Huh7 cells expressing the apolipoproteins were determined by focus-forming assay at 72 h post-infection. In all cases, asterisks
indicate significant differences (*, P,0.05; **, P,0.01) versus the results for control cells. doi:10.1371/journal.ppat.1004534.g001 Figure 1. Several apolipoproteins participate in HCV propagation. (A) Relative mRNA expression of the ap Figure 1. Several apolipoproteins participate in HCV propagation. (A) Relative mRNA expression of the apolipoproteins in the liver tissues
(left columns) was determined using the NextBio Body Atlas application. The median expression (right columns) was calculated across all 128 human
tissues from 1,068 arrays using the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. mRNA expression for each gene was log10
transformed. (B) Log10 transformed, normalized signal intensity of the apolipoproteins in Huh7 (left columns) and HepG2 (right columns) cells were
extracted from previously published expression microarray dataset GSE32886. MTTP participates in HCV particle formation through the
maturation of ApoB (C) A subgenomic HCV RNA replicon of the JFH1 strain was electroporated into BE-KO1 and BE-KO2 cells
with/without expression of ApoE by lentiviral vector (ApoE-res), and the colonies were stained with crystal violet at 31 days post-electroporation after
selection with 400 mg/ml of G418. Huh7, BE-KO1 and BE-KO2 cells were infected with HCVcc at an MOI of 1, and intracellular HCV RNA (D) and
infectious titers in the supernatants (E) were determined at 72 h post-infection by qRT-PCR and focus-forming assay, respectively. (F) Exogenous
expression of ApoE in BE-KO1 and BE-KO2 cells by lentiviral vector was determined by immunoblotting analysis. (G) Infectious titers in the culture
supernatants of BE-KO1 (gray bars) and ApoE-res cells (red bars) infected with HCVcc at an MOI of 1 were determined at 72 h post-infection by focus-
forming assay. doi:10.1371/journal.ppat.1004534.g002 apolipoproteins on the infectious particle formation, the effects of
exogenous expression of these apolipoproteins on the propagation
of HCVcc in BE-KO1 cells were determined. ApoA1, ApoA2,
ApoC1, ApoC2, ApoC3, ApoE and ApoH were expressed by
lentiviral vector in BE-KO1 cells (Fig. 4B upper panel). The
expressions of ApoA1, ApoA2, ApoC1, ApoC2, ApoC3 and ApoE
but not of ApoH enhanced extracellular HCV RNA, while they
exhibited no effect on intracellular HCV RNA (Fig. 4C). In
addition, the expressions of these exchangeable apolipoproteins
enhanced the infectious particle formation in the supernatants of
BE-KO1 cells (Fig. 4B lower panel). On the other hand, the
expression of nonhepatic apolipoproteins, including ApoD, ApoL1,
and ApoO, exhibited no effect on HCV particle formation in BE-
KO1 cells (Figure S6). These results suggest that exogenous
expression of not only the ApoE but also the ApoA and ApoC
families can compensate for the impairment of HCV particle
formation in BE-KO1 cells. Interestingly, specific infectivity
(infectious titers/viral RNA levels in supernatants) was also
enhanced by the expression of ApoA1, ApoA2, ApoC1, ApoC2,
ApoC3 and ApoE, suggesting that these apolipoproteins participate
in the infectious but not non-infectious particle formation of HCV
(Fig. 4D). Previous reports have suggested that the expressions of
Claudin1 (CLDN1), miR-122 and ApoE facilitate the production of
infectious particles in nonhepatic 293T cells [16]. Therefore, the
effects of exogenous expression of exchangeable apolipoproteins on
particle formation were examined in 293T cells expressing CLDN1
and miR-122 (293T-CLDN/miR-122 cells). MTTP participates in HCV particle formation through the
maturation of ApoB Exogenous expression
of ApoA1, ApoA2, ApoC1, ApoC2, ApoC3 and ApoE, but not of
ApoH by lentiviral vector facilitated the production of infectious in the HCV assembly through the regulation of ApoB secretion. To further confirm the roles of MTTP in HCV assembly through
ApoB secretion, the effects of exogenous expression of MTTP in
EM-KO cells on the infectious particle formation of HCV were
determined. Immunoblotting and ELISA analyses revealed that
exogenous expression of MTTP rescued the secretion of ApoB
into the supernatants of EM-KO cells (Fig. 3D and E), while
expression of ApoE or MTTP in both BE-KO and EM-KO cells
exhibited no effect on the intracellular RNA replication (Fig. 3F). Although exogenous expression of ApoE rescued the infectious
particle formation of HCV in both BE-KO and EM-KO cells,
expression of MTTP rescued the particle formation in EM-KO
cells but not in BE-KO cells (Fig. 3G), supporting the notion that
MTTP plays a crucial role in the HCV assembly through the
maturation of ApoB. MTTP participates in HCV particle formation through the
maturation of ApoB It is difficult to determine the roles of ApoB in the particle
formation of HCV, because ApoB is too large (550 kDa) to obtain
cDNA for expression. However, previous reports have shown that
expression of MTTP facilitates the secretion of ApoB [21]. To December 2014 | Volume 10 | Issue 12 | e1004534 PLOS Pathogens | www.plospathogens.org PLOS Pathogens | www.plospathogens.org 3 Exchangeable Apolipoproteins in HCV Assembly Figure 2. ApoB and ApoE redundantly participate in the formation of infectious HCV particles. (A) Huh7 and E-KO1 cells were infected
with HCVcc at an MOI of 1 at 6 h post-transfection with siRNAs targeting ApoB or ApoE, and infectious titers in the culture supernatants were
determined by focus-forming assay at 72 h post-infection. (B) HCVpp were inoculated into Huh7, BE-KO1 and BE-KO2 cells, and luciferase activities
were determined at 48 h post-infection. (C) A subgenomic HCV RNA replicon of the JFH1 strain was electroporated into BE-KO1 and BE-KO2 cells
with/without expression of ApoE by lentiviral vector (ApoE-res), and the colonies were stained with crystal violet at 31 days post-electroporation after
selection with 400 mg/ml of G418. Huh7, BE-KO1 and BE-KO2 cells were infected with HCVcc at an MOI of 1, and intracellular HCV RNA (D) and
infectious titers in the supernatants (E) were determined at 72 h post-infection by qRT-PCR and focus-forming assay, respectively. (F) Exogenous
expression of ApoE in BE-KO1 and BE-KO2 cells by lentiviral vector was determined by immunoblotting analysis. (G) Infectious titers in the culture
supernatants of BE-KO1 (gray bars) and ApoE-res cells (red bars) infected with HCVcc at an MOI of 1 were determined at 72 h post-infection by focus-
forming assay. doi:10.1371/journal.ppat.1004534.g002 . ApoB and ApoE redundantly participate in the formation of infectious HCV particles. (A) Huh7 and E-KO Figure 2. ApoB and ApoE redundantly participate in the formation of infectious HCV particles. (A) Huh7 and E-KO1 cells were infected
with HCVcc at an MOI of 1 at 6 h post-transfection with siRNAs targeting ApoB or ApoE, and infectious titers in the culture supernatants were
determined by focus-forming assay at 72 h post-infection. (B) HCVpp were inoculated into Huh7, BE-KO1 and BE-KO2 cells, and luciferase activities
were determined at 48 h post-infection. PLOS Pathogens | www.plospathogens.org Exchangeable apolipoproteins redundantly participate in
the assembly of infectious HCV particles Next, to examine the roles played in HCV particles formation by
other apolipoproteins highly expressed in the liver (Fig. 1A), the
expressions of ApoA1, ApoA2, ApoC1, ApoC2, ApoC3 and ApoH
in BE-KO1 cells were suppressed by siRNAs (Fig. 4A and Figure
S5). While knockdown of ApoA1, ApoC3 and ApoH exhibited no
effect, that of ApoA2, ApoC1 and ApoC2 significantly inhibited the
release of infectious particles, which was consistent with the
expression pattern of endogenous apolipoproteins except for ApoH
in Huh7 cells (Fig. 1B), suggesting that not only ApoB and ApoE
but also other exchangeable apolipoproteins participate in HCV
particle formation. To confirm the redundant role of these December 2014 | Volume 10 | Issue 12 | e1004534 PLOS Pathogens | www.plospathogens.org 4 Exchangeable Apolipoproteins in HCV Assembly Figure 3. MTTP participates in the formation of infectious HCV particles through the maturation of ApoB. (A) Expressions of ApoB,
ApoE and MTTP in Huh7, B-KO1, M-KO1, E-KO1, BE-KO1 and EM-KO1 cells were determined by immunoblotting analysis. Cells were infected with
HCVcc at an MOI of 1, and intracellular HCV RNA (B) and infectious titers in the supernatants (C) were determined at 72 h post-infection by qRT-PCR
and focus-forming assay, respectively. The expressions of ApoB, ApoE and MTTP in BE-KO1 and EM-KO1 cells with/without expression of ApoE or
MTTP by lentiviral vector were determined by immunoblotting (D) and ELISA (E). Cells were infected with HCVcc at an MOI of 1, and intracellular HCV
RNA (F) and infectious titers in the supernatants (G) were determined at 72 h post-infection by qRT-PCR and focus-forming assay, respectively. doi:10.1371/journal.ppat.1004534.g003 Figure 3. MTTP participates in the formation of infectious HCV particles through the maturation of ApoB. (A) Expressions of ApoB,
ApoE and MTTP in Huh7, B-KO1, M-KO1, E-KO1, BE-KO1 and EM-KO1 cells were determined by immunoblotting analysis. Cells were infected with
HCVcc at an MOI of 1, and intracellular HCV RNA (B) and infectious titers in the supernatants (C) were determined at 72 h post-infection by qRT-PCR
and focus-forming assay, respectively. The expressions of ApoB, ApoE and MTTP in BE-KO1 and EM-KO1 cells with/without expression of ApoE or
MTTP by lentiviral vector were determined by immunoblotting (D) and ELISA (E). Cells were infected with HCVcc at an MOI of 1, and intracellular HCV
RNA (F) and infectious titers in the supernatants (G) were determined at 72 h post-infection by qRT-PCR and focus-forming assay, respectively. Exchangeable apolipoproteins redundantly participate in
the assembly of infectious HCV particles doi:10.1371/journal.ppat.1004534.g003 HCV particles in 293T-CLDN/miR-122 cells (Fig. 4E). On the
other hand, the expression of ApoE exhibited no effect on the
propagation of Japanese encephalitis virus (JEV) and dengue virus
(DENV) (Figure S7) in BE-KO1 cells. These results suggest that the
exchangeable apolipoproteins and ApoB redundantly and specifi-
cally participate in the formation of HCV particles. freeze
and
thaw
method. Not
only
intracellular
but
also
extracellular infection titers were impaired in BE-KO1 cells
compared with those in parental and ApoE-res cells (Fig. 6A),
suggesting that intracellular particle formation is impaired by
deficiencies in the expression of ApoB and ApoE. Previous reports
have shown that the recruitment of viral proteins around LD and
redistribution of LD are essential for HCV assembly [24]. To
clarify the roles of the exchangeable apolipoproteins on HCV
assembly in more detail, we examined the intracellular localization
of viral proteins, LD and ER in BE-KO1 and ApoE-res cells. The
localization of core proteins around LD and the membranous-web
structure forming the replication complex were observed in BE-
KO1 cells upon infection with HCVcc, as reported in parental
Huh7 cells (Fig. 6B, 6C and Figure S8). However, greater
accumulation of core proteins and LD around the perinuclear
region was detected in BE-KO1 cells in comparison with ApoE-res
cells (Fig. 6C and 6D), supporting the notion that apolipoproteins
participate in the infectious particle formation in HCV rather than
viral RNA replication. Previous studies revealed that core proteins
were mainly localized on the ER membrane upon infection with
the genotype 2a Jc1 strain-based HCVcc (HCVcc/Jc1), and
inhibition of capsid assembly and envelopment caused accumu-
lation of core proteins on the surface of LD [25–27]. In ApoE-res
cells, core proteins of HCVcc/Jc1 were mainly localized on the To examine the role of exchangeable apolipoproteins in the
formation of other genotypes of HCV, the effect of exogenous
expression of these apolipoproteins on the propagation of
genotype 1b and 3a chimeric HCVcc, TH/JFH1 and S310/
JFH1 viruses in BE-KO1 cells was determined (Fig. 5) [22,23]. As
seen in infection with HCVcc (JFH1), expression of ApoA1,
ApoA2, ApoC1, ApoC2, ApoC3 and ApoE enhanced the
formation of infectious particles of TH/JFH1 and S310/JFH1
chimeric viruses. These results suggest that ApoA1, ApoA2,
ApoC1, ApoC2, ApoC3 and ApoE redundantly participate in the
efficient formation of infectious HCV particles of genotypes 1b, 2a
and 3a. Apolipoproteins participate in the post-envelopment
step of particle formation To determine the details of the assembly of infectious HCV
particles in the BE-KO1 cells, intracellular infectious titers were
determined in Huh7, BE-KO1 and ApoE-res cells by using the December 2014 | Volume 10 | Issue 12 | e1004534 PLOS Pathogens | www.plospathogens.org 5 Exchangeable Apolipoproteins in HCV Assembly xchangeable apolipoproteins redundantly participate in the formation of infectious HCV particles. (A) BE-KO1 cells infected
at an MOI of 1 at 6 h post-transfection with siRNAs targeting ApoA1 (A1), ApoA2 (A2), ApoC1 (C1), ApoC2 (C2), ApoC3 (C3) and ApoH (H)
us titers in the culture supernatants were determined by focus-forming assay at 72 h post-infection. (B) ApoA1, ApoA2, ApoC1, ApoC2,
E and ApoH were exogenously expressed in BE-KO1 cells by infection with lentiviral vectors, and then infected with HCVcc at an MOI of 1. f the apolipoproteins was determined by immunoblot analysis (upper), and infectious titers in the culture supernatants were determined
-infection by focus-forming assay (lower). (C) Extracellular and intracellular HCV RNA in BE-KO1 cells expressing apolipoproteins and
h HCVcc were determined at 72 h post-infection by qRT-PCR. (D) Specific infectivity was calculated as extracellular infectious titers/
HCV RNA copies in BE-KO1 cells expressing apolipoproteins at 72 h post-infection. (E) 293T cells stably expressing CLDN1 and miR-122
miR-122 cells) were infected with the lentiviral vectors, and the expressions of the apolipoproteins were determined by immunoblot
er). These cells were infected with HCVcc at an MOI of 1, and infectious titers in the supernatants were determined at 72 h post-infection
ming assay (lower). In all cases, asterisks indicate significant differences (*, P,0.05; **, P,0.01) versus the results for control cells. ournal.ppat.1004534.g004 Figure 4. Exchangeable apolipoproteins redundantly participate in the formation of infectious HCV particles. (A) BE-KO1 cells infected
with HCVcc at an MOI of 1 at 6 h post-transfection with siRNAs targeting ApoA1 (A1), ApoA2 (A2), ApoC1 (C1), ApoC2 (C2), ApoC3 (C3) and ApoH (H)
and infectious titers in the culture supernatants were determined by focus-forming assay at 72 h post-infection. (B) ApoA1, ApoA2, ApoC1, ApoC2,
ApoC3, ApoE and ApoH were exogenously expressed in BE-KO1 cells by infection with lentiviral vectors, and then infected with HCVcc at an MOI of 1. Expression of the apolipoproteins was determined by immunoblot analysis (upper), and infectious titers in the culture supernatants were determined
at 72 h post-infection by focus-forming assay (lower). PLOS Pathogens | www.plospathogens.org Apolipoproteins participate in the post-envelopment
step of particle formation (C) Extracellular and intracellular HCV RNA in BE-KO1 cells expressing apolipoproteins and
infected with HCVcc were determined at 72 h post-infection by qRT-PCR. (D) Specific infectivity was calculated as extracellular infectious titers/
extracellular HCV RNA copies in BE-KO1 cells expressing apolipoproteins at 72 h post-infection. (E) 293T cells stably expressing CLDN1 and miR-122
(293T-CLDN/miR-122 cells) were infected with the lentiviral vectors, and the expressions of the apolipoproteins were determined by immunoblot
analysis (upper). These cells were infected with HCVcc at an MOI of 1, and infectious titers in the supernatants were determined at 72 h post-infection
by focus-forming assay (lower). In all cases, asterisks indicate significant differences (*, P,0.05; **, P,0.01) versus the results for control cells. doi:10.1371/journal.ppat.1004534.g004 were analyzed by buoyant density ultracentrifugation (Fig. 7A–B)
[28]. Secretion of viral capsids in the supernatants was severely
impaired in BE-KO1 cells in comparison with that in ApoE-res
cells (Fig. 7A upper), in contrast to the detection of abundant
intracellular capsids in both cell lines (Fig. 7B upper). Although
peak levels of the core proteins and infectious titers were detected
around 1.08 g/ml in both cell lines, the infectious titers in all
fractions of BE-KO1 cells were significantly lower than those in
ApoE-res
cells,
supporting
the
notion
that
apolipoproteins
participate in the post-assembly process of HCV capsids which ER membrane, in contrast to the co-localization of core proteins
of HCVcc (JFH1) with LD (Fig. 6E upper). However, core
proteins were accumulated around LD in BE-KO1 cells infected
with HCVcc/Jc1, as seen in those infected with HCVcc (JFH1)
(Fig. 6E
lower). These
results
suggest
that
apolipoproteins
participate in the steps of HCV particle formation occurring after
HCV protein assembly on the LD. To further examine the involvement of apolipoproteins in the
infectious particle formation of HCV, culture supernatants and
cell lysates of BE-KO1 and ApoE-res cells infected with HCVcc December 2014 | Volume 10 | Issue 12 | e1004534 6 Exchangeable Apolipoproteins in HCV Assembly Figure 5. Exchangeable apolipoproteins participate in the formation of infectious HCV particles of genotype 1 and 3. ApoA1, ApoA2,
ApoC1, ApoC2, ApoC3, ApoE and ApoH were exogenously expressed in BE-KO1 cells by infection with lentiviral vectors, and then infected with
genotype 1b and 3a chimeric HCVcc, TH/JFH1 (A) and S310/JFH1 (B) at an MOI of 0.5. Apolipoproteins participate in the post-envelopment
step of particle formation Intracellular HCV RNA and infectious titers in the culture
supernatants were determined at 72 h post-infection by qRT-PCR (upper) and focus-forming assay (lower). Asterisks indicate significant differences
(**, P,0.01) versus the results for control cells. doi:10.1371/journal.ppat.1004534.g005 Figure 5. Exchangeable apolipoproteins participate in the formation of infectious HCV particles of genotype 1 and 3. ApoA1, ApoA2,
ApoC1, ApoC2, ApoC3, ApoE and ApoH were exogenously expressed in BE-KO1 cells by infection with lentiviral vectors, and then infected with
genotype 1b and 3a chimeric HCVcc, TH/JFH1 (A) and S310/JFH1 (B) at an MOI of 0.5. Intracellular HCV RNA and infectious titers in the culture
supernatants were determined at 72 h post-infection by qRT-PCR (upper) and focus-forming assay (lower). Asterisks indicate significant differences
(**, P,0.01) versus the results for control cells. doi:10.1371/journal.ppat.1004534.g005 is required to confer infectivity. Next, to examine the involvement
of apolipoproteins in the envelopment of HCV particles, lysates of
BE-KO1 and ApoE-res cells infected with HCVcc were treated
with proteinase K in the presence or absence of Triton X [26]. Protection of HCV core proteins from the protease digestion was
observed in both cell lysates (Fig. 7C), suggesting that apolipopro-
teins are not involved in the envelopment of HCV particles. Collectively, these results suggest that exchangeable apolipopro-
teins participate in the post-envelopment step of HCV particle
formation. in the production of infectious HCV particles. In addition, more
abundant full-length and truncated ApoE were detected in the
precipitates of the culture supernatants of cells infected with
HCVcc than those of mock-infected cells concentrated by
ultracentrifugation, suggesting that the amphipathic a-helices of
apolipoproteins
are
directly
associated
with
HCV particles
(Fig. 8D and E). Taken together, the data in this study strongly
suggest that exchangeable apolipoproteins redundantly participate
in the infectious particle formation of HCV through the
interaction between amphipathic a-helices and viral particles. PLOS Pathogens | www.plospathogens.org December 2014 | Volume 10 | Issue 12 | e1004534 Exchangeable Apolipoproteins in HCV Assembly These data suggest that the roles of the exchangeable
apolipoproteins in HCV assembly are independent from the
production of VLDL. MTTP plays crucial roles in the lipoprotein
formation through the incorporation of triglyceride into growing
lipoprotein and secretion of ApoB [21]. Although it has been
shown that treatment with an MTTP inhibitor impairs the
production of HCV particles [11], in this study, we found that
knockout of MTTP abrogated the secretion of ApoB but not the
production of infectious HCV particles. Collectively, these data
suggest that exchangeable apolipoproteins redundantly participate
in the infectious particle formation of HCV independently from
lipoprotein secretion machinery. addition, exogenous expression of ApoE facilitated the infectious
particle formation of HCV in 293T cells stably expressing CLDN1
and miR-122 [16], suggesting that ApoE-mediated particle
formation is independent from VLDL production. Furthermore,
exogenous expression of ApoA1, a major apolipoprotein of HDL,
also facilitated the production of HCV particles as shown in
Fig. 4E. These data suggest that the roles of the exchangeable
apolipoproteins in HCV assembly are independent from the
production of VLDL. MTTP plays crucial roles in the lipoprotein
formation through the incorporation of triglyceride into growing
lipoprotein and secretion of ApoB [21]. Although it has been
shown that treatment with an MTTP inhibitor impairs the
production of HCV particles [11], in this study, we found that
knockout of MTTP abrogated the secretion of ApoB but not the
production of infectious HCV particles. Collectively, these data
suggest that exchangeable apolipoproteins redundantly participate
in the infectious particle formation of HCV independently from
lipoprotein secretion machinery. p p
p
g
Although the gene-knockout technique is essential to obtain
reproducible and reliable data, and many knockout mice have
been produced in various research areas, the development of
experimental tools for HCV study has also been hampered by the
narrow cell tropism [47,48]. A humanized mouse model in which
human liver cells were xenotransplanted into immunodeficient
mouse was developed and provided an important platform for the
analysis of pathogenesis and the development of antivirals for
HCV [49]. However, the exogenous expression of human receptor
molecules required for HCV entry and impairment of innate
immunity are required for the complete propagation of HCV in
mice [50]. Exchangeable Apolipoproteins in HCV Assembly Gene-knockout techniques using a CRISPR/Cas9
system composed of guide RNA and Cas9 nuclease that form
RNA-protein complexes to cleave the target sequences [19] have
allowed quick and easy establishment of gene-knockout mice and
cancer cell lines [51,52], and indeed, such MTTP-knockout cell
lines were established also in this study. Recently, the high-
throughput screening of host factors involved in several conditions
was reported by using a CRISPR/Cas9 system [53]. Together,
these novel genome-editing techniques are expected to reveal the
precise roles of host factors involved in the HCV life cycle. p p
y
Production of HCV capsids in the culture supernatants is
impaired in 293T cells expressing miR-122 due to lack of ApoE
expression, but envelopment of viral capsids is observed [37],
suggesting that ApoE is involved in the post-envelopment step. Coller et al. suggested that ApoE is associated with de novo
formation of HCV particles during secretory pathway based on an
experiment using HCV possessing a tetracysteine-tag in the core
protein [38]. In this study, ApoA1, ApoA2, ApoC1, ApoC2,
ApoC3 and ApoE enhanced the formation of HCV particles in the
post-envelopment step. These results suggest that a direct
interaction between exchangeable apolipoproteins and enveloped
particles in the ER lumen facilitates an efficient secretion of
infectious
HCV
particles. Ultrastructural
analysis
of
HCV
particles has shown that large amounts of apolipoproteins,
including ApoA1, ApoB and ApoE, bind to the surface of viral
particles [39]. Interestingly, ApoE-specific antibodies were more
efficient in capturing viral particles than a-E1/E2 antibodies, and
significantly large numbers of gold particles reacting with ApoE
were observed per virion than those with E2, suggesting that viral
envelope proteins are masked by a large amount of apolipopro-
teins. The unique characteristics of interaction between apolipo-
proteins and HCV particles might be applied for visualization of
entry and purification of HCV particles by using GFP- or affinity-
tagged amphipathic a-helices of apolipoproteins. In the previous
report, virocidal amphipathic helical peptides impaired the
infectivity of viral particles [40]. There is a possibility that such
peptide influences on the interaction between apolipoproteins and
viral particles, and might be a new therapeutic approach. In summary, we have shown that apolipoproteins, including
ApoA1, ApoA2, ApoC1, ApoC2, ApoC3, ApoE and ApoB,
possess redundant roles in the assembly of HCV through the
interaction of the amphipathic a-helices in the apolipoproteins
with viral particles in the post-envelopment step. Exchangeable Apolipoproteins in HCV Assembly It is hoped that
these findings will provide clues to the life cycle of HCV and assist
in the development of novel antivirals targeting the assembly
process of HCV. Amphipathic a-helices in exchangeable apolipoproteins
participate in the formation of infectious HCV particles
through the interaction with viral particles In this study, we demonstrated the redundant roles of ApoB and
the
exchangeable
apolipoproteins
ApoA1,
ApoA2,
ApoC1,
ApoC2, ApoC3 and ApoE in the assembly of infectious HCV
particles. The deficiencies of both ApoB and ApoE inhibited the
production of infectious HCV particles in Huh7 cells, and
exogenous expression of exchangeable apolipoproteins rescued
the particle formation. cDNA microarray revealed that the
expression patterns of exchangeable apolipoproteins in hepatic
cancer cell lines are widely different from those in liver tissue. In
previous reports, ApoE and ApoB were identified as important
host factors for the assembly of infectious HCV particles [10,11],
and knockdown of ApoE and ApoB expression also inhibited the
production of infectious particles in this study. Because ApoB and
ApoE are major apolipoproteins in VLDL, several reports have
suggested that the VLDL production machinery participates in the
production of HCV particles. Furthermore, density gradient
analyses revealed co-fractionation of HCV RNA with lipoproteins,
with the resulting complexes being termed lipoviroparticles (LVP)
[12,35]. However, it has been reported that there is no correlation
between secretion of VLDL and production of LVP [36]. In To determine the structural relevance of apolipoproteins
involved in the HCV assembly, the secondary structures of the
apolipoproteins were deduced by using a CLC Genomics
Workbench and previous reports (Fig. 8A) [29–34]. Tandem
repeats of amphipathic a-helices were observed in the apolipo-
proteins capable of rescuing HCV assembly in BE-KO1 cells, but
not in those lacking this activity, suggesting that amphipathic a-
helices in the apolipoproteins participate in the assembly of HCV. To examine the involvement of the amphipathic a-helices of the
exchangeable apolipoproteins in the particle formation of HCV,
we constructed expression plasmids encoding deletion mutants of
ApoE and ApoC1, and then these deletion mutants were
exogenously expressed in BE-KO1 cells by lentiviral vectors
(Fig. 8B and C upper panels). The expression of all of the deletion
mutants of ApoE and ApoC1 containing either N-terminal or C-
terminal amphipathic a-helices rescued the particle formation of
HCV in BE-KO1 cells (Fig. 8B and C lower panels), suggesting
that amphipathic a-helices in the apolipoproteins play crucial roles December 2014 | Volume 10 | Issue 12 | e1004534 PLOS Pathogens | www.plospathogens.org 7 Exchangeable Apolipoproteins in HCV Assembly plospathogens.org
8
December 2014 | Volume December 2014 | Volume 10 | Issue 12 | e1004534 PLOS Pathogens | www.plospathogens.org PLOS Pathogens | www.plospathogens.org 8 Exchangeable Apolipoproteins in HCV Assembly Figure 6. Accumulation of core proteins around lipid droplets in BE-KO1 cells. (A) Extracellular and intracellular infectious titers in Huh7,
BE-KO1 and ApoE-restored cells infected by lentiviral vector (ApoE-res) were determined at 72 h post-infection with HCVcc at an MOI of 1 by focus-
forming assay. Asterisks indicate significant differences (**, P,0.01) versus the results for parental cells. (B) BE-KO1 cells infected with HCVcc at an
MOI of 1 were stained with anti-Core antibody at 72 h post-infection and examined by fluorescence microscopy. Identical fields were observed under
electron microscopy by using the correlative FM-EM technique. The boxed areas are magnified and displayed. Huh7, BE-KO1 and ApoE-res cells
infected with HCVcc at an MOI of 1 were subjected to immunofluorescence analyses by using anti-Core antibody (C), and immunoblotting by using
antibodies against Core, NS3, ApoE, and actin at 72 h post-infection (D). Lipid droplets and cell nuclei were stained by BODIPY and DAPI, respectively. (E) BE-KO1 and ApoE-res cells infected with Jc1 strain-based HCVcc (HCVcc/Jc1; left panel) or JFH1 strain-based HCVcc (HCVcc/JFH1; right panel) at an
MOI of 1 were subjected to immunofluorescence analysis by using anti-Core antibody at 72 h post-infection. Lipid droplets and cell nuclei were
stained by BODIPY and DAPI, respectively. doi:10.1371/journal.ppat.1004534.g006 interacts with ApoE and hypervariable region 1 (HVR1) in the
envelope protein of HCV [43]. In this study we have shown that
exchangeable apolipoproteins including not only ApoE but also
ApoA and ApoC facilitate the production of infectious HCV
particles, and that exchangeable apolipoproteins directly associate
with viral particles. Meunier et al. reported that ApoC1 associates
intracellularly with viral particles during particle morphogenesis
and enhances the entry of HCV through an interaction of the C-
terminal region of ApoC1 with heparan sulfate [45]. Another
group also showed that ApoC1 enhances HCV infection through
the triple interplay among HVR1, ApoC1, and SR-B1 [46]. These
results suggest that the interaction of HCV particles with
apolipoproteins also participates in the entry through the binding
of lipoprotein receptors including SR-B1 and LDLR. addition, exogenous expression of ApoE facilitated the infectious
particle formation of HCV in 293T cells stably expressing CLDN1
and miR-122 [16], suggesting that ApoE-mediated particle
formation is independent from VLDL production. Furthermore,
exogenous expression of ApoA1, a major apolipoprotein of HDL,
also facilitated the production of HCV particles as shown in
Fig. 4E. PLOS Pathogens | www.plospathogens.org Plasmids Lysates were separated into 3 parts and incubated for 1 h on ice in the
presence or absence of 50 mg/ml proteinase K with/without pretreat-
ment with 5% Triton-X and then subjected to immunoblotting. doi:10.1371/journal.ppat.1004534.g007 Figure 7. Apolipoproteins participate in the post-envelopment Cell lines All cell lines were cultured at 37uC under the conditions of a
humidified atmosphere and 5% CO2. The human hepatocellular
carcinoma-derived Huh7 and human embryonic kidney-derived
293T cells were obtained from Japanese Collection of Research
Bioresources (JCRB) Cell Bank (JCRB0403 and JCRB9068), and
maintained in DMEM (Sigma) supplemented with 100 U/ml
penicillin, 100 mg/ml streptomycin, and 10% fetal calf serum (FCS). The Huh7-derived cell line Huh7.5.1 was kindly provided by F. Chisari. Huh7 cells harboring JFH1-based HCV-SGR were prepared
according to the method of a previous report [54] and maintained in
DMEM containing 10% FCS and 1 mg/ml G418 (Nakalai Tesque). Exchangeable Apolipoproteins in HCV Assembly Figure 7. Apolipoproteins participate in the post-envelopme
t
f th
HCV lif
l
Th
t
t (A)
d l
t
(B)
f measurements are generated from all available RNA expression
studies that used Affymetrix U133 Plus or U133A Genechip
Arrays for human studies. The results from 128 human tissue
samples were incorporated from 1,067 arrays; 157 human cell
types from 1,474 arrays; and 359 human cancer cell lines from 376
arrays. Gene queries return a list of relevant tissues or cell types
rank-ordered by absolute gene expression and grouped by body
systems or across all body systems. In the current analysis, we
determined the expression levels of the apolipoproteins ApoA1,
ApoA2, ApoB, ApoC1, ApoC2, ApoC3, ApoD, ApoE, ApoH,
ApoL1, ApoL2 and ApoO in liver tissue. We used an analysis
protocol developed by NextBio, the details of which have been
described previously [20]. Figure 7. Apolipoproteins participate in the post-envelopment
step of the HCV life cycle. The supernatants (A) and lysates (B) of BE-
KO1 and ApoE-restored (ApoE-res) cells infected with HCVcc at an MOI
of 1 were subjected to density gradient fractionation. Each fraction was
subjected to immunoblotting using anti-Core antibody (upper). The
infectious titers and densities of each fraction were determined (lower). (C) The lysates of BE-KO1 and ApoE-res cells infected with HCVcc at an
MOI of 1 were subjected to proteinase K digestion protection assay. Lysates were separated into 3 parts and incubated for 1 h on ice in the
presence or absence of 50 mg/ml proteinase K with/without pretreat-
ment with 5% Triton-X and then subjected to immunoblotting. doi:10.1371/journal.ppat.1004534.g007 cDNA microarray Expression profiling was generated using the 4 x 44 K whole
human genome oligo-microarray ver.2.0 G4845A (Agilent Tech-
nologies) as previously described [54]. Raw data were imported into
Subio platform ver.1.12 (Subio) for database management and
quality control. Raw intensity data were normalized against GAP-
DH expression levels for further analysis. These raw data have been
accepted by GEO (a public repository for microarray data, aimed at
storing MIAME). Access to data concerning this study may be
found under GEO experiment accession number GSE32886. Figure 7. Apolipoproteins participate in the post-envelopme NextBio Body Atlas In previous reports, the importance of the interaction between
lipoprotein receptors and ApoE in the entry of HCV has been well
established. Lipoprotein receptors including scavenger receptor
class B type 1 (SR-B1) and low-density lipoprotein receptor
(LDLR) are involved in HCV entry into the target cells [41,42]. LDLR is thought to mediate cell attachment of HCV through an
interaction with virus associated ApoE [43,44]. SR-B1 also The NextBio Body Atlas application presents an aggregated
analysis of gene expression across various normal tissues, normal
cell types, and cancer cell lines [20]. It enables us to investigate the
expression of individual genes as well as gene sets. Samples for
Body Atlas data are obtained from publicly available studies that
are internally curated, annotated, and processed. Body Atlas December 2014 | Volume 10 | Issue 12 | e1004534 PLOS Pathogens | www.plospathogens.org 9 Exchangeable Apolipoproteins in HCV Assembly Plasmids The cDNA clones of pri-miR-122, ApoA1, ApoA2, ApoC1,
ApoC2, ApoC3, ApoE, ApoH, and AcGFP were inserted between
the XhoI and XbaI sites of lentiviral vector pCSII-EF-RfA, which
was kindly provided by M. Hijikata, and the resulting plasmids
were designated pCSII-EF-miR-122, pCSII-EF-MT-apolipopro-
teins, and pCSII-EF-AcGFP, respectively. The deletion mutants of
ApoC1 and ApoE were amplified by PCR and introduced into
pCSII-EF. pHH-JFH1-E2p7NS2mt contains three adaptive mu-
tations in pHH-JFH1 [55]. The pFL-J6/JFH1 plasmid that
encodes the entire viral genome of the chimeric strain of HCV-
2a, J6/JFH1, was kindly provided by Charles M. Rice [8]. pTH/
JFH1 (genotype 1b) and pS310/JFH1 (genotype 3a) were used for
the production of chimeric viruses [22,23]. The plasmid pX330,
which encodes hCas9 and sgRNA, was obtained from Addgene
(Addgene plasmid 42230). The fragments of guided RNA
targeting the MTTP gene were inserted into the Bbs1 site of
pX330 and designated pX330-MTTP. The plasmids used in this
study were confirmed by sequencing with an ABI 3130 genetic
analyzer (Life Technologies). Figure 7. Apolipoproteins participate in the post-envelopment
step of the HCV life cycle. The supernatants (A) and lysates (B) of BE-
KO1 and ApoE-restored (ApoE-res) cells infected with HCVcc at an MOI
of 1 were subjected to density gradient fractionation. Each fraction was
subjected to immunoblotting using anti-Core antibody (upper). The
infectious titers and densities of each fraction were determined (lower). (C) The lysates of BE-KO1 and ApoE-res cells infected with HCVcc at an
MOI of 1 were subjected to proteinase K digestion protection assay. Lysates were separated into 3 parts and incubated for 1 h on ice in the
presence or absence of 50 mg/ml proteinase K with/without pretreat-
ment with 5% Triton-X and then subjected to immunoblotting. doi:10.1371/journal.ppat.1004534.g007 Figure 7. Apolipoproteins participate in the post-envelopment
step of the HCV life cycle. The supernatants (A) and lysates (B) of BE-
KO1 and ApoE-restored (ApoE-res) cells infected with HCVcc at an MOI
of 1 were subjected to density gradient fractionation. Each fraction was
subjected to immunoblotting using anti-Core antibody (upper). The
infectious titers and densities of each fraction were determined (lower). (C) The lysates of BE-KO1 and ApoE-res cells infected with HCVcc at an
MOI of 1 were subjected to proteinase K digestion protection assay. Antibodies Mouse monoclonal antibodies to HCV core, b-actin and
Calnexin were purchased from Thermo Scientific and Sigma
Aldrich, respectively. Mouse anti-ApoA1, ApoB, ApoC1, ApoE Mouse monoclonal antibodies to HCV core, b-actin and
Calnexin were purchased from Thermo Scientific and Sigma
Aldrich, respectively. Mouse anti-ApoA1, ApoB, ApoC1, ApoE December 2014 | Volume 10 | Issue 12 | e1004534 PLOS Pathogens | www.plospathogens.org December 2014 | Volume 10 | Issue 12 | e1004534 10 Exchangeable Apolipoproteins in HCV Assembly Amphipathic a-helices in apolipoproteins participate in the infectious particle formation of HCV. (A) Predicted o
ally determined secondary structures of apolipoproteins. Secondary structures of the helices and sheets in the apolipoproteins are colore
n, respectively. The three-dimensional structures of ApoA1 (Protein Data Bank (PDB) ID, 3R2P), ApoC1 (PDB ID, 1IOJ), ApoD (PDB ID, 2HZR
ID, 2L7B), ApoH (PDB ID, 1C1Z) and ApoM (PDB ID, 2XKL) are also shown in a ribbon model using the same color code of secondar
n cases in which the structure was not available, the secondary structure was predicted by using a CLC Genomics Workbench. (B,C
of the ApoE- and ApoC1-deletion mutants (upper). Deletion mutants with HA tags expressed in BE-KO1 cells by lentiviral vectors wer
immunoblotting (middle). BE-KO1 cells expressing the WT or deletion mutants of ApoE or ApoC1 were infected with HCVcc at an MOI o
tious titers in the culture supernatants were determined by focus-forming assay at 72 h post-infection. Asterisks indicate significan
(**, P,0.01) versus the results for control cells. (D) Schematic of the concentration of viral particles from HCV-infected cells usin
gation. (E) BE-KO1 cells expressing the WT or deletion mutants of ApoE were infected with HCVcc at an MOI of 1. Culture supernatant
t 72 h post-infection were concentrated by ultracentrifugation at 32,000 rpm for 2 h at 4uC, and subjected to immunoblotting. /journal.ppat.1004534.g008 gure 8. Amphipathic a-helices in apolipoproteins participate in the infectious particle formation of HCV. (A) Predicted o
perimentally determined secondary structures of apolipoproteins. Secondary structures of the helices and sheets in the apolipoproteins are colore
d and cyan, respectively. The three-dimensional structures of ApoA1 (Protein Data Bank (PDB) ID, 3R2P), ApoC1 (PDB ID, 1IOJ), ApoD (PDB ID, 2HZR
E (PDB ID 2L7B) A
H (PDB ID 1C1Z)
d A
M (PDB ID 2XKL)
l
h
i
ibb
d l
i
h
l
d
f
d Figure 8. Amphipathic a-helices in apolipoproteins participate in the infectious particle formation of HCV. Lipofection and lentiviral gene transduction The lentiviral vectors and ViraPower Lentiviral Packaging Mix
(Life Technologies) were co-transfected into 293T cells by Trans
IT LT-1 (Mirus), and the supernatants were recovered at 48 h
post-transfection. The lentivirus titer was determined by the Lenti-
XTM qRT-PCR Titration Kit (Clontech), and the expression
levels and AcGFP were determined at 48 h post-inoculation. Immunofluorescence assay Cells cultured on glass slides were fixed with 4% paraformal-
dehyde (PFA) in phosphate buffered saline (PBS) at room
temperature for 30 min, permeabilized for 20 min at room
temperature with PBS containing 0.2% Triton after being washed
three times with PBS, and blocked with PBS containing 2% FCS
for 1 h at room temperature. The cells were incubated with PBS
containing the appropriate primary antibodies at room temper-
ature for 1 h, washed three times with PBS, and incubated with
PBS containing AF488- or AF594-conjugated secondary antibod-
ies at room temperature for 1 h. For lipid-droplet staining, cells
incubated in medium containing 20 mg/ml BODIPY for 20 min
at 37uC were washed with pre-warmed fresh medium and
incubated for 20 min at 37uC. Cell nuclei were stained with
DAPI. Cells were observed with a FluoView FV1000 laser
scanning confocal microscope (Olympus). Preparation of viruses Upon transfection of pHH-JFH1-E2p7NS2mt or in vitro
transcribed TH/JFH1, J6/JFH1 and S310/JFH1 RNA into
Huh7.5.1 cells, HCV in the supernatant was collected after serial
passages, and infectious titers were determined by a focus-forming
assay and expressed in focus-forming units (FFU) [22,23,54]. To
compare the localization of core protein, J6/JFH1 was used in
Fig. 6E. Pseudoparticles expressing HCV envelope glycoprotein
were generated in 293T cells as previously reported [5], and
infectivity was assessed by luciferase expression using the Bright-
Glo Luciferase assay system (Promega) and expressed in relative
light units (RLU). Exchangeable Apolipoproteins in HCV Assembly (Life Technologies). Fluorescent signals were analyzed with the
ViiA7 system. geting siRNA were purchased from Dharmacon, and transfected
into cells using Lipofectamine RNAi MAX (Life Technologies)
according to the manufacturer’s protocol. A human shRNA
library was purchased from Takara Bio Inc. (Life Technologies). Fluorescent signals were analyzed with the
ViiA7 system. Immunoblotting Cells lysed on ice in lysis buffer (20 mM Tris-HCl [pH 7.4],
135 mM NaCl, 1% Triton-X 100, 10% glycerol) supplemented
with a protease inhibitor mix (Nacalai Tesque) were boiled in
loading buffer and subjected to 5–20% gradient SDS-PAGE. The
proteins were transferred to polyvinylidene difluoride membranes
(Millipore) and reacted with the appropriate antibodies. The
immune complexes were visualized with SuperSignal West Femto
Substrate (Pierce) and detected by the LAS-3000 image analyzer
system (Fujifilm). In vitro transcription, RNA transfection, and colony
formation The plasmid pSGR-JFH1 was linearized with XbaI, and treated
with mung bean exonuclease. The linearized DNA was tran-
scribed in vitro by using the MEGAscript T7 kit (Life Technol-
ogies) according to the manufacturer’s protocol. The in vitro
transcribed RNA (10 mg) was electroporated into Huh7 cells at 107
cells/0.4 ml under conditions of 190 V and 975 mF using a Gene
Pulser (Bio-Rad) and plated on DMEM containing 10% FCS. The
medium was replaced with fresh DMEM containing 10% FCS
and 1 mg/ml G418 at 24 h post-transfection. The remaining
colonies were cloned by using a cloning ring (Asahi Glass) or fixed
with 4% PFA and stained with crystal violet at 4 weeks post-
electroporation. Enzyme-linked immunosorbent assay (ELISA) Protein concentrations of ApoB or ApoE in the culture
supernatants were determined by using ELISA immunoassay kits
(Alercheck Inc.) according to the manufacturer’s protocol. Electron microscopy and correlative FM-EM analysis Electron microscopy and correlative FM-EM analysis
Correlative fluorescence microscopy-electron microscopy (FM-
EM) allows individual cells to be examined both in an overview
with fluorescence microscopy and in a detailed subcellular-
structure view with electron microscopy. Cells infected with
HCVcc were examined by the correlative FM-EM method as
described previously [57]. Enzyme-linked immunosorbent assay (ELISA)
Protein concentrations of ApoB or ApoE in the culture
supernatants were determined by using ELISA immunoassay kits
(Alercheck Inc.) according to the manufacturer’s protocol. Intracellular infectivity Intracellular viral titers were determined according to a method
previously reported [10]. Briefly, cells were extensively washed
with PBS, scraped, and centrifuged for 5 min at 10006 g. Cell
pellets were resuspended in 500 ml of DMEM containing 10%
FCS and subjected to three cycles of freezing and thawing using
liquid nitrogen and a thermo block set to 37uC. Cell lysates were
centrifuged at 10,0006g for 10 min at 4uC to remove cell debris. Cell-associated infectivity was determined by a focus-forming
assay. Antibodies (A) Predicted or
experimentally determined secondary structures of apolipoproteins. Secondary structures of the helices and sheets in the apolipoproteins are colored
red and cyan, respectively. The three-dimensional structures of ApoA1 (Protein Data Bank (PDB) ID, 3R2P), ApoC1 (PDB ID, 1IOJ), ApoD (PDB ID, 2HZR),
ApoE (PDB ID, 2L7B), ApoH (PDB ID, 1C1Z) and ApoM (PDB ID, 2XKL) are also shown in a ribbon model using the same color code of secondary
structures. In cases in which the structure was not available, the secondary structure was predicted by using a CLC Genomics Workbench. (B,C)
Schematics of the ApoE- and ApoC1-deletion mutants (upper). Deletion mutants with HA tags expressed in BE-KO1 cells by lentiviral vectors were
detected by immunoblotting (middle). BE-KO1 cells expressing the WT or deletion mutants of ApoE or ApoC1 were infected with HCVcc at an MOI of
1, and infectious titers in the culture supernatants were determined by focus-forming assay at 72 h post-infection. Asterisks indicate significant
differences (**, P,0.01) versus the results for control cells. (D) Schematic of the concentration of viral particles from HCV-infected cells using
ultracentrifugation. (E) BE-KO1 cells expressing the WT or deletion mutants of ApoE were infected with HCVcc at an MOI of 1. Culture supernatants
harvested at 72 h post-infection were concentrated by ultracentrifugation at 32,000 rpm for 2 h at 4uC, and subjected to immunoblotting. doi:10.1371/journal.ppat.1004534.g008 antibodies, and AF594-conjugated anti-mouse IgG2a antibodies
were purchased from Life Technologies. antibodies, and AF594-conjugated anti-mouse IgG2a antibodies
were purchased from Life Technologies. and ApoH antibodies were purchased from Cell Signaling,
ALerCHEK Inc., Abnova, NOVUS Biologicals, and Santa Cruz
Biotechnology, respectively. Rat anti-ApoA2 and Sheep anti-
ApoC2 antibodies were purchased from R&D systems. Rabbit
anti-NS5A antibody was prepared as described previously [54]. Alexa Fluor (AF) 488-conjugated anti-rabbit or mouse IgG Gene silencing
A small interfering RNA (siRNA) pool targeting various
apolipoproteins (siGENOME SMARTpool) and control nontar- December 2014 | Volume 10 | Issue 12 | e1004534 PLOS Pathogens | www.plospathogens.org PLOS Pathogens | www.plospathogens.org 11 Exchangeable Apolipoproteins in HCV Assembly Generation of gene-knockout Huh7 cell lines Custom ZFN plasmids were designed to bind and cleave the
ApoB, ApoE and MTTP genes and were obtained from Sigma
Aldrich. Huh7 cells were transfected with in vitro transcribed
ZFNs mRNA or pX330-MTTP by Lipofectamine 2000 (Life
Technologies), and single cell clones were established by the single
cell isolation technique. To screen for gene-knockout Huh7 cell
clones, mutations in target loci were determined by using a
Surveyor assay as previously described [56]. Frameshift of the
genes and deficiencies of protein expression were confirmed by
direct sequencing and immunoblotting analysis, respectively. PLOS Pathogens | www.plospathogens.org Buoyant density fractionation Total RNA was extracted from cells by using an RNeasy
minikit (Qiagen) and the first-strand cDNA synthesis and qRT-
PCR were performed with TaqMan EZ RT-PCR core reagents
and a ViiA7 system (Life Technologies), respectively, according to
the manufacturer’s protocol. The primers for TaqMan PCR
targeted to the noncoding region of HCV RNA were synthesized
as previously reported [54]. Taqman Gene expression assays
were used as the primers and probes targeting to apolipoproteins Culture supernatants of cells infected with HCVcc were
concentrated 50 times by using Spin-X UF concentrators
(Corning), and the intracellular proteins collected after freeze-
and-thaw were applied to the top of a linear gradient formed from
10–40% OptiPrep (Axis-Shield) in PBS and spun at 32,000 rpm
for 16 h at 4uC by using an SW41 Ti rotor (Beckman Coulter). Aliquots of 10 consecutive fractions were collected, and the
infectious titer and density were determined. December 2014 | Volume 10 | Issue 12 | e1004534 PLOS Pathogens | www.plospathogens.org December 2014 | Volume 10 | Issue 12 | e1004534 12 Exchangeable Apolipoproteins in HCV Assembly antibodies. Expression of ApoB (D) and ApoE (E) in the culture
supernatants of 293T, Huh7, BE-KO1 and BE-KO2 cells was
determined by ELISA. (TIF) Proteinase K digestion protection assay Proteinase K digestion protection assay The proteinase K digestion protection assay was performed as
described previously [37]. Briefly, cells were extensively washed
with PBS, scraped, and centrifuged for 5 min at 10006g. The cell
pellets were resuspended in 500 ml of PBS and subjected to three
cycles of freezing and thawing using liquid nitrogen and a thermo
block set to 37uC. The cell lysates were centrifuged at 10,0006 g
for 10 min at 4uC to remove cell debris. The cell lysates were then
incubated with 50 mg/ml proteinase K (Life Technologies) in the
presence or absence of 5% Triton-X for 1 h on ice, and the
digestion was terminated by addition of PMSF (Wako Chemical
Industries). Figure S4
Establishment of MTTP-knockout (M-KO)
and ApoE and MTTP double-knockout (EM-KO) Huh7
cell lines. (A) Gene knockout by the ZFN in the 2 alleles of the
MTTP gene in the knockout cell lines, M-KO1 and M-KO2, is
shown. (B) Expression of MTTP in Huh7, M-KO1 and M-KO2
cells was determined by immunoblotting. Expression of ApoB (C)
and ApoE (D) in the culture supernatants of Huh7, M-KO1, M-
KO2 and 293T cells was determined by ELISA. Acknowledgments We thank M. Tomiyama for her secretarial work and M. Ishibashi and Y. Sugiyama for their technical assistance. We also thank M. Hijikata, T. Wakita, R. Bartenschlager, F. Chisari, and M. Whitt for providing
experimental materials. We thank M. Tomiyama for her secretarial work and M. Ishibashi and Y. Sugiyama for their technical assistance. We also thank M. Hijikata, T. Wakita, R. Bartenschlager, F. Chisari, and M. Whitt for providing
experimental materials. Figure S3
Establishment of ApoB and ApoE double-
knockout (BE-KO) Huh7 cell lines. Gene knockout by the
ZFN in the 2 alleles of the ApoB and ApoE genes in the double-
knockout cell lines, BE-KO1 (A) and BE-KO2 (B), is shown. Deletion and insertion of the sequences are indicated by dotted
lines and blue characters in brackets, respectively. (C) The absence
of the expressions of ApoB and ApoE in BE-KO1 and BE-KO2
was confirmed by immunoblotting using anti-ApoB and -ApoE (TIF) Figure S5
Gene silencing of apolipoproteins. BE-KO1
cells infected with HCVcc at an MOI of 1 at 6 h post-transfection
with siRNAs targeting ApoA1, ApoA2, ApoC1, ApoC2, ApoC3
and ApoH, and the expression levels of these apolipoproteins were
determined by q-RT PCR using specific primers and probes. (TIF) Figure S6
ApoD, ApoL1, and ApoO do not participate in
the formation of infectious HCV particles. Exogenous
expression of ApoD, ApoE, ApoL1 and ApoO in BE-KO1 cells by
lentiviral vector was determined by immunoblotting analysis
(upper), and infectious titers in the culture supernatants of cells
infected with HCVcc at an MOI of 1 were determined at 72 h
post-infection by focus-forming assay (lower). (TIF) Figure S2
Both ApoB and ApoE are involved in the
formation of infectious HCV particles. (A) HCVpp were
inoculated into Huh7, B-KO1, B-KO2, E-KO1 and E-KO2 cells,
and luciferase activities were determined at 48 h post-infection. (B)
A subgenomic HCV RNA replicon of the JFH1 strain was
electroporated into Huh7, B-KO1 and E-KO1 cells, and colonies
were stained with crystal violet at 31 days post-electroporation
after selection with 400 mg/ml of G418. HCVcc were inoculated
into Huh7, B-KO1, B-KO2, E-KO1 and E-KO2 cells at an MOI
of 1 and intracellular HCV RNA at 12, 24, 36 and 60 h post-
infection (C), and infectious titers in the culture supernatants at
72 h post-infection (D) were determined by qRT-PCR and focus-
forming assay, respectively. (E) Exogenous expression of ApoE in
E-KO1 and E-KO2 cells by lentiviral vector was determined by
immunoblotting analysis (upper), and infectious titers in the
culture supernatants of cells infected with HCVcc at an MOI of 1
were determined at 72 h post-infection by focus-forming assay
(lower). Figure S7
BE-KO1 cells permit propagation of JEV and
DENV. Huh7, BE-KO1 and ApoE-restored (ApoE-res) cells were
infected with JEV and DENV at an MOI of 0.1, and infectious
titers in the culture supernatants were determined by focus-
forming assay at 48 h post-infection. (TIF) Figure S8
Localization of core, NS5A proteins and ER in
BE-KO Huh7 cells. BE-KO1 cells infected with HCVcc at an
MOI of 1 were subjected to immunofluorescence analyses by using
antibodies against core, NS5A and Calnexin. (TIF) Statistics The data for statistical analyses are the average of three
independent experiments. Results were expressed as the means 6
standard deviation. The significance of differences in the means
was determined by Student’s t-test. Buoyant density fractionation (E) Gene
knockout in the 2 alleles of the MTTP genes by the CRISPR/
Cas9 system and in one allele of the ApoE gene by the ZFN in the
double-knockout cell lines, EM-KO1 and EM-KO2, is shown. (F)
Expression of MTTP in Huh7, EM-KO1 and EM-KO2 cells was
determined by immunoblotting. Expression of ApoB (G) and
ApoE (H) in the culture supernatants of Huh7, EM-KO1, EM-
KO2 and 293T cells was determined by ELISA. (I) Expression of
ApoB mRNA in Huh7, M-KO1, M-KO2, EM-KO1, EM-KO2
and 293T cells was determined by qRT-PCR. (TIF) Supporting Information Figure S1
Establishment of ApoB- or ApoE-knockout
Huh7 cell lines. Target sequences of ZFNs to ApoB (A) and
ApoE (B) are indicated by red characters inside a red box at the
top of the panel. Gene knockout by the sequence modification in
the 2 alleles of the ApoB (A) or ApoE (B) gene in knockout cell
lines (B-KO1 and B-KO2, or E-KO1 and E-KO2) is shown. Deletion and insertion of the sequences are indicated by dotted
lines and blue characters in brackets, respectively. Absence of the
expressions of ApoB (C) and ApoE (D) in the knockout cell lines
was confirmed by immunoblotting using anti-ApoB and -ApoE
antibodies. Expression of ApoB (E) and ApoE (F) in the culture
supernatants of 293T, Huh7 and the knockout cell lines was
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containing particles. J. Virol. 76: 6919–6928. 40. Cheng G, Montero A, Gastaminza P, Whitten-Bauer C, Wieland SF, et al. (2008) A virocidal amphipathic a-helical peptide that inhibits hepatitis C virus
infection in vitro. Proc. Natl. Acad. Sci. U. S. A. 105: 3088–3093. 13. Saito H, Lund-Katz S, Phillips MC. (2004) Contribution of domain structure
and lipid interaction to the functionality of exchangeable human apolipopro-
teins. Prog. Lipid Res. 43: 350–380. 41. Scarselli E, Ansuini H, Cerino R, Roccasecca RM, Acali S, et al. Author Contributions Conceived and designed the experiments: TF SN MY IS TW KK YM. Performed the experiments: TF MW CO MS SY TM. Analyzed the data:
TF MW SN TO DO YM. Contributed reagents/materials/analysis tools:
KK YM. Wrote the paper: TF SN DO YM. Conceived and designed the experiments: TF SN MY IS TW KK YM. Performed the experiments: TF MW CO MS SY TM. Analyzed the data:
TF MW SN TO DO YM. Contributed reagents/materials/analysis tools:
KK YM. Wrote the paper: TF SN DO YM. December 2014 | Volume 10 | Issue 12 | e1004534 PLOS Pathogens | www.plospathogens.org 13 References Shavinskaya A, Boulant S, Penin F, McLauchlan J, Bartenschlager R. (2007)
The lipid droplet binding domain of hepatitis C virus core protein is a major
determinant for efficient virus assembly. J. Biol. Chem. 282: 37158–37169. p
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55. Russell RS, Meunier JC, Takikawa S, Faulk K, Engle RE, et al. Advantages of a
single-cycle production assay to study cell culture-adaptive mutations of hepatitis
C virus. Proc. Natl. Acad. Sci. U. S. A. 105: 4370–4375 (2008). 28. Ai L, Lee Y, Chen SS (2009) Characterization of hepatitis C virus core protein
multimerization and membrane envelopment: revelation of a cascade of core-
membrane interactions. J. Virol. 83: 9923–9939. 56. Guschin YD, Waite AJ, Katibah GE, Miller JC, Holmes MC, et al. (2010) A
rapid and general assay for monitoring endogenous gene modification. Methods
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dimerization. J. Biol. Chem. 286: 38570–38582. 57. Rieder CL, Bowser SS (1985). Correlative immunofluorescence and electron
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Study of Various General-Purpose Technologies and Their Comparison Towards Developing Sustainable Society
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International journal of management, technology, and social science
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International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018
SRINIVAS
PUBLICATION
Study of Various General-Purpose Technologies
and Their Comparison Towards Developing
Sustainable Society
P. S. Aithal1 & Shubhrajyotsna Aithal 2
1Srinivas Institute of Management Studies, Srinivas University, Mangalore – 575001, INDIA
2 College of Engineering & Technology, Srinivas University, Mangalore – 574146, India
E-mail: psaithal@gmail.com
Type of the Paper: Research Paper.
Type of Review: Peer Reviewed.
Indexed in: OpenAIRE.
DOI: http://doi.org/10.5281/zenodo.1409476.
Google Scholar Citation: IJMTS
International Journal of Management, Technology, and Social Sciences (IJMTS)
A Refereed International Journal of Srinivas University, India.
How to Cite this Paper:
Aithal, P. S. & Aithal, Shubhrajyotsna. (2018). Study of Various General-Purpose
Technologies and Their Comparison Towards Developing Sustainable Society. International
Journal of Management, Technology, and Social Sciences (IJMTS), 3(2), 16-33 (2581-6012).
DOI: http://doi.org/10.5281/zenodo.1409476. International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 SRINIVAS
PUBLICATION Study of Various General-Purpose Technologies
and Their Comparison Towards Developing
Sustainable Society How to Cite this Paper:
Aithal, P. S. & Aithal, Shubhrajyotsna. (2018). Study of Various General-Purpose
Technologies and Their Comparison Towards Developing Sustainable Society. International
Journal of Management, Technology, and Social Sciences (IJMTS), 3(2), 16-33 (2581-6012). DOI: http://doi.org/10.5281/zenodo.1409476. International Journal of Management, Technology, and Social Sciences (IJMTS)
A Refereed International Journal of Srinivas University, India. A Refereed International Journal of Srinivas University, India. © With Authors. © With Authors. ABSTRACT Technology is used in many ways to solve many complicated challenges in the society. Certain
technologies have grown and expanded their branches to many areas and sectors of practice in
such a way that they have been designated as General-Purpose Technologies. General Purpose
Technologies’ (GPT) are characterized by pervasiveness where they have an inherent potential
for technical improvements, and innovation complementarities, meaning that the productivity
through research and development in related sectors increases due to the consequence of
innovative applications through such general-purpose technologies. Thus, as general-purpose
technologies progress, they spread throughout the economy, eventually bringing about
generalized productivity gains. Examples include the steam engine, railroad, interchangeable
parts, electricity, electronics, materialhandling, mechanization,
control
theory (automation),
the automobile, the computer, the Internet, and nanotechnology. In this paper, we have
identified, analysed, and compared Information Communication and Computation Technology
(ICCT), and Nanotechnology (NT) as two most important general-purpose technologies due to
their abilities to solve both basic problems and advanced need of the society. The paper also
contains a conceptual and predictive proposal on how various general-purpose technologies
including ICCT and NT are potentially contributing towards creating a techno-society and based
on further progress and spread of such technologies to every dimension of human life to reach
the ultimate level of civilization in or around this earth. The analysis finally leads to the
development of the concept of ‘Universal Technology’ model. Keywords: General purpose technologies, Information Communication and Computation
Technology (ICCT), Nanotechnology (NT), Technologies for social development, Universal
technology. Keywords: General purpose technologies, Information Communication and Computation
Technology (ICCT), Nanotechnology (NT), Technologies for social development, Universal
h
l Study of Various General-Purpose Technologies and Their
Comparison Towards Developing Sustainable Society P. S. Aithal1 & Shubhrajyotsna Aithal 2
1Srinivas Institute of Management Studies, Srinivas University, Mangalore – 575001, INDIA
2 College of Engineering & Technology, Srinivas University, Mangalore – 574146, India
E-mail: psaithal@gmail.com This work is licensed under a Creative Commons Attribution-Non Commercial 4.0 International
License subject to proper citation to the publication source of the work. Disclaimer: The scholarly papers as reviewed and published by the Srinivas Publications (S.P.),
India are the views and opinions of their respective authors and are not the views or opinions of
the SP. The SP disclaims of any harm or loss caused due to the published content to any party. PAGE 16 PAGE 16 P. S. Aithal et al. (2018); www.srinivaspublication.com International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 SRINIVAS
PUBLICATION 1. INTRODUCTION: they have an inherent potential for technical
improvements,
and
innovation
complementarities,
meaning
that
through
research and development the productivity in
sub-sectors increases as a consequence of such
innovation in the GPT [1].Many of the killer
applications invented in the history are though
costly and less productive initially, emerged
eventually as General Purpose Technologies. Examples include the steam engine, railroad,
interchangeable parts, electricity, semiconductor
electronics, material
handling, mechanization,
control
theory (automation),
the automobile, they have an inherent potential for technical
improvements,
and
innovation
complementarities,
meaning
that
through
research and development the productivity in
sub-sectors increases as a consequence of such
innovation in the GPT [1].Many of the killer
applications invented in the history are though
costly and less productive initially, emerged
eventually as General Purpose Technologies. Examples include the steam engine, railroad,
interchangeable parts, electricity, semiconductor
electronics, material
handling, mechanization,
control
theory (automation),
the automobile, Technology by itself is an application of science
is used in many ways to solve many complicated
challenges in the society to make human life
comfortable and enjoyable. Certain technologies
have grown and expanded their branches to
many areas and sectors of practice in such a way
that they have been designated as General-
Purpose Technologies. Such technologies are
identified and used by many branches of
engineering to solve or simplify the problems of
those fields. General Purpose Technologies’
(GPT) are characterized by pervasiveness where PAGE 17 PAGE 17 P. S. Aithal et al. (2018); www.srinivaspublication.com International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 SRINIVAS
PUBLICATION SRINIVAS
PUBLICATION As listed in table 1, some of the killer
applications transformed into GPT’s and many
of them are making an impact only in one or two
related areas and replaced by other innovative
technologies or die-down eventually. In this
paper, we have identified and analysed two
killer applications and compared Information
Communication and Computation Technology
(ICCT), and Nanotechnology (NT) as two most
important general-purpose technologies due to
their abilities to solve both basic problems and
advanced need of the society. We also studied
the similarities and differences between these
two GPT’s in terms of their specially identified
characteristics of Pervasiveness, Improvement,
and Innovation spawning abilities. The paper
also contains a conceptual and predictive
proposal on how various general-purpose
technologies including ICCT and NT are
potentially contributing towards creating a
techno-society and based on further progress and
spread of such technologies to every dimension
of human life to reach the ultimate level of
civilization in or around this earth. •
The Internet
•
The space industries
Based on his reading of the histories of these
technologies, Ruttan finds that military and
defense-related procurement has been a major
source of technology development. He believes
that the current technological landscape would
look very different in the absence of military and
defense-related contributions to commercial
technology development. However, from his
research, Ruttan determines that commercial
technology development would have occurred in
the absence of military procurement but more
slowly, e.g., the aircraft, computer, and Internet
industries. He cites nuclear power as an example
of a general-purpose technology that would not
have developed in the absence of military and
defense-related procurement [2]. p
Economist Richard Lipsey and Kenneth Carlaw
[3] suggested that there have only been 24
technologies in history that have been identified
as true GPTs. They define a transforming GPT
follows four criteria which are listed below:
(1) GPT is a single, recognisable generic
technology. Economist Richard Lipsey and Kenneth Carlaw
[3] suggested that there have only been 24
technologies in history that have been identified
as true GPTs. They define a transforming GPT
follows four criteria which are listed below:
(1) GPT is a single, recognisable generic
technology. (1) GPT is a single, recognisable generic
technology. 2. GENERAL PURPOSE TECHNOLOGIES (2)
Initially
GPT
has
much
scope
for
improvement but comes to be widely used
across the economy. A general-purpose technology or GPT is a term
coined to describe a new method of producing
and inventing that is important enough to have a
protracted aggregate impact. Electricity and
information technology (IT) probably are the
two most important GPTs till 20th century. A
GPT can be a product, a process, technology or
an organisational system. y
(3) GPT has many different uses in many areas
to solve problems or to provide comfortability. (4) GPT creates many spill over effects to spread
its base to many sectors. (3) GPT has many different uses in many areas
to solve problems or to provide comfortability. (4) GPT creates many spill over effects to spread
its base to many sectors. SRINIVAS
PUBLICATION the computer,
the Internet,
Information
Communication and Computation Technology
(ICCT), and Nanotechnology (NT). The major
stakeholders of Information Communication and
Computation Technology (ICCT) as general
purpose technology are Analytics and Big data,
Cloud
Technology,
Artificial
Intelligence,
Internet of Things (IoT), Digital Marketing, 3D
Printing, Virtual Reality and optical computing. The major stakeholders of Nanotechnology as
general-purpose technology are Agriculture with
improved
and
sustained
yield,
Universal
drinking water system, Universal Renewable
energy system, Optical computation, Embedded
intelligence, Chameleon chips, Flying cars,
Space travel, Nanomedicine, and anticipated
Immortality. Table 1 :Killer Applications and their transformation into GPT
S. No. Killer Technology
Spillover Effect
Era
1
Wheel
Mechanization
4000-3000 BC
2
Bronze
Tools & Weapons
2800 BC
3
Printing
Knowledge Economy
16th Century
4
Steam Engine
Industrial Revolution
18th Century
5
Electricity
Power generation & Usage
19th Century
6
Automobile
Long distance commuting & Transportation
20th Century
7
Airplane
International Travel & Transportation
20th Century
8
Telephone
Distance communication
20th Century
9
Television
Video communication
20th Century
10
Computer
Data Processing
20th Century
11
Internet
Data & Information Communication, E-business 20th Century
12
Mobile Communication
Ubiquitous communication
20Th Century
13
Biotechnology
Bio-engineering, Gene Therapy,
20th Century
14
Information Communication
& Computation Technology
(ICCT)
Ubiquitous computing & Communication
21st Century
15
Nanotechnology (NT)
Solutions to nutritious food, drinking water,
renewable energy, Nanomedicine& Therapy
21st Century
16
Artificial Intelligence (AI)
Total automation
21st Century
P. S. Aithal et al. (2018); www.srinivaspublication.com
PAGE 18 PAGE 18 International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 (ii) Cloud Technology: (ii) Cloud Technology:
Cloud computing is one of the advances in
computer technology and is uses information
communication technology as well. Due to the
ubiquity of cloud computing facility with
flexibility in scaling it has become an important
topic of research and provides the value for
computing processes in the business. The cloud
computing model offers so-called Business
Intelligence (BI) for any kind of business
decisions
via
the
Internet. Using
cloud
computing model, one can offer a rented
hardware as well as software to process the data
online. Thus, cloud computing model has three
variations as Software as a Service (SaaS),
Infrastructure-as-a-Service (IaaS) and Platform-
as-a-Service (PaaS) to provide ubiquitous
computing service solutions to the business. The
cloud computing solution to any business will
allow companies to reduce their investment cost
and maintenance cost for without compromising
to have access to BI solution which will give the
business an edge on their competition [8-15]. Could computing is subfield of the Information
Communication and Computation Technology
(ICCT). p
p
g
(i) Big data and business Analytics: ( )
g
y
The emerging subfield of ICCT named big data
and business analytics focus on handling huge
amount of data continuously generated in any
business or data capturing process and analyses
it
using
various
quantitative
analytical
techniques and mathematical models to study
the
pattern
and
descriptive
information,
predictive
information,
and
prescriptive
information for supporting the decision makers
to take optimum decisions to the problems
related to future aspects of the business. Predictive analytics in various functional areas
like Marketing analytics, Retail Analytics
(Customer Analytics / Supply Chain Analytics),
Pricing Analytics, Financial analytics, Social
media analytics, sports analytics, and Healthcare
analytics are finding importance in the business
environment for effective decision making. Further Prescriptive Analytics foroptimizing the
decisions with multiple objectives / portfolio
analytics, optimizing complex decisions /
salesforce analytics, and Retail Analytics etc are
also have futuristic impact on effective business (iii) Artificial Intelligence : Artificial intelligence (AI) is an area of
computer science which focus on the creation of
intelligent machines that makes decisions like
human beings. The main functions of artificial
intelligence machines are to recognize the
environment
such
as
speech
recognition,
Learning, Planning, Problem solving, and hence
decision making. Artificial intelligence machine
mimics cognitive functions of human beings
associated with other human minds, such as
learning & memorising and decision making for
problem solving. ICCT has created a platform
for AI to be introduced and developed for
adding intelligent thinking components in
electronic systems used in any industrial sectors
[16]. 4.1 ICCT as GPT : It is observed that Information Communication
and
Computation
Technology
(ICCT)
is
showing all the three characteristics of GPT. In
the 21st century, ICCT is grown and spread its
roots to all industries and industry sectors from
A to Z due to its pervasiveness property as
shown in Table 2. The Improvement and
Innovation spawning properties of Information
Communication and Computation Technology
(ICCT) as general purpose technology are
created major stake holding areas including Big
data and business Analytics, Cloud Technology,
Artificial Intelligence, Internet of Things (IoT),
Digital Marketing, 3D Printing, Virtual Reality,
and optical computing. p
p
g
(i) Big data and business Analytics: 3. Whole eras of technical progress and growth
appear to be driven by a few ‘General Purpose
Technologies’ (GPT's), such as the steam
engine, the electric motor, and semiconductors. GPT's are characterized by pervasiveness to
many sectors, inherent potential for technical
improvements,
and
innovation
complementarities to many applications, giving
i
i
i
l
f
i
A
bl Whole eras of technical progress and growth
appear to be driven by a few ‘General Purpose
Technologies’ (GPT's), such as the steam
engine, the electric motor, and semiconductors. General purpose technologies have the potential
to reshape the economy of the world and boost
productivity across all sectors and industries. Such transformations are far more than simple
technical
innovation,
or
anew
discovery. However, such technologies often require a
wholesale
remaking
of
infrastructure
environments, of business models, and of
cultural norms. There are three fundamental
features of GPTs that differentiate them from
other technologies which are (1) Pervasiveness –
The GPT should spread to most sectors. (2)
Improvement – The GPT should get better over
time and, hence, should keep lowering the costs GPT's are characterized by pervasiveness to
many sectors, inherent potential for technical
improvements,
and
innovation GPT's are characterized by pervasiveness to
many sectors, inherent potential for technical
improvements,
and
innovation complementarities to many applications, giving
rise to increasing scale of operation. A notable
writer Ruttan[2] identified the development of
six general-purpose technologies: •
Interchangeable parts and mass production •
Military and commercial aircraft •
Nuclear energy •
Computers and semi-conductors PAGE 19 P. S. Aithal et al. (2018); www.srinivaspublication.com International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 SRINIVAS
PUBLICATION of its users. (3) Innovation spawning – The GPT
should support to invent and produce new
products or processes. Most technologies
possess each of these characteristics to some
degree,
and
thus
a
GPT
cannot
differ
qualitatively from these other technologies [1]. (v) Digital Marketing : ICCT created a new business model called E-
business/
M-business
model. This
model
consists of ubiquitous selling proposition. Digital marketing is the marketing of products or
services using digital technologies as per such
new business model using mainly on the
Internet, but also including mobile phones,
display
advertising,
and
any
other digital medium. At a high level, digital
marketing refers
to
advertising
delivered
through digital channels such as search engines,
websites, social media, email, and mobile apps. Digital marketing is emerged as an essential
future marketing activity using ICCT general
purpose technology [18]. p
(vii) Virtual Reality : Virtual reality is an artificial environment that is
created with the help of computer-based
software and presented to the user in such a way
that the user suspends belief and accepts it as a
real environment. On a computer, virtual reality
is primarily experienced through two of the five
senses: sight and sound. Currently, the virtual
reality is mainly developed and used in
simulated training and education as well as the
simulated game environment. But it may further
find its applications in many other areas
including business as augmented reality and may
enter the group of general purpose technology
[20]. (viii) Optical computing: High speed computers based on optical signal
switching and optical signal processing are
expected to breakthrough with their full
potentials and capabilities using optical logic
gates
and
flip-flops
fabricated
by
nanocomposites are expected to breakthrough in
this century. High speed computation and data
storage using nanotechnology based optical
computers are going to revolutionize the entire
computer industry. Optical computation is
joining both general purpose technologies of
Nanotechnology
and
ICCT
through
the
processes of design & production as well as
operation & applications respectively [21]. SRINIVAS
PUBLICATION data and information. These objects are provided
with unique identifiers (UIDs) and are capable to
transfer data and information over a network
without requiring human-to-human or human-to-
computer interaction by using IOT technology. Such a connection of physical things/objects to
the Internet makes it possible to access remote
sensor data and to control the physical world
from a distance. The mash-up of captured data
with data retrieved from other sources, eg, with
data that is contained in the Web, gives rise
to new synergistic services that go beyond the
services that can be provided by an isolated
embedded system [17]. Internet-of-Things
(IoT) is not in any new disruptive technology
but is the pervasive deployment and innovation
of ICCT. created. 3D printing can be divided into metal,
fabrics, bio and a whole host of other industries
with many applications in many industries
worldwide.3D printing is a variant of ICCT
general purpose technology and has wide scope
in various industrial automation and home
automation processes. 3D printing comprises of
many other technologies along with ICCT. Some
of the 3D printers make use of nanomaterials
and nanocomposites [19]. ( ii) Vi
l R
li (iv) Internet of Things (IoT) : It is a network of various electronic, computing,
and optical devices/objects including human
beings connected virtually by means of internet
or intranet for enabling them to send and receive PAGE 20 P. S. Aithal et al. (2018); www.srinivaspublication.com International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 SRINIVAS
PUBLICATION (vi) 3D Printing : 3D printing is an ICCT application where
various materials are joined or solidified using
various processes under the control of computer
to create a three-dimensional object. In 3D
printing, an object is created by laying down
successive layers of material until the object is Table 2 :Industries and Industry sectors which use and benefit from ICCT
S. No. Industries
Industry Sectors/Segments
ICCT Applications
1
Agricultural & Allied
Industries
Agricultural products,
Forestry & logging, Fishery. Remote sensing using
satellite technologies,
Geographical information systems,
Agronomy and soil sciences, Weather
prediction& forecasting etc. 2
Auto components
Engine &Drive
Product Lifecycle Management,
P S Aithal et al (2018); www srinivaspublication com
PAGE 21 PAGE 21 PAGE 21 P. S. Aithal et al. (2018); www.srinivaspublication.com International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 SRINIVAS
PUBLICATION SRINIVAS
PUBLICATION Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018
PUBLICATION
Transmission Parts, Suspension
& Braking Parts,
Electricals,
Body and Chassis Parts,
Equipment etc. Research & Development
Manufacturing automation
Sales automation
Post sales solution
3
Automobiles
Commercial vehicles, Passenger
cars,
Three & two-wheelers. Use of IT in new vehicle design,
systems control, manufacturing,
sourcing, and marketing. 4
Aviation
Civil aviation,
Military aviation
Airport operations, Air-cargo
operations, Airline management, Air-
ticketing, Security etc. 5
Banking& Insurance
Public Banking
Private banking
International banking
Life insurance
General insurance
E-banking, Online banking, Online
and mobile services, ERP and
networking, Transactional services,
ATM management etc. 6
Cement
Cement Production,
Cement transportation
Energy efficiency in production
Transportation management, Quality
monitoring etc. 7
Consumer Durables
Consumer electronics (brown
goods) Consumer appliances
(white goods)
Production automation, Supply chain
management, Online sales, Online
payment, Online customer support,
etc. 8
E-Commerce
E-procurement
E-marketing
E-payment
E-cart, Internet, Secured online
payment, Customer support, E-
advertisement
9
Education & Training Education,
Training
Online education, Online training, E-
gadgets, online evaluation etc. 10
Engineering &
Capital Goods
Transport equipment,
Capital goods, other
machinery/equipment and
light engineering products such
as castings, forgings and
fasteners. Made to order, Order processing,
Supply chain management, Quality
control systems
Online customer management, ERP
etc. 11
Financial Services
Credit unions, Banks,
Credit-card companies,
Insurance companies,
accountancy companies,
Consumer-finance companies,
stock brokerages,
Investment funds, etc. Operational automation, Online
transactions, Online services,
electronic business, etc. 12
FMCG
Packaged foods, Beverages,
Toiletries, Over-the-counter
drugs, Other consumables. Retail management
Supply chain management
E-distribution
Business to Business E-Commerce
13
Healthcare
Hospitals, Medical devices,
Clinical trials, Outsourcing,
Telemedicine, Medical
tourism, Health insurance
Communication,
Computerization of medical records,
Networking of various departments in
a hospital,
P. S. Aithal et al. (2018); www.srinivaspublication.com
PAGE 22 PAGE 22 PAGE 22 SRINIVAS
PUBLICATION SRINIVAS
PUBLICATION International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018
PUBLICATION
and Medical equipment
Tele-medicine services
14
Infrastructure
Transportation, Communication,
Sewage, Water and electric
systems
Basic Communication and
Computation
15
IT & ITES
E-Communication
Cloud computing,
Mobile applications
E-services
E-Business
Business Automation
Business process outsourcing
Resource sharing
Ubiquitous business
16
Manufacturing
Raw materials,
Food manufacturing,
Textiles
Automated procurement
Quality control
Online marketing
17
Media &
Environment
Print media, Radio,
TV media, Social media, and
film,
Digital techniques
Online media
Internet based social networks etc
18
Pharmaceuticals
Pharmacy
Drug production
Clinical practice
Online pharmacy
Drug information
Clinical information sharing
19
Ports
Ports
Terminal management
Shipping
Automation of all activities
20
Power
Coal
Hydro
Renewable energy
Automation of all activities
Control of wastage
21
Railways
Goods trains
Passenger trains
Railways Infrastructure
Online booking
E-communication
Safety
22
Real Estate
Residential sector
Commercial sector
Tele communication
Entertainment
23
Retail
Convenience Stores,
Specialty Stores,
Department Stores,
Supermarkets, Hypermarkets,
Discount Stores,
Multichannel Stores. Communication
Online transactions
Online store
Online payment
E-inventory etc. 24
Science &
Technology
Research & Development
Emerging technologies
Bio-technology
Space technology
Nanotechnology etc. Back ground support
Online Journals
Research data
Space data & Travel
Design 7 development
25
Services
Warehousing and
truck transportation services,
Information sector services,
Commodities, Securities and
other investment services
Process Automation
Customer service
Information
E-Business models
Online payment
26
Telecommunications
Telecom equipment (the
largest),
Telecom services (next largest),
Wireless communication. Electronic signal
Radiofrequency signal
Wireless data
Video and audio communication
P. S. Aithal et al. (2018); www.srinivaspublication.com
PAGE 23 PAGE 23 PAGE 23 International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018
SRINIVAS
PUBLICATION
27
Tourism &
Hospitality
Food and beverages,
Travel and Tourism,
Lodging,
Recreation. Geographical Positioning systems,
Online booking
Audio & Video recording &
Processing. International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018
SRINIVAS
PUBLICATION
27
Tourism &
Hospitality
Food and beverages,
Travel and Tourism,
Lodging,
Recreation. Geographical Positioning systems,
Online booking
Audio & Video recording &
Processing. International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 4.2 Nanotechnology as GPT : The major stakeholders of Nanotechnology as
general-purpose
technology
are
Universal
drinking water system, Universal Renewable
energy system, Optical computation, Embedded
intelligence, Chameleon chips, Flying cars,
Space travel, and anticipated Immortality [25-
26]. The major stakeholders of Nanotechnology as
general-purpose
technology
are
Universal
drinking water system, Universal Renewable
energy system, Optical computation, Embedded
intelligence, Chameleon chips, Flying cars,
Space travel, and anticipated Immortality [25-
26]. (iii) Automobiles :Automobiles is one of the
largest industries in the world. Automobile
industry is trying to make a breakthrough in
improving the efficiency, durability, and cost of
vehicles using nanotechnology solutions along
with decreasing the pollution by using hydrogen
fuel or electric engines. The major expected
impact of nanotechnology innovations on
Automobile sector in order to solve the problems
in automobile efficiency, durability, cost, and
environmental
pollution
to
produce
electric/pollution
free
vehicles
with
nanotechnology-based auto-components, auto-
engines, auto-tyres, auto-electronics, auto-seat
materials, auto-bodies, aeroplanes, space crafts,
and rockets [29]. ]
(i)
Nanotechnology
treated
Seeds
for
Innovations in Agriculture :NT solutions in
agriculture
reduce
applications
of
plant
protection products, minimize nutrient losses in
fertilization, and increase agricultural yields
through optimized nutrient management. It also
provides nanotech-based tools to detect diseases
in a rapid manner, improve the ability of plants
to absorb nutrients and promote the molecular
treatment of diseases. Use of nano-sensors
supports
the
use
of
precision
farming
methodologies to a multifold increase of crop
yield. Nanotech-enabled “smart” devices can
preventive and warn to choose diseased plant
even before they detected by the farmers and
simultaneously provide remedial measures. These nanotech systems can also be used to
monitor the delivery of chemicals [27]. (ii)
Universal
drinking
water
system
:Nanotechnology has shown opportunity in
solving another fundamental problem of many
people of the world which is the scarcity of
drinking water. Though we have abundant water
in the sea, it is non-potable and hence not useful
for drinking and irrigation. Nanotechnology
filters are promising solutions to filter soluble (i)
Nanotechnology
treated
Seeds
for
Innovations in Agriculture :NT solutions in
agriculture
reduce
applications
of
plant
protection products, minimize nutrient losses in
fertilization, and increase agricultural yields
through optimized nutrient management. It also
provides nanotech-based tools to detect diseases
in a rapid manner, improve the ability of plants
to absorb nutrients and promote the molecular
treatment of diseases. Use of nano-sensors
supports
the
use
of
precision
farming
methodologies to a multifold increase of crop
yield. P. S. Aithal et al. (2018); www.srinivaspublication.com SRINIVAS
PUBLICATION Geographical Positioning systems,
Online booking
Audio & Video recording &
Processing. ICCT has made a major impact due its
associated new business models called e-
business model and m-business models. These
business
models
provided
both
business
organizations and customers to use ubiquitous
concept of selling and buying the goods and
services from anywhere, anytime and any
amount of time. The business model developed
using ICCT general purpose technology is very
close to ideal business model [22-24]. and insoluble impurities mixed from the water
both in small scale and large scale. This
innovation leads to scalable universal drinking
water system. Nanotechnology will provide a
solution for this challenge of providing abundant
drinking water through inexpensive methods of
water purification by detection of the molecular
level
of
contaminants,
and
improved
nanomembrane based filtration systems. This
helps the conversion of seawater to drinking
water at a very low cost [28]. and insoluble impurities mixed from the water
both in small scale and large scale. This
innovation leads to scalable universal drinking
water system. Nanotechnology will provide a
solution for this challenge of providing abundant
drinking water through inexpensive methods of
water purification by detection of the molecular
level
of
contaminants,
and
improved
nanomembrane based filtration systems. This
helps the conversion of seawater to drinking
water at a very low cost [28]. (iii) Automobiles :Automobiles is one of the
largest industries in the world. Automobile
industry is trying to make a breakthrough in
improving the efficiency, durability, and cost of
vehicles using nanotechnology solutions along
with decreasing the pollution by using hydrogen
fuel or electric engines. The major expected
impact of nanotechnology innovations on
Automobile sector in order to solve the problems
in automobile efficiency, durability, cost, and
environmental
pollution
to
produce
electric/pollution
free
vehicles
with
nanotechnology-based auto-components, auto-
engines, auto-tyres, auto-electronics, auto-seat
materials, auto-bodies, aeroplanes, space crafts,
and rockets [29]. (iv)
Renewable
energy
system
:The
nanotechnology impact on seven areas of energy
sector including solar energy, wind energy,
nuclear energy oil-fuel based energy artificial 4.2 Nanotechnology as GPT : 4.2 Nanotechnology as GPT : Space exploration also helps us to monitor the
health of our planet, a source of resources and an
outlet for our imagination. Using carbon
nanotubes to make the cable needed for the
space
elevator,
a
system
which
could
significantly reduce the cost of sending material
into orbit. Nanotechnology will create the ability
for humans to operate in space more safely. Applications, where nanotechnology will impact
space exploration, are propulsion fuels, coatings,
structural materials, smart uniforms, electronics
and life support environments. These will be
more efficient, stronger, self-healing and lighter
than what is currently available [37-40]. (ix) Anticipated Immortality: The dream of
every human being is to a leave long time with
good health. This can be achieved using
nanotechnology innovations. There are two
ways in which nanotechnology may be able to
extend our lives. One is by helping to eradicate
life-threatening diseases such as cancer, and the
other is by repairing damage to our bodies at the
cellular level - a nano version of the fountain of to each other with many computational units
which can process signals simultaneously. While
reconfiguring these chips as per desired, the
connections are automatically changed. i.e., the
connections between blocks and inside blocks
are changed. After loading the software, the old
hardware design will be erased and a new
hardware design is generated by activating some
connections
and
inactivating
some
other
connections. Hence the system defines the
configuration of hardware for loaded software. Chameleon chips can be realized using dye
doped nanocomposite materials [25]. nanocomposites are expected to breakthrough in
this century. High speed computation and data
storage using nanotechnology based optical
computers are going to revolutionize the entire
computer industry. Optical computation is
joining both general purpose technologies of
Nanotechnology
and
ICCT
through
the
processes of design & production as well as
operation & applications respectively. (vi)
Embedded
intelligence
:Embedded
intelligence is a technique to modify the ability
of a product, process or service to reflect on its
own operational performance, usage load, or in
relation to the end-user or environment in terms
of
satisfactory
experience
and
smart
improvement. This improvement through self-
reflection, facilitated by information collected
by sensors and processed locally or remotely,
must be considered from the design stage such
as to improve the product features, enhance the
product lifetime and performance, increase the
quality of process or service delivery, or ensure
customer satisfaction and market acceptance
[33-36]. 4.2 Nanotechnology as GPT : Nanotech-enabled “smart” devices can
preventive and warn to choose diseased plant
even before they detected by the farmers and
simultaneously provide remedial measures. These nanotech systems can also be used to
monitor the delivery of chemicals [27]. (ii)
Universal
drinking
water
system
:Nanotechnology has shown opportunity in
solving another fundamental problem of many
people of the world which is the scarcity of
drinking water. Though we have abundant water
in the sea, it is non-potable and hence not useful
for drinking and irrigation. Nanotechnology
filters are promising solutions to filter soluble (iv)
Renewable
energy
system
:The
nanotechnology impact on seven areas of energy
sector including solar energy, wind energy,
nuclear energy, oil-fuel based energy, artificial
photosynthesis, energy storage and effective
energy
management
to
promote
nanotechnology-based energy as ubiquitous
energy are discussed and reviewed. The paper
includes possible innovations and research
opportunities in nano-modified solar cells,
Nano-influenced
Fuel
storage
cells,
and
nanotech based artificial photosynthesis [30-32]. (v) Optical computation :High speed computers
based on optical signal switching and optical
signal processing are expected to breakthrough
with their full potentials and capabilities using
optical logic gates and flip-flops fabricated by (ii)
Universal
drinking
water
system
:Nanotechnology has shown opportunity in
solving another fundamental problem of many
people of the world which is the scarcity of
drinking water. Though we have abundant water
in the sea, it is non-potable and hence not useful
for drinking and irrigation. Nanotechnology
filters are promising solutions to filter soluble P. S. Aithal et al. (2018); www.srinivaspublication.com International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 SRINIVAS
PUBLICATION SRINIVAS
PUBLICATION to each other with many computational units
which can process signals simultaneously. While
reconfiguring these chips as per desired, the
connections are automatically changed. i.e., the
connections between blocks and inside blocks
are changed. After loading the software, the old
hardware design will be erased and a new
hardware design is generated by activating some
connections
and
inactivating
some
other
connections. Hence the system defines the
configuration of hardware for loaded software. Chameleon chips can be realized using dye
doped nanocomposite materials [25]. (viii) Space Travel : The growing population
and diminished resources on earth suggest
identifying an opportunity for space exploration. P. S. Aithal et al. (2018); www.srinivaspublication.com 4.2 Nanotechnology as GPT : Embedded
intelligence
aims
at
delivering smarter products, systems or services
to industry through their integration and
purposeful
use
for
a
given
application. Embedded
intelligence
(EI)
system/service
application
contains
various
components/
processes
which
include
design
for
EI,
intelligent software, packaging & interconnect,
manufacturing solutions and/or system services. Nanotechnology supports the integration of
embedded processors with sensors, intelligence,
wireless connectivity and other components with
high level operating systems, middleware and
system integration services. (viii) Space Travel : The growing population
and diminished resources on earth suggest
identifying an opportunity for space exploration. Space exploration also helps us to monitor the
health of our planet, a source of resources and an
outlet for our imagination. Using carbon
nanotubes to make the cable needed for the
space
elevator,
a
system
which
could
significantly reduce the cost of sending material
into orbit. Nanotechnology will create the ability
for humans to operate in space more safely. Applications, where nanotechnology will impact
space exploration, are propulsion fuels, coatings,
structural materials, smart uniforms, electronics
and life support environments. These will be
more efficient, stronger, self-healing and lighter
than what is currently available [37-40]. (ix) Anticipated Immortality: The dream of
every human being is to a leave long time with
good health. This can be achieved using
nanotechnology innovations. There are two
ways in which nanotechnology may be able to
extend our lives. One is by helping to eradicate
life-threatening diseases such as cancer, and the
other is by repairing damage to our bodies at the
cellular level - a nano version of the fountain of
youth. The most exciting possibility exists in the
potential for repairing our bodies at the cellular
level. Research in this regard is very active at
the laboratory level to achieve and realize these
dreams in the 21st century itself [25]. (vii) Chameleon chips :A chameleon chip is a
self-configurable electronic or optical circuit to
modify the output signal characteristics as per
the system requirements. It has an erasable
hardware configuration. It is also possible to
rewire it by itself through adapted programming
tasks. Chameleon chip consists of many
functional blocks which are connected parallel P. S. Aithal et al. (2018); www.srinivaspublication.com PAGE 25 PAGE 25 International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018
SRINIVAS
PUBLICATION
Table 3 : Industries and Industry sectors which use and benefit from Nanotechnology
S. No. 4.2 Nanotechnology as GPT : Industries
Industry Sectors/Segments
NT Applications
1
Agricultural &
Allied Industries
Agricultural products,
Forestry & logging, Fishery. Nanotechnology based
optimized nutrient management,
Nanosensors,
Nanotechnology based genetic
transformation,
Nanoencapsulation of
nutraceuticals,
Nanofertilizers
2
Automobiles
Commercial vehicles, Passenger cars,
Three & two-wheelers. Nanomaterials for energy
storage, increasing body
strength, efficient engine and
body parts
3
Aviation
Civil aviation,
Military aviation
Light & strong materials for
airplane
4
Banking &
Insurance
Public Banking
Private banking
International banking
Life insurance
General insurance
Increased efficiency of ICT
components and devices
5
Cement
Cement Production,
Cement transportation
Nanomaterials for increasing
strength, controlling hardness
6
Consumer Durables
Consumer electronics (brown goods)
Consumer appliances (white goods)
Increased performance
efficiency of goods and devices
7
E-Commerce
E-procurement
E-marketing
E-payment
Increased efficiency of ICT
components and devices
8
Education &
Training
Education,
Training
Increased efficiency of ICT
components and devices
9
Engineering &
Capital Goods
Transport equipment,
Capital goods, other
machinery/equipment and
light engineering products such as
castings, forgings and fasteners. Nanomaterial based light
weight components with
enhanced durability. Nanocomposite based battery
and solar panels to enhance
efficiency. 10
FMCG
Packaged foods, Beverages, Toiletries,
Over-the-counter drugs, Other
consumables. Nanomaterial based cosmetics,
paints, food packaging, drugs
&sports equipment etc. 11
Gems & Jewellery
Gems
Jewellery
Nanotechnology based artificial
gems and jewellery
12
Healthcare
Hospitals, Medical devices, Clinical
trials, Outsourcing,
Telemedicine, Medical
tourism, Health insurance
and Medical equipment
Nanomaterial based building
materials, Nanomedicine,
Nanotechnology based
controlled drug delivery
13
Infrastructure
Transportation, Communication,
Nanomaterial based parts, International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 SRINIVAS
PUBLICATION SRINIVAS
PUBLICATION PAGE 26 SRINIVAS
PUBLICATION International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 SRINIVAS
PUBLICATION Nanotechnology supports industries to do
innovations in material and manufacturing
processes to improve the performance quality
towards optimum systems whereas the ICCT
supports industries to do innovations at
application and service side of businesses. Thus,
both NT and ICCT works like complementary to
each other instead of competitive technologies. Thus, both technologies are expanding with time
to many industries by showing all the three
characteristics of GPT with Pervasiveness,
Improvement
over
time,
and
Innovation
spawning abilities. .3 Similarities between the ICCT and NT : 2. ICCT is spreading more slowly than did
nanotechnology, due to the fact that ICCT is an
application
type
technology
rather
than
Nanotechnology which is a manufacturing
technology. 2. ICCT is spreading more slowly than did
nanotechnology, due to the fact that ICCT is an
application
type
technology
rather
than
Nanotechnology which is a manufacturing
technology. 3. The productivity slowdown is stronger in the
Nanotechnology era than the ICCT era. 4. Nanotechnology supports various products
preparation and quality improvement whereas
ICCT improves many applications in various
industrial sectors. 5. Nanotechnology has anticipated hidden
environmental problems and challenges which is
not a case in ICCT. 1. In both technologies, productivity growth
rates are below those attained in the decades
immediately preceding the GPT’s arrival. The differences seem to be quite important. But
overall the evidence clearly supports the view
that technological progress is uneven, that it
does entail the episodic arrival of GPTs, and that
these GPTs bring on turbulence and lower
growth early on and higher growth and
prosperity later. 2. Measures of reallocation and invention – the
entry and exit of firms to these business market,
investment by new firms relative to incumbents,
and grants of patents and trademarks – are all
higher during both the GPT’s. 4.5 Measuring the three characteristics of
ICCT as GPT : SRINIVAS
PUBLICATION Sewage, Water and electric systems
filters, power storage devices
14
IT & ITES
E-Communication
Cloud computing,
Mobile applications
E-services
E-Business
Nanomaterial based electronic
and photonic components
15
Manufacturing
Raw materials,
Food manufacturing,
Textiles
Nanomaterials used for
fabrication to improve the
strength and performance
16
Media &
Environment
Print media, Radio,
TV media, Social media, and film,
Nano ink,
Nanotechnology based
electronic and photonic
components
17
Pharmaceuticals
Pharmacy
Drug production
Clinical practice
Nanomaterial based medicines,
Nanomaterial based controlled
drug delivery
18
Ports
Ports
Terminal management
Shipping
Improved mechanical tensile
strength
19
Power
Coal
Hydro
Renewable energy
Improved technology of power
generation,
Improved performance of
turbines,
Solar panel with improved
efficiency,
Wind turbines with increased
durability
20
Railways
Goods trains
Passenger trains
Railways Infrastructure
Nanomaterial based improved
performance
21
Real Estate
Residential sector
Commercial sector
Nanomaterial based quality and
durable construction materials
22
Renewable Energy
Solar Energy
Wind Energy
Nuclear energy
Improved efficiency
Improved strength
Improved safety
23
Retail
Convenience Stores,
Specialty Stores,
Department Stores,
Supermarkets, Hypermarkets,
Discount Stores,
Multichannel Stores. Quality and efficient products
24
Roads
Roads
Bridges
Nanomaterials for durable roads
and bridge construction
25
Science &
Technology
Research & Development
Emerging technologies
Bio-technology
Space technology
Nanotechnology etc. Research for new smart
materials, new smart
components, new and improved
devices through improved
properties
26
Steel
Iron Ore
Parts with improved strength
P. S. Aithal et al. (2018); www.srinivaspublication.com
PAGE 27 PAGE 27 P. S. Aithal et al. (2018); www.srinivaspublication.com International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018
SRINIVAS
PUBLICATION
Steel
Steel products
and
increased durability
27
Telecommunications Telecom equipment (the largest),
Telecom services (next largest),
Wireless communication. Components with Improved
efficiency, Improved strength,
small size. 28
Textiles
Dying,
Treads,
Weaving machines
Improved colouration,
Improved strengths,
Stain-free fabrics,
Durable Weaving machines,
etc. SRINIVAS
PUBLICATION International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 ICCT era, and the price of IT is falling 100 times
faster, at least, than did the price of
Nanotechnology. 4.3 Similarities between the ICCT and NT :
Even though both ICCT and NT are emerging as
general-purpose technologies, they are not
seeming
to
be
competing
technologies. 4.5 Measuring the three characteristics of
ICCT as GPT : 3. Expansion to various industries rises
gradually during each GPT time. (1) Pervasiveness of the GPT : The first
characteristic is the technology’s pervasiveness. The spread of ICCT in various industry segment
is immense and the details are given in table 2. (2) Improvement :As per this characteristics, the
ICCT should get better over time and, hence,
should keep lowering the costs of its users. ICCT is playing this role more effectively. The
technology through its digital model lowered the
cost of various processes and their applications
to a minimum level due to the strategy of (1) Pervasiveness of the GPT : The first
characteristic is the technology’s pervasiveness. The spread of ICCT in various industry segment
is immense and the details are given in table 2. (2) Improvement :As per this characteristics, the
ICCT should get better over time and, hence,
should keep lowering the costs of its users. ICCT is playing this role more effectively. The
technology through its digital model lowered the
cost of various processes and their applications
to a minimum level due to the strategy of (1) Pervasiveness of the GPT : The first
characteristic is the technology’s pervasiveness. 4. Both technologies are supporting each other
and hence interrelated and complementary to
solve many problems in the organizations and in
the society. The spread of ICCT in various industry segment
is immense and the details are given in table 2. 4.4 Differences between the Information
Communication &Computer Technology and
Nanotechnology: 4.6 Measuring the three characteristics of NT
as GPT : (1) Pervasiveness of the GPT : The first
characteristic is the technology’s pervasiveness. Nanotechnology finds its applications in almost
every industrial sector as shown in table 3. It has
spread its roots in solving both fundamental
problems of human beings and offering
luxurious facilities in every part and parcel of
human life. 3. Entry, exit, and mergers should rise – This
feature is also observed in the case of both
technologies as the alternative modes for the
reallocation of assets. 3. Entry, exit, and mergers should rise – This
feature is also observed in the case of both
technologies as the alternative modes for the
reallocation of assets. 4. Stock prices should initially fall – This also
applicable in case of both technologies. The
speed of such fall depends on the way that the
market learns of the GPT’s arrival. 4. Stock prices should initially fall – This also
applicable in case of both technologies. The
speed of such fall depends on the way that the
market learns of the GPT’s arrival. (2) Improvement : The GPT should get better
over time and, hence, should keep lowering the
costs
of
its
users. This
is
happening
nanotechnological solutions. The improvements
in material properties and hence the overall
performance of devices in almost all industrial
sectors support this property of nanotechnology. 5. Young and small firms should do better – The
ideas and products associated with both
technologies are often be brought to market by
new firms. The market share and market value
of young firms should, therefore, rise relative to
old firms. (3) Innovation spawning : The nanotechnology
as GPT supports to invent and produce new
products or processes. In agriculture, it supports
to develop new sensors, a new way of
controlling genetic transformation, optimization
of nutrient management, nanoencapsulation of
nutraceuticals, nano-fertilizers etc. It also helps
to produce potable water from seawater. It
supports to fabricate highly efficient long-life
batteries for electric vehicles and allows to
fabricate high efficiency solar panels and strong
and durable windmills. It also helps to develop
an innovative process of controlled drug
delivery. Finally, it also supports the most
anticipated technology of lifespan expansion of 6. Interest rates and the trade deficit – The
sudden increase in the output due to these
technologies cause a rise in interest rates or to
worsen the trade balance. 7. SRINIVAS
PUBLICATION human beings so that is predicted as the ideal
technology and technology of the 21st century. attaining
the
cost
leadership
by
various
competitors
in
different
industries. The
continuous improvement in both minimizing the
size and enhancing the speed of ICCT devices,
the customers and hence the society is
continuously getting benefits. 5. CONTRIBUTION OF GPT’S TOWARDS
DEVELOPING SUSTAINABLE SOCIETY The symptoms of a GPT proposed by different
authors with the three characteristics – its
pervasiveness, its rate of improvement, and its
innovation-spawning
tendency
are
clearly
observed in both the technologies ICCT and NT. Apart from these, additional symptoms as listed
below are also observed in the case of these two
technologies : The symptoms of a GPT proposed by different
authors with the three characteristics – its
pervasiveness, its rate of improvement, and its
innovation-spawning
tendency
are
clearly
observed in both the technologies ICCT and NT. (3) Innovation spawning: The ICCT as GPT
supports to invent and produce new products or
processes. ICCT based innovative new products
and processes in high speed computing,
electronic communication, business analytics,
virtual reality, artificial intelligence, 3D printing,
digital marketing, cloud computing, Internet of
things technologies are becoming more popular
and hence ICCT is growing beyond general
purpose technology and marching towards so-
called universal technology. Apart from these, additional symptoms as listed
below are also observed in the case of these two
technologies : 1. Productivity slows down initially – The new
technology may not be user-friendly at first, and
output may fall for a while as the economy
adjusts. 2. The skill premium rises with time – If the
GPT is not user-friendly at first, skilled people
will be in greater demand when the new
technology arrives, and their earnings should
rise compared to those of the unskilled. This is
true in both cases. 4.4 Differences between the Information
Communication &Computer Technology and
Nanotechnology: 1. Innovation measures are growing much faster
for ICCT than for Nanotechnology – patents and
trademarks surge much more strongly during the PAGE 28 P. S. Aithal et al. (2018); www.srinivaspublication.com International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 Nanotechnology Nanotechnology
supported
Life
extension science, also known as anti-aging
medicine, indefinite life extension, experimental
gerontology, and biomedical gerontology, is the
study of slowing down or reversing the
processes of aging to extend both the maximum
and average lifespan. Artificial intelligence machine mimics cognitive
functions of human beings associated with
other human minds, such as learning &
memorizing and decision making for problem
solving. 6. FUTURE OF ICCT & NT •
GPS systems which simplify the complexity
of millions of routes to find the best suitable
one based on the user’s preference. Both ICCT and NT as general purpose
technologies are further expanding their roots to
many other industries and becoming so-called
Universal Technologies. Universal technology is
a technology more than general purpose
technology where apart from (1) Pervasiveness,
(2) Improvement, and (3) Innovation spawning
characteristics, additional characteristics like (4)
Universal applicability, and (5) Ideal solutions to
problems. The advents of both technologies
ICCT
and
Nanotechnology
are
presently
expanding together in many areas including
three very important futuristic technologies
which are virtual reality, artificial intelligence,
and human life expansion. •
Smart phones understand human speech and
mobile applications like Siri, Cortana, and
Google
Now
are
getting
better
at
understanding
user
intentions
through
improved AI techniques. •
Cars from Google and Tesla can drive
themselves using currently available AI
systems, autopilot systems based on AI
technology direct airplanes around the
world, and robotic surgeons are operating
more exactly and high speed than their
human counterparts. It is predicted that AI enabled super-smart
computer devices may allow the blind to see, the
deaf to hear, and the disabled and the elderly to
walk, run, and even dance. It is also predicted
that artificial intelligence is the pivotal step in
addressing the grand challenges of humanity and
our brains may be able to connect directly with
the cloud via nanobots by the year 2030. On the
negative side, the uncontrolled and unregulated
advents in AI could be a threat to humanity and
may have disastrous effects leading to the end of
the world. 6.2 Artificial Intelligence : Artificial intelligence (AI) is a branch of
computer science which focus on the creation of
intelligent machines that makes decisions like
human beings. The main functions of artificial
intelligence machines are to recognize the
environment
such
as
speech
recognition,
Learning, Planning, Problem-solving, and hence
decision making. 6.1 Virtual Reality : Virtual reality is an artificial environment that is
created with the help of computer-based
software and presented to the user in such a way
that the user suspends belief and accepts it as a
real environment. On a computer, virtual reality
is primarily experienced through two of the five
senses: sight and sound. Currently, the virtual
reality is mainly developed and used in
simulated training and education as well as the
simulated game environment. But it may further
find its applications in many other areas
including business as augmented reality and may
enter the group of general purpose technology. The integrated version of ICCT and NT
applications may support high level advanced
and personalized healthcare, virtual reality, and
artificial intelligence making human life more
comfort and connecting dream and real life of
human being on the earth in particular and in the
entire universe in general. 4.6 Measuring the three characteristics of NT
as GPT : Improvements in the living standard of
people in the society–The contribution of these
two GPT’s is expected to raise the living
standard of people in the society due to their
abilities to solve problems related to both
fundamental and luxurious facilities for happy
life leading. PAGE 29 PAGE 29 P. S. Aithal et al. (2018); www.srinivaspublication.com International Journal of Management, Technology, and Social
Sciences (IJMTS), ISSN : 2581-6012, Vol. 3, No. 2, August 2018 SRINIVAS
PUBLICATION Steven
N. Durlauf,
Elsevier
B.V. DOI:
10.1016/S1574-0684(05)01018-X. Technology and Nanotechnology are, to most
observers, the two most important GPTs to date,
according to the three criteria that Bresnahan
and
Trajtenberg
proposed. These
two
technologies
emerging
as
independent,
integrated and complementary technologies with
two additional characteristics proposed in this
paper that is (1) Universal applicability, and (2)
Ideal solutions to problems. Thus, these two
technologies
are
emerging
as
universal
technologies of the 21st century. In this paper,
we have analyzed how these two GPTs are
spreading to many areas of society and changing
the lifestyle of human beings. Having discussed
in detail these two GPTs we believe that the
technological changes and their effects are
spilling over the world. The ICCT and NT
innovations differ in some important ways but
have made and going to make a further impact
on many areas of the society. ICCT is more
broadly adopted, whereas NT seems to be
technologically more revolutionary and yet to be
commercialized so that these two technologies
together
lead
to
complete
revolution
of
civilization in this world by solving both
fundamental and advanced challenges for human
prosperity. The productivity slowdown is
stronger in the NT era, but the ongoing spread of
NT and its continuing proposed precipitous price
decline are reasons for optimism about growth in
the coming decades relative to what happened at
the end of the 20th century following the spread
of ICCT. But it is the similarities between the
two epochs that are the most instructive and that
will guide our expectations about how the next
universal technologies will affect the economy
of the world when it comes along [1]. Based on
the analysis in this paper, it is also concluded
that both the technologies ICCT and NT are
potentially contributing towards creating a
techno-society and based on further progress and
spread of such technologies to every dimension
of human life to reach the ultimate level of
civilization in or around this earth. REFERENCES: [2] Ruttan, Vernon (2006). Is War Necessary for
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PUBLICATION International Journal of Applied and Advanced
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Delhi Publishers, 2018, pp. 1-170, ISBN No. :
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PUBLICATION Senthamarai, R. (2018 April). Mobile Cloud
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PUBLICATION P. S. Aithal et al. (2018); www.srinivaspublication.com P. S. Aithal et al. (2018); www.srinivaspublication.com International Journal of Management, Technology, and Social
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Faizullabhoy. (1992). Embedded intelligence. BYTE 17(3), 195. P. S. Aithal et al. (2018); www.srinivaspublication.com PAGE 33
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LEXICAL SIMILARITIES AND DIFFERENCES IN THE MATHEMATICS, SCIENCE AND ENGLISH LANGUAGE TEXTBOOKS
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K@ta
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English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING Corpus Research Insights from corpus research have revolutionized the way language is
viewed, especially words and their relationship with each other in context
(Schmitt, 2000). Corpus research allows researchers and learners to gain
insights into the language, particularly the interconnection of lexical and
grammatical patterns, collocations, colligations, the frequency of words
and the use and functional behavior of these words (Tognini-Bonelli, 2001,
Schmitt, 2000, & Sinclair, 1991). One of the ways to improve the understanding of the Science and
Math discourse and texts in the classroom and to learn about the specific
sentence structures, lexis and grammar, is to get the students to engage with
the data or texts. This would require not only the creation of a specific
database or corpus, but also learning how to control it and incorporating it
into the teaching and learning process (Tognini-Bonelli, 2001). There is reasonable consensus that a corpus will not just provide
insights into the contents but also that the results of the analyses will be
claimed to be typical of the language from which the corpus was selected. Through corpora, teachers and learners would be able to check prescribed
rules and generalizations against linguistic data and to make their own
interpretations and generalizations of these patterns (Tognini- Bonelli,
2001). Corpora allow researchers, teachers and learners to use great
amounts of real data in their study of language, instead of having to rely on
intuition and made-up examples. VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 92 time to reach the level they would have achieved if the medium of
instruction had been in a language they are familiar with, such as Bahasa
Malaysia (national language). Therefore, there is a need for both the
students and the teachers to recognize and understand Scientific and
Mathematical concepts and also understand and have knowledge of the
different types of lexical and grammatical constructions used in Science
and Math. Jayakaran Mukundan
Department of Language and Humanities Education, Faculty of Educational
Studies, University of Putra Malaysia, Selangor, Malaysia
e-mail: jaya@educ.upm.edu.my Jayakaran Mukundan
Department of Language and Humanities Education, Faculty of Educational
Studies, University of Putra Malaysia, Selangor, Malaysia
e-mail: jaya@educ.upm.edu.my Sujatha Menon
English Language Department, Academy of Language Studies, UITM
Melaka, Melaka, Malaysia
e-mail: sjathark@yahoo.com Abstract: The teaching of Science and Math in English in Malaysia is
an area of great concern to educators and students alike. This study
looks, in particular, at the common word classes among keywords
identified in the Science, Math and English language Form One
textbooks used in Malaysia and the differences in language use
identified in the Science and Math textbooks. Key words: corpus, science, math, word class, keyword With the sudden re-introduction in Malaysia of the teaching of Math
and Science in English in January 2003, in standard One and forms One
and lower Six, concerns regarding the effect of English language
proficiency on the performance of students in these subjects were raised by
various sectors of the community. It cannot be denied that there is a need to study the language used and
needed in the teaching of Math and Science in schools. In fact, in a study
conducted on 88 Math and Science teachers in Perak regarding their
attitudes and perceived readiness to teach Math and Science in English, it
was found that most of these teachers were not clear about the linguistic
features of their content subjects and had difficulty to communicate the
linguistic elements of this form of discourse to their students (Pandian &
Ramiah, 2004). According to Holme (2006), children who have been
identified by their teachers as being intellectually able to learn Math and
Science but are not proficient in English language, would take a longer 91 VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 Language of Science The languages of Science and Math are different from the languages
that students use socially at home and with their peers, and in other subject
areas at school (Laplante, 1997). Everyday words may mean something
else in Math and Science, for example the words ‘average’ and ‘divide’ 93 Mukunda, Lexical Similarities and Differences in the Mathematics may be everyday words but they acquire a more precise meaning in Math
(Carlson, 2000, Khisty, 1995, & Thompson & Rubenstein, 2000). Trimble
(1985) in his description of English for Science and Technology state that
English for Science and Technology range from English for Occupational
Purposes (EOP) to English for Academic Purposes (EAP) with a great deal
of overlapping between the two. He states that there are two areas of
problems concerning language in English for Science and Technology
discourse for non-native learners. The first being the rhetorical-
grammatical relationships and the second being the lexical elements of sub-
technical vocabulary and the noun compounds. Trimble (1985) restricts his discussion of lexis to three lexical areas,
that is, the technical vocabulary, sub-technical vocabulary and the noun
compounds. He believes that non-native learners do not usually have a
problem with technical vocabulary as it is taught explicitly by content
matter teachers. Sub-technical vocabulary is also considered not a very
problematic one as they can be understood quickly with the use of
specialist dictionaries. However, the most problematic area for students is
compounds (Trimble, 1985). p
Sub-technical vocabulary according to Trimble (1985) mean both
context-independent words that occur with a high frequency across
different disciplines of science, retaining the same meaning across these
scientific disciplines and also words that have one or more ‘general’
English meanings and which in technical contexts take on extended
meanings. The vocabulary of Science has been discussed and categorized
by many other linguists. The most notable and one of the earliest
categorization of the lexis of Science was by Cowan (1974), who was
widely attributed with the introduction of the concept of sub-technical
vocabulary. It was Cowan’s definition of sub-technical vocabulary that
Trimble extended in 1985. Cowan (1974, p. 391) describes four categories of vocabulary ranging
from highly technical words to sub-technical vocabulary which he defined
as ‘context independent’ words which occur with high frequency across
disciplines to semi-technical and finally, non-technical words, such as
hospital, medicine, disease, which he grouped together making no clear
distinction between them. Nation (2001, p. English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 94 The first category is the most technical with the words appearing
rarely outside its particular field such as ‘morpheme’ in the field of applied
linguistics and ‘pixel’ in the computing field. This category can be
described as being similar to Cowan’s (1974) highly technical words. The second category consists of words that are used both inside and
outside this particular field but with different meanings, such as the sub-
technical vocabulary of Trimble’s (1985) and Cowan’s (1974). The third category consists of words that are used both inside and
outside this particular field but the majority of its uses with a specific
meaning are related to this field. The specialized meaning it has in this field
is readily understood outside the field, such as the word ‘accused’ in the
field of Law and ‘memory’ in the computing field (Nation, 2001, p. 199). This category is similar to that described by Cowan (1974) as semi-
technical. The final category consists of words that are more common in this
field than elsewhere, similar to Cowan’s (1974) non-technical words. There is little specialization of meaning, example ‘judge’ in the field of
Law and ‘print’ in the field of computing. The first step into looking at the type of language used and required of
students for the study of Science and Math in English is to create a corpus
of the language used in these subjects. A corpus would provide a
convenient source from which to obtain evidence of the behavior of many
different facets of language: lexical, grammatical and pragmatic (Schmitt,
2000). Once a corpus has been compiled then the language in it can be
analyzed. This work, therefore, aims to analyze the language used in a prescribed
Form 1 Science textbook and Math textbook in Malaysian schools, as the
first step to identifying the type of language students are required to
understand and grasp in the process of learning Science and Math in
Malaysian schools. This language will then be compared to the general
English language used in the prescribed Form 1 English language
textbook. The distribution of prescribed textbooks in Malaysia is decided by the
Textbook Bureau of the Malaysian Education Ministry. English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING Language of Science 198) adds to these categories by declaring that there
are degrees of ‘technicalness’ depending on how restricted a word is to a
particular area. These degrees were categorized into four groups: VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 To standardize the
type of textbooks used in schools and to allow more opportunities for
different publishers to be involved in the production of textbooks for
schools, the Bureau has divided all the schools in Malaysia into five
textbook zones. These textbook zones are divided according to the states in 95 Mukunda, Lexical Similarities and Differences in the Mathematics Malaysia. If schools from different states fall under one zone, then these
schools would use the same textbooks. The five textbook zones are the
northern, central, eastern, southern and east Malaysia zones. This study
focuses on the prescribed Form 1 textbooks from the southern zone in
Malaysia. Malaysia. If schools from different states fall under one zone, then these
schools would use the same textbooks. The five textbook zones are the
northern, central, eastern, southern and east Malaysia zones. This study
focuses on the prescribed Form 1 textbooks from the southern zone in
Malaysia. In order to find out what constitutes Scientific and Mathematical
English in the textbooks used, and to see how it differs from the language
used in the English language textbook, two research questions were
formulated. The research questions posed were: 1 What are the most common word class among the keywords identified
in the Science, Math and English language textbooks? 1 What are the most common word class among the keywords identified
in the Science, Math and English language textbooks? 2 What are the differences in language use identified in the Science and
Math textbooks? 2 What are the differences in language use identified in the Science and
Math textbooks? Math textbooks? Research Design The methodological base of a corpus research is diverse as it not only
covers the fields of corpus linguistics but also involves looking into
grammatical and lexical relationships and discourse analysis. The study is
concerned with data of language used in textbooks and uses corpora to
investigate the language of Science and Math. English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING The books used were: 1 Abdullah, K. I., Wee, D., & Teo, H, B. (2002). English form 1. Pustaka
Alhas (M) Sdn. Bhd.: Kuala Lumpur. 2 Mat Saat, A. S., Salehin, M. S., & Wan Zaid, W. Z. (2002). Science
form 1. Pustaka Sistem pelajaran Sdn. Bhd. Malaysia: Kuala Lumpur
3 Chua, M. K., Teh, E. K., & Ooi, S. H. (2002). Mathematics form 1
(Vols. 1-2). Penerbitan Pelangi Sdn. Bhd. : Kuala Lumpur 1 Abdullah, K. I., Wee, D., & Teo, H, B. (2002). English form 1. Pustaka
Alhas (M) Sdn. Bhd.: Kuala Lumpur. 1 Abdullah, K. I., Wee, D., & Teo, H, B. (2002). English form 1. Pustaka Alhas (M) Sdn. Bhd.: Kuala Lumpur. 2 Mat Saat, A. S., Salehin, M. S., & Wan Zaid, W. Z. (2002). Science
form 1. Pustaka Sistem pelajaran Sdn. Bhd. Malaysia: Kuala Lumpur 2 Mat Saat, A. S., Salehin, M. S., & Wan Zaid, W. Z. (2002). Science
form 1. Pustaka Sistem pelajaran Sdn. Bhd. Malaysia: Kuala Lumpur
3 Chua, M. K., Teh, E. K., & Ooi, S. H. (2002). Mathematics form 1 form 1. Pustaka Sistem pelajaran Sdn. Bhd. Malaysia: Kuala Lumpur
3 Chua, M. K., Teh, E. K., & Ooi, S. H. (2002). Mathematics form 1
(Vols. 1-2). Penerbitan Pelangi Sdn. Bhd. : Kuala Lumpur 3 Chua, M. K., Teh, E. K., & Ooi, S. H. (2002). Mathematics form 1
(Vols. 1-2). Penerbitan Pelangi Sdn. Bhd. : Kuala Lumpur The Use of Textbooks The textbook is and has always been an important aspect of teaching
in Malaysian schools. Students, while enjoying the benefits of the textbook
as a teaching device that works alongside a teacher, would appreciate the
role of the book as reference, for this enables the learner to revise and work
on consolidation both inside and outside the classroom (Mukundan, 2004). The criticisms leveled against textbooks are plenty and the fact
remains that the textbook in most cases is indispensable and while teachers
complain about them, they cannot do without them (Ansary & Babaii,
2003). As textbooks are used by students daily in their schools, it is
important to analyze the language contained in the textbooks as this would
be the language that would challenge the students the most, in the course of
learning the English language used in Science and Math. English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 96 English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING Population and Sampling in the Science, Math and English language
Corpus For the purpose of this study, the population for the Science, Math and
English language corpus is defined as the prescribed Science, Math and
English language textbooks used by Form 1 students in the southern zone
of Malaysia. The books used were: Data Collection For this study, all the textbooks were first scanned, page by page, and
then converted into text files. As there was distortion to text scanned,
manual correction was carried out by entering (typed) words or phrases left
out or undetected by the scanner. These text files were then analyzed using
the WordSmith 4.0 software program. Instrumentation: WordSmith Tools 4 WordSmith Tools was designed by Mike Scott (1996, 1997, & 1999)
for students and researchers to be able to access and analyze corpora at
their convenience on their PCs (Scott, 2001). The reliability of WordSmith
Tools has been verified by numerous studies on various corpora which
have used these tools to analyze texts (Flowerdew, 2003, Nelson, 2000,
Mukundan, 2004, Scott, 2001, Henry & Roseberry, 2001, & Bondi, 2001). Other than these studies using WordSmith Tools, the reliability and wide
capability of the software was verified by Mukundan, in his unpublished
thesis (2004), in his exploration for suitable software to analyze prescribed
textbooks. The researchers decided to use the latest WordSmith Tools, version 4
for the purpose of text analysis in this study as this latest version has a
larger capacity for concordancing and creation of word lists with more
details, an improved word list cluster handling, enhanced tag handling and
enhanced statistical functions for collocation, to name a few
(http://www.lexically.net/wordsmith/version4.htm). English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING 97 Mukunda, Lexical Similarities and Differences in the Mathematics Analysis of Key Words A more accurate picture of any language can be gained by analysis of
words that occur significantly more often in a particular linguistic area, in
comparison to general language usage, rather than by looking at words that
have high occurrence in terms of overall frequency. These words have
been termed key words (Scott, 1997 &1999). Key words were arrived at in
this study by using the key word function of WordSmith 4. A word will get
into the list if it is unusually frequent (or unusually infrequent) in
comparison to a larger word list. English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING Key Words by Chapters This section of the discussion analyses the three largest word class
categories found among the key words by chapters for each subject. Tables
1, 2 and 3 are a summary of the key words by chapters. Table 1. Percentage of Word Class Distribution among Key Words:
Science Text
Chapter
Nouns
Verbs
Adjectives
1
79
7
14
2
86
0
14
3
65
18
5
4
89
5
0
5
73
3
19
6
59
9
27
7
34
20
27
Total Average
69
9
15 Table 1. Percentage of Word Class Distribution among Key Words:
S i
T le 1. Percentage of Word Class Distribution among Key Words:
Science Text English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 98 English Department Faculty of Letters Petra Christian University
Table 2. Percentage of Word Class Distribution among Key Words:
Math Text
Chapter
Nouns
Verbs
Adjectives
1
17
0
33
2
41
6
29
3
43
14
29
4
75
25
0
5
64
18
5
6
71
0
14
7
43
21
29
8
80
7
0
9
35
4
48
10
70
20
5
11
81
10
0
12
84
5
11
Total Average
57
11
17
Table 3. Percentage of Word Class Distribution among Key Words:
English Language Text
Chapter
Nouns
Verbs
Adjectives
1
40
0
10
2
50
10
10
3
33
33
33
4
56
22
22
5
100
0
0
6
67
11
11
7
50
50
0
8
75
25
0
9
60
0
20
10
100
0
0
11
50
17
33
12
25
0
25
13
50
17
17
14
100
0
0
15
43
0
43
16
100
0
0
17
80
20
0
18
50
25
25
Total Average
63
12
14 Table 2. Percentage of Word Class Distribution among Key Words:
Math Text
Chapter
Nouns
Verbs
Adjectives
1
17
0
33
2
41
6
29
3
43
14
29
4
75
25
0
5
64
18
5
6
71
0
14
7
43
21
29
8
80
7
0
9
35
4
48
10
70
20
5
11
81
10
0
12
84
5
11
Total Average
57
11
17 Table 2. Percentage of Word Class Distribution among Key Words:
Math Text English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING
Table 3. Key Words by Chapters Percentage of Word Class Distribution among Key Words:
English Language Text
Chapter
Nouns
Verbs
Adjectives
1
40
0
10
2
50
10
10
3
33
33
33
4
56
22
22
5
100
0
0
6
67
11
11
7
50
50
0
8
75
25
0
9
60
0
20
10
100
0
0
11
50
17
33
12
25
0
25
13
50
17
17
14
100
0
0
15
43
0
43
16
100
0
0
17
80
20
0
18
50
25
25
Total Average
63
12
14 English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING
Table 3. Percentage of Word Class Distribution among Key Words:
English Language Text
Chapter
Nouns
Verbs
Adjectives
1
40
0
10
2
50
10
10
3
33
33
33
4
56
22
22
5
100
0
0
6
67
11
11
7
50
50
0
8
75
25
0
9
60
0
20
10
100
0
0
11
50
17
33
12
25
0
25
13
50
17
17
14
100
0
0
15
43
0
43
16
100
0
0
17
80
20
0
18
50
25
25
Total Average
63
12
14 Table 3. Percentage of Word Class Distribution among Key Words:
English Language Text English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING 99 Mukunda, Lexical Similarities and Differences in the Mathematics Overall, the three largest word class categories among the key words
by chapters for all three subjects were similar, that is, nouns. This was
followed by adjectives and then verbs. The Science text had the highest
average percentage of nouns as keywords (69%), followed by the English
language text (63%) and Math text (57%). The average percentage of
adjectives as key words in all the subjects was quite similar, ranging from
14% (English language) to 17% (Math). There was not much difference in
the percentage of verbs among the three subjects. The English language
text had the highest average percentage of verbs as key words (12%),
followed by Math (11%) and then Science (9%). What seems obvious is
that in all the three texts, the percentage of key words which were nouns far
outnumbered the percentage of key words from other word classes. Key Words by Chapters As the key words in this analysis were derived by using the entire text
as a reference corpus, a complete picture of the lexis specific to Math and
Science and which is different from general English language cannot be
obtained. For this purpose, a key word analysis of the Science and Math
word lists against the English language word list as reference corpus was
carried out. English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 100 Key Words in Math and Science This section of the discussion delves into the specialized vocabulary or
lexis of Math and Science. The three largest word class categories among
the key words found in the Math and Science texts are analyzed and
presented as percentages of word class distribution and according to
whether they were positive or negative key words, as seen in the table
below. Table 4. Percentage of Word Class Distribution among Key Words:
Science and Math against Reference Corpus
SCIENCE Positive NOUNS Negative Positive VERBS
Negative Positive ADJECTIVE Negative
55%
63%
8%
7%
13%
6%
9%
11%
2%
MATH
Positive NOUNS Negative Positive VERBS
Negative Positive ADJECTIVE Negative
37%
44%
7%
12%
24%
12%
8%
9%
1% Table 4. Percentage of Word Class Distribution among Key Words:
Science and Math against Reference Corpus
SCIENCE Positive NOUNS Negative Positive VERBS
Negative Positive ADJECTIVE Negative
55%
63%
8%
7%
13%
6%
9%
11%
2%
MATH
Positive NOUNS Negative Positive VERBS
Negative Positive ADJECTIVE Negative
37%
44%
7%
12%
24%
12%
8%
9%
1% Table 4. Percentage of Word Class Distribution among Key Words:
Science and Math against Reference Corpus For the purpose of discussion, only the positive key words were
looked into as these were the specialized words which occurred frequently
in the Math and Science texts in comparison to the general English
language text. Thus, these were words which could be considered
specialized language used in Math and Science texts. Major Word Classes Similar to the findings in the previous keyword analysis, the main
word class category in the Science and Math texts was nouns, 63%
(Science) and 44% (Math). Positively keyed nouns (55%-Science, 37%-
Math) occurred more than negatively keyed nouns. The next word class
categories were verbs, 13% (Science) and 24% (Math), followed by
adjectives (11%-Science, 9%-Math). Though there does not seem to be a
great difference in the occurrence of verbs and adjectives in the Science
text, there seems to be a large difference between the use of verbs and
adjectives in the Math text. There seems to be an equal or near equal percentage of positive and
negative key verbs in both of the texts. Looking at positive key verbs, there
is only 7% positively keyed verbs in the Science text in comparison to 12%
positive key verbs in the Math text. The percentage of positively keyed
adjectives (9%-Science, 8%-Math) nearly equals that of positively keyed
verbs. English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING Nouns The positive key nouns were further categorized into technical and
sub-technical vocabulary as categorized by Cowan (1974) and Nation
(2001). Tables 5 and 6 below list the nouns from the Math and Science lists
according to the four categories: highly technical, sub-technical, semi-
technical and non-technical. English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING
Table 5. Categorization of Positively Keyed Nouns in the Science List
SCIENCE TEXT
HIGHLY
TECHNICAL
SUB-
TECHNICAL
SEMI-
TECHNICAL
NON-
TECHNICAL
Bunsen burner
cell
apparatus
activity
cytoplasm
combustion
beaker
air
hydroxide
composition
bicarbonate
animal
litmus
fossil
carbon dioxide
ball
photosynthesis
nucleus
chemical
body
sodium
conical
candle
copper
colour
cylinder
conclusion Table 5. Categorization of Positively Keyed Nouns in the Science List
SCIENCE TEXT
HIGHLY
TECHNICAL
SUB-
TECHNICAL
SEMI-
TECHNICAL
NON-
TECHNICAL
Bunsen burner
cell
apparatus
activity
cytoplasm
combustion
beaker
air
hydroxide
composition
bicarbonate
animal
litmus
fossil
carbon dioxide
ball
photosynthesis
nucleus
chemical
body
sodium
conical
candle
copper
colour
cylinder
conclusion Table 5. VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111
104 104 As can be seen from tables 5 and 6 above, the majority of the
positively keyed nouns consisted of non-technical words or common
general English language terms (Science-58%, Math-52%), such as
‘water’, ‘air’, ’solution’, ‘value’, ‘figure’. The next largest sub-category
was the semi-technical words (Science-32%, Math-25%) or words which
are usually used both inside and outside the Science and Math fields but the
majority of its uses with a specific meaning are related to these two fields
(Nation, 2001). This specialized meaning in its particular field is readily
understood outside the field, such as words like ‘triangle’, ‘mass’,
‘percentage’, ‘oxygen’ and ‘density’. Only a small number of nouns were highly technical and sub-technical
words. This finding indicates that the Math and Science texts contained
more general English language and common nouns, which is appropriate
for this level of students. However, semi-technical nouns may need to be
given more attention to especially for L2 learners of low proficiency in
English language. Nouns Categorization of Positively Keyed Nouns in the Science List English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING 101 Mukunda, Lexical Similarities and Differences in the Mathematics E
li h D
t
t F
lt
f L tt
P t
Ch i ti
U i
it
SCIENCE TEXT
HIGHLY
TECHNICAL
SUB-
TECHNICAL
SEMI-
TECHNICAL
NON-
TECHNICAL
density
earth
experiment
elements
filter
energy
fuels
example
gas
figure
gauze
flame
kerosene
flask
laboratory
function
lever
glass
mass
heat
matter
hot
mercury
human
metal
ice
microorganisms
indicator
microscope
input
minerals
investigation
nitrogen
jar
organisms
length
oxygen
level
particles
life
pendulum
light
radian
lime
respiration
liquid
rod
material
rubber
measurement
science
method
space
metre
substance
mixture
sulphur
movement
thermometer
object
vapour
observation
variable
option
volume
outcomes
paper English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 102 SCIENCE TEXT
HIGHLY
TECHNICAL
SUB-
TECHNICAL
SEMI-
TECHNICAL
NON-
TECHNICAL
photo
physical
plant
procedure
process
properties
quantity
red
report
resources
salt
soil
solid
solution
sources
splinter
state
structure
sun
surface
system
temperature
test
things
tube
unit
vocabulary
water
weight SCIENCE TEXT
HIGHLY
TECHNICAL
SUB-
TECHNICAL
SEMI-
TECHNICAL
NON-
TECHNICAL
photo
physical
plant
procedure
process
properties
quantity
red
report
resources
salt
soil
solid
solution
sources
splinter
state
structure
sun
surface
system
temperature
test
things
tube
unit
vocabulary
water
weight English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING 103 Mukunda, Lexical Similarities and Differences in the Mathematics Table 6. Categorization of Positively Keyed Nouns in the Math List
MATH TEXT
HIGHLY
TECHNICAL
SUB-
TECHNICAL
SEMI-
TECHNICAL
NON-
TECHNICAL
cuboid
angle
addition
activity
decimal
factor
cube
aim
denominator
fraction
digit
application
integers
prime
division
area
numerator
estimate
cost
parallelogram
mass
difference
perimeter
multiplication
example
protractor
number
faces
simulator
percentage
figure
trapezium
rectangle
finding
vertices
square
height
subtraction
hour
sum
length
tonnes
line
triangle
minutes
volume
months
zero
page
plan
problem
procedure
process
question
sides
solution
system
temperature
term
terms
unit
unknown
value Table 6. Categorization of Positively Keyed Nouns in the Math List English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING Verbs The positive verb key words in both texts were examined and
categorized as lexicalized and delexicalised verbs as categorized by
Sinclair (1991). Lexicalised verbs are verbs which carry specific meaning
in relation to the field and delexicalised verbs are verbs which carry general
meaning that is equally common to both the Science and Math and non-
Science and non-Math language use. In short, lexicalized verbs are more
technical and specialized, while delexicalised verbs are non-technical
common English language verbs. Table 7 shows the categories according
to text type. The categories show that there is an equal number of lexicalized and
delexicalised verbs in both the Science and Math texts. Even though, the
verbs may be familiar to the students, the lexicalized verbs carry specific
meanings when used in their respective fields and thus, need to be given
more attention in class. A point to note is that there could be more
delexicalised verbs used in these texts, but as they are equally common in
general English language use, they are not found as key words because
their usage is similar in both the English language text and the Science and
Math texts. English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING 105 Mukunda, Lexical Similarities and Differences in the Mathematics Table 7. Delexicalised and Lexicalised Verbs by Text
SCIENCE
MATH
Lexicalised
Delexicalised
Lexicalised
Delexicalised
balance
contains
angled
copy
change
happen
calculate
determine
effect
is
carry
draw
float
learn
convert
find
flow
need
devise
follow
measure
show
divided
increasing
observe
understand
evaluate
involving
record
measure
is
multiply
mixed
perform
represent
shaded
try
simplify
understand
solve Table 7. Delexicalised and Lexicalised Verbs by Text Adjectives The adjectives were analyzed to find out if they were adjectives
derived from changing nouns, changing verbs, adding suffixes to nouns
and verbs, adding prefixes to verbs, or simple modifiers such as ‘hot’, ‘red’
(Quirk & Greenbaum, 1973, & Master, 1996). Table 8 lists the adjectives
types. yp
The adjectives used both in the Science and Math texts mostly consist
of modifiers such as ‘red’, ‘hot’, ‘multiple’, ‘obtuse’. The Science text had
more adjectives which were changed from nouns using suffixes such as
‘science+ic’,’wood+en’ and adjectives which were changed from verbs
using suffixes such as ‘renew+able’, ‘avail+able’. The difference in adjective use between the Math and Science texts is
that the adjectives used in the Science text is more complex ranging from
simple modifiers to adjectives with different word class base forms. The
Math text, on the other hand, is less complex involving basic modifiers
related to this specific field. However, as the modifiers are specifically
related to the Math field such as ‘obtuse angle’, ‘acute angle’, ‘parallel VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 106 line’, these words can also be considered sub-technical and semi-technical
words (Nation, 2001, & Cowan, 1974). In summary, the analysis of key words has clearly identified that both
Math and Science texts have more nouns as key words, with adjectives and
verbs as the next two word classes more commonly used, and that the
majority of the words used are semi-technical and non-technical words
which are appropriate for this level of students. Table 9 shows the specific
similarities and differences in language use between the Math, Science and
English language texts. Table 8. Adjective Types by Text
SCIENCE
MATH
Word
Adj Type
Word
Adj Type
renewable
suffix to verbs
total
modifiers
available
suffix to verbs
straight
modifiers
soluble
suffix to nouns
multiple
modifiers
scientific
suffix to nouns
adjacent
modifiers
wooden
suffix to nouns
common
modifiers
natural
suffix to nouns
parallel
modifiers
basic
suffix to nouns
parallel
modifiers
potential
modifiers
acute
modifiers
kinetic
modifiers
lowest
modifiers
various
modifiers
obtuse
modifiers
convestion
modifiers
unknown
prefix+verbs
blue
modifiers
improper
prefix+modifier
red
modifiers
algebraic
suffix to nouns
dull
modifiers
moist
modifiers
hot
modifiers
measuring
changing verb forms
living
changing verb forms Table 8. Adjective Types by Text Table 8. Adjective Types by Text Nouns Semi-Technical
and Non-
Technical
Semi-Technical
and Non-
Technical
Equal number of
lexicalized and
delexicalised
verbs
Equal number
of lexicalized
and
delexicalised
verbs
Modifiers
Equal number
f
difi Major
positively
keyed
adjectives Table 8. Adjective Types by Text English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING 107 Mukunda, Lexical Similarities and Differences in the Mathematics Table 9. Similarities and Differences in Language Use in Math,
Science and English Language Texts
ENGLISH
MATH
SCIENCE
Major key
word , word
class category,
by chapters
Nouns
Nouns
Nouns
Major key
word , word
class category,
against English
language list as
reference
corpus
Nouns
Nouns
Major
positively
keyed nouns
Semi-Technical
and Non-
Technical
Semi-Technical
and Non-
Technical
Major
positively
keyed verbs
Equal number of
lexicalized and
delexicalised
verbs
Equal number
of lexicalized
and
delexicalised
verbs
Major
positively
keyed
adjectives
Modifiers
Equal number
of modifiers
and adjectives
from noun and
verb changes
CONCLUSION AND PEDAGOGICAL IMPLICATIONS English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING Table 9. Similarities and Differences in Language Use in Math,
Science and English Language Texts Nouns
Nouns CONCLUSION AND PEDAGOGICAL IMPLICATIONS There is a need to determine the type of language the students are
required to have, whether it is general English or technical language, to be
able to cope with the everyday learning of Math and Science in English. This small corpus study on the language used in the Form 1 Math and
Science textbooks used in one zone in Malaysia has provided insights into VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 108 the type of language students are required to know and need to be able to
read and understand their textbooks. As textbooks are an integral part of
teaching and learning in schools in Malaysia, it is important for these
textbooks to be analyzed and assessed. This study which focuses on key words has shown that there is a
greater emphasis on the learning of nouns in the three texts, followed by
verbs and adjectives. The nouns used in the Math and Science texts were
mostly semi-technical and non-technical words, which are simple and
considered to be appropriate for this level of students. This implies that
there would not be much difficulty in learning new terms or words in the
Math and Science texts as most of the words should be quite familiar to
them. However, semi-technical words, such as ‘density’, ‘vapour’, ‘digit’
and ‘respiration’, could pose a problem to L2 learners who have a low
proficiency in English language; therefore, these words would have to be
given more attention in class. There should also be caution in handling
these words or nouns as individual words in Math and Science as many of
them appear more as multi-word units in the texts and thus become more
technical in appearance, for example, the words ‘line’ and ‘angle’ may be
familiar but when in collocate form such as ‘acute angle’ and ‘parallel line’,
these words then become sub-technical and semi-technical words which
then need to be taught explicitly and learnt intentionally (Nation, 2001). In the analysis of the verbs used in the Math and Science texts, it was
found that there were an equal number of lexicalized and delexicalised
verbs in both the Science and Math texts. Delexicalised verbs may not be a
problem for students as these are common general English language verbs. However, special attention should be given to lexicalized verbs as these
verbs carry specific meanings related to the Math and Science fields. English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING CONCLUSION AND PEDAGOGICAL IMPLICATIONS It is
only through small corpus studies such as this that differences in language
can be observed and it is these differences which have to be brought into
the classrooms and taught specifically. The analysis of the adjective key words provided gainful insights into
the language of Science and Math. The type of adjectives used in the
Science text was more complex than the type used in the Math text. Overall, there were more simple modifiers used in both these texts but the
Science text had many more derived adjectives. This implies that students
would have to understand lexical derivatives, as this seems to be one of the 109 Mukunda, Lexical Similarities and Differences in the Mathematics features of Scientific lexis (Thirumalai, 2003), to be able to produce them
correctly. In conclusion, this study reaffirms previous studies carried out on
learning and teaching vocabulary for Math and Science which emphasizes
the need for students to learn technical and sub-technical words and to
recognize the differences between words in general English and their
meanings in Math and Science (Khisty, 1995, Bernhardt, Hirsch, Teemant
& Rodriguez-Munoz, 1996). There is no doubt that there is a need for the
integration of language and Science and Math instruction for second
language learners as the language demands facing Science and Math
learners are very complex and different. It is through corpus studies like this that teachers and material writers
would be able to check and understand the differences in Scientific and
Mathematical English from the general English language and be able to
apply this knowledge to the teaching of Science and Math in English and
the creation of better Science and Math textbooks. Corpora studies have
allowed researchers, teachers and learners to use great amounts of real data
in their study of language, instead of having to rely on intuition and made-
up examples. This study proves that there is a need for small corpus studies
to be carried out, especially on language for specific purposes, as these
types of studies provide insights which would help in the production of
better learning materials and in the teaching and learning process. REFERENCES Ansary, H., & Babaii, E. (2003). A ‘Bell-Jar’ consensus-reached set of universal
characteristics of ELT textbooks. In J. Mukundan (Ed.), Readings on ELT
materials (pp. 69-83). Serdang: Universiti Putra Malaysia Press. Bernhardt, E., Hirsch, G., Teemant, A., & Rodriguez-Munoz, M. (1996). Language diversity and science: Science for limited English proficiency
students. Science Teacher, 63(2), 24-27. Bondi, M. (2001). Small corpora and language variation: reflexivity across genres. In M. Ghadessy, A. Henry, & R. L. Roseberry (Eds.), Small corpus studies
and ELT (pp. 135-174). Amsterdam/Philadelphia: John Benjamins
Publishing Co. Carlson, C. (2000). Scientific literacy for all: Helping English language learners
make sense of academic language. Science Teacher, 62(3), 48-52. English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111
110 VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 110 Cowan, J. R. (1974). Lexical and syntactic research for the design of EFL reading
materials. TESOL Quarterly, 8(4), 389-399. Flowerdew, L. (2003). A combined corpus and systemic-functional analysis of the
problem-solution pattern in a student and professional corpus of technical
writing. TESOL Quarterly, 37(3), 489-511. Henry, A., & Roseberry, R. L. (2001). Using a small corpus to obtain data for
teaching a genre. In M. Ghadessy, A. Henry, R. L. Roseberry (Eds.), Small
corpus studies and ELT (pp. 93-113). Amsterdam/Philadelphia: John
Benjamins Publishing Co. Holme, R. (2006). English for science or science for English: Designing content-
based materials for language teaching. In J. Mukundan (Ed.), Readings on
ELT materials (pp. 17-34). Serdang: Universiti Putra Malaysia Press. Khisty, L. L. (1995). Making inequality: Issues of language and meanings in
mathematic teaching with Hispanic students. In W.G. Secada, E. Fennema,
& L.B. Adajian (Eds.), New directions for equality in mathematics
education (pp. 279-297). New York, NY: Cambridge University Press. Laplante, B. (1997). Teaching science to language minority students in elementary
classrooms. NYSABE Journal, 12, 62-83. Master, P. (1996). Systems in English grammar: An introduction for language
teachers. New Jersey: Prentice Hall. Mukundan, J. (2004). A composite framework for ESL textbook evaluation. Unpublished doctoral dissertation. Universiti Putra Malaysia, Serdang. Nation, I. S. P. (2001). Learning vocabulary in another language. Cambridge:
Cambridge University Press. Nelson, M. (2000). A corpus based study of business English and business English
teaching materials. Unpublished doctoral dissertation. University of
Manchester, Manchester. Pandian, A., & Ramiah, R. (2004). Mathematics and science in English: Teacher
voice. The English Teacher, 33, 50-61. Quirk, R., & Greenbaum, S. (1973). A university grammar of English. London:
Longman. Schmitt, N. (2000). Vocabulary in language teaching. Cambridge: Cambridge
University Press. English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING 111 Mukunda, Lexical Similarities and Differences in the Mathematics Scott, M. (2001). Comparing corpora and identifying key words, collocations and
frequency distributions through the WordSmith Tools suite of computer
programs. In M. Ghadessy, A. Henry, & R. L. Roseberry, (Eds.), Small
corpus studies and ELT (pp. 47-67). Amsterdam/Philadelphia: John
Benjamins Publishing Co. Scott, M. (1996, 1997, 1999). WordSmith tools. Versions (1.0, 2.0, 3.0, 4.0). Oxford: Oxford University Press Sinclair, J. M. (1991). Corpus, concordance and collocation. Oxford: Oxford
University Press. Thirumalai, M. S. (2003). Language in science. Electronic Journal of Language in
Science, 3(1). Retrieved July 6, 2006, from http://www. VOLUME 9, NUMBER 2, DECEMBER 2007: 91-111 languageinin-
dia.com/jan2003/languageinscience.html on 6 July 2006 Thompson, D. R., & Rubenstein, R.N. (2000). Learning mathematics vocabulary:
Potential pitfalls and instructional strategies. Mathematic Teacher, 93(7),
568-573 Tognini-Bonelli, E. (2001). Corpus linguistics at work. Philadelphia: John
Benjamins Trimble, L. (1985). English for science and technology: A discourse approach. New York, NY: Cambridge University Press. English Department, Faculty of Letters, Petra Christian University
http://www.petra.ac.id/~puslit/journals/dir.php?DepartmentID=ING
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https://research-information.bris.ac.uk/files/283419999/elife_68829_v1.pdf
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Analysis of the PcrA-RNA polymerase complex reveals a helicase interaction motif and a role for PcrA/UvrD helicase in the suppression of R-loops
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bioRxiv (Cold Spring Harbor Laboratory)
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Urrutia-Irazabal, I., Ault, J. R., Sobott, F., Savery, N. J., & Dillingham,
M. S. (2021). Analysis of the PcrA-RNA polymerase complex reveals
a helicase interaction motif and a role for PcrA/UvrD helicase in the
suppression of R-loops. eLife, 10, Article e68829. https://doi.org/10.7554/eLife.68829 Publisher's PDF, also known as Version of record
License (if available):
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PDF-document This is the final published version of the article (version of record). It first appeared online via eLife Sciences
Publications at 10.7554/eLife.68829. Please refer to any applicable terms of use of the publisher. Urrutia-Irazabal, I., Ault, J. R., Sobott, F., Savery, N. J., & Dillingham,
M. S. (2021). Analysis of the PcrA-RNA polymerase complex reveals
a helicase interaction motif and a role for PcrA/UvrD helicase in the
suppression of R-loops. eLife, 10, Article e68829.
https://doi.org/10.7554/eLife.68829 Urrutia-Irazabal, I., Ault, J. R., Sobott, F., Savery, N. J., & Dillingham,
M. S. (2021). Analysis of the PcrA-RNA polymerase complex reveals
a helicase interaction motif and a role for PcrA/UvrD helicase in the
suppression of R-loops. eLife, 10, Article e68829. https://doi.org/10.7554/eLife.68829 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/ Analysis of the PcrA-RNA polymerase complex reveals a helicase
1
interaction motif and a role for PcrA/UvrD helicase in the
2
suppression of R-loops. 3
4
Urrutia-Irazabal, I.1, Ault, JR. 2, Sobott, F.2, Savery, N.J.1 and Dillingham, M.S.1*
5
6
1DNA:Protein Interactions Unit, School of Biochemistry, University of Bristol. Biomedical
7
Sciences Building, University Walk, Bristol BS8 1TD, UK. 8
2Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology,
9
University of Leeds, Leeds LS2 9JT, UK. 10
*To whom correspondence should be addressed: mark. dillingham@bristol.ac.uk
11
12 12 Introduction
28 Helicases are conserved proteins found in all kingdoms of life. They are involved in a wide
29
variety of DNA transactions including replication, repair, recombination and transcription. 30
Structural and bioinformatic analyses have classified helicases into several superfamilies (SF)
31
(Gorbalenya and Koonin, 1993; Singleton et al., 2007). Amongst the best-studied systems,
32
particularly from a mechanistic point of view, are the UvrD-like family of SF1 enzymes,
33
which include UvrD and its close cousin Rep from E. coli, as well as PcrA (a UvrD
34
orthologue) from B. subtilis and related organisms. Structural and biochemical analyses of the
35
helicases in this family revealed conserved mechanisms for ATP hydrolysis, DNA
36
translocation and DNA unwinding among the different members (Lee and Yang, 2006;
37
Velankar et al., 1999). 38 The PcrA/UvrD helicase has roles in rolling circle replication, nucleotide excision repair
39
(NER), mismatch repair (MMR) and homologous recombination (HR) (Bruand and Ehrlich,
40
2000; Husain et al., 1985; Lahue et al., 1989; Merrikh et al., 2015; Petit et al., 1998; Zieg et
41
al., 1978). This adaptability is provided by the many different interaction partners, for
42
example RepC/D, UvrB, MutL and RecA, which recruit the helicase to their respective
43
pathways via physical interactions (Hall et al., 1998; Machón et al., 2009; Manelyte et al.,
44
2009; Veaute et al., 2005). However, the structural basis for these interactions with different
45
partner proteins remains largely unclear. Most recently, an interaction between PcrA/UvrD
46
and RNAP has been identified, implying a role for PcrA in transcription, but its function is
47
not understood (Delumeau et al., 2011; Epshtein et al., 2014; Gwynn et al., 2013). Studies in
48
E. coli showed that UvrD can act as an accessory helicase which helps to minimise conflicts
49
between the replisome and replication fork barriers including transcription complexes
50
(Boubakri et al., 2010; Guy et al., 2009; Hawkins et al., 2019). The interaction was also
51
reported to facilitate an alternative pathway for transcription-coupled nucleotide excision
52
repair (TC-NER) in which UvrD removes RNAP from lesions by promoting backtracking
53
and then helps to recruit NER factors to repair the damage (Epshtein et al., 2014; Kamarthapu
54
and Nudler, 2015). Abstract
13 13 The PcrA/UvrD helicase binds directly to RNA polymerase (RNAP) but the structural basis
14
for this interaction and its functional significance have remained unclear. In this work we
15
used biochemical assays and hydrogen-deuterium exchange coupled to mass spectrometry to
16
study the PcrA-RNAP complex. We find that PcrA binds tightly to a transcription elongation
17
complex in a manner dependent on protein:protein interaction with the conserved PcrA C-
18
terminal Tudor domain. The helicase binds predominantly to two positions on the surface of
19
RNAP. The PcrA C-terminal domain engages a conserved region in a lineage-specific insert
20
within the β subunit which we identify as a helicase interaction motif present in many other
21
PcrA partner proteins, including the nucleotide excision repair factor UvrB. The catalytic
22
core of the helicase binds near the RNA and DNA exit channels and blocking PcrA activity in
23
vivo leads to the accumulation of R-loops. We propose a role for PcrA as an R-loop
24
suppression factor that helps to minimise conflicts between transcription and other processes
25
on DNA including replication. 26 27 Introduction
28 However, the existence of this new TC-NER pathway (separate from that
55
promoted by the canonical transcription-repair coupling factor Mfd) is debated, because high-
56
throughput sequencing of the oligonucleotides excised during NER showed no evidence for
57
strand-specific repair promoted by UvrD (Adebali et al., 2017a). In the Gram-(+)ve model
58
organism Bacillus subtilis, PcrA was found to be enriched at highly transcribed regions of the
59
genome and, in common with its orthologue UvrD, shown to alleviate replication-
60 transcription conflicts (Merrikh et al., 2015). Furthermore, genetic analyses of PcrA place this
61
helicase at the interface of replication, repair, transcription and chromosome segregation
62
(Moreno-del Alamo et al., 2020; Petit et al., 1998). However, the mechanistic details of how
63
PcrA/UvrD might help to promote the replication and/or repair of DNA on actively
64
transcribed DNA remain unclear. 65 transcription conflicts (Merrikh et al., 2015). Furthermore, genetic analyses of PcrA place this
61
helicase at the interface of replication, repair, transcription and chromosome segregation
62
(Moreno-del Alamo et al., 2020; Petit et al., 1998). However, the mechanistic details of how
63
PcrA/UvrD might help to promote the replication and/or repair of DNA on actively
64
transcribed DNA remain unclear. 65 One nucleic acid intermediate that impacts on each of these key DNA transactions is the R-
66
loop; a three-stranded structure in which a single RNA strand hybridises to a DNA duplex
67
displacing an equivalent region of ssDNA (Aguilera and García-Muse, 2012; Crossley et al.,
68
2019). R-loops can be formed when an RNA transcript re-hybridises to template DNA behind
69
RNA polymerase. Once formed, R-loops are relatively stable structures which require active
70
removal by enzymes such as RNases to prevent interference with other processes on DNA
71
and preserve genome integrity. Interestingly, recent publications have shown that UvrD, Rep,
72
Mfd and the single-stranded DNA binding protein SSB are all important for R-loop
73
homeostasis via unknown mechanisms (Wimberly et al., 2013; Wolak et al., 2020). 74 Although the function of PcrA/UvrD in transcription is still unclear, details are beginning to
75
emerge about the interface formed between the helicase and RNAP which may provide some
76
important clues. in vitro and that blocking PcrA activity in vivo, by either overexpressing a dominant negative
93
mutant or by preventing PcrA-RNAP association, results in increased R-loop levels. On the
94
basis of these observations we develop a model for PcrA as an R-loop surveillance helicase,
95
where it acts to unwind DNA:RNA hybrids and minimise the potentially toxic effects of
96
transcription on DNA replication and repair (Epshtein et al., 2014; Matson, 1989; Wimberly
97
et al., 2013).
98 Results
100 Interaction between PcrA/UvrD and RNAP is well-documented in the literature, but
102
little is known about which nucleic acid scaffolds or transcription states are the most
103
favourable for association. In E. coli, a complex between RNAP and UvrD was reported to be
104
stable enough to coelute during size-exclusion chromatography (SEC) (Epshtein et al., 2014). 105
In the B. subtilis system, although purified PcrA and RNAP do clearly interact in the absence
106
of nucleic acids, we found that a DNA/RNA scaffold which mimics an elongation complex
107
was required to form a sufficiently stable complex to resolve using SEC (Figure 1 – figure
108
supplement 1A-B). To rule out the possibility that the enhanced interaction was due simply
109
to the helicase binding to the DNA, the complex formation was analysed further by
110
electrophoretic mobility shift assays (EMSA) (Figure 1 – figure supplement 1C). PcrA
111
efficiently supershifted the transcription elongation complex (TEC; a pre-formed complex
112
between RNAP and a DNA/RNA scaffold) whereas it was barely able to bind to the scaffold
113
alone. Based upon EMSA, the apparent dissociation constant was ~500 nM and the
114
interaction was dependent on the C-terminal Tudor domain of PcrA (CTD; Figure 1A). 115
Removal of the CTD almost eliminated complex formation at concentrations of up to 1.5 µM
116
of PcrA. Moreover, the purified CTD alone supershifted the TEC efficiently, albeit to a lower
117
position in the gel. Although the UvrD CTD has been shown to possess a weak DNA binding
118
activity (Kawale and Burmann, 2020), in our hands the PcrA CTD does not detectably bind
119
to either single- or double-stranded DNA (Kd >> 5 µM) (Sanders et al., 2017). Therefore,
120
this result further supports the idea that the PcrA-TEC interaction (at least with this scaffold)
121
is substantially mediated by protein-protein interactions. Additional support for this assertion
122
was provided by EMSA supershift experiments in which the structure of the scaffold was
123
varied (Figure 1B). PcrA efficiently shifted the TEC regardless of the presence or length of
124
the upstream and downstream DNA in the scaffold. Results
100 PcrA binds tightly to a transcription elongation complex using its C-terminal Tudor
101
domain. Interaction between PcrA/UvrD and RNAP is well-documented in the literature, but
102
little is known about which nucleic acid scaffolds or transcription states are the most
103
favourable for association. In E. coli, a complex between RNAP and UvrD was reported to be
104
stable enough to coelute during size-exclusion chromatography (SEC) (Epshtein et al., 2014). 105
In the B. subtilis system, although purified PcrA and RNAP do clearly interact in the absence
106
of nucleic acids, we found that a DNA/RNA scaffold which mimics an elongation complex
107
was required to form a sufficiently stable complex to resolve using SEC (Figure 1 – figure
108
supplement 1A-B). To rule out the possibility that the enhanced interaction was due simply
109
to the helicase binding to the DNA, the complex formation was analysed further by
110
electrophoretic mobility shift assays (EMSA) (Figure 1 – figure supplement 1C). PcrA
111
efficiently supershifted the transcription elongation complex (TEC; a pre-formed complex
112
between RNAP and a DNA/RNA scaffold) whereas it was barely able to bind to the scaffold
113
alone. Based upon EMSA, the apparent dissociation constant was ~500 nM and the
114
interaction was dependent on the C-terminal Tudor domain of PcrA (CTD; Figure 1A). 115
Removal of the CTD almost eliminated complex formation at concentrations of up to 1.5 µM
116
of PcrA. Moreover, the purified CTD alone supershifted the TEC efficiently, albeit to a lower
117
position in the gel. Although the UvrD CTD has been shown to possess a weak DNA binding
118
activity (Kawale and Burmann, 2020), in our hands the PcrA CTD does not detectably bind
119
to either single- or double-stranded DNA (Kd >> 5 µM) (Sanders et al., 2017). Therefore,
120
this result further supports the idea that the PcrA-TEC interaction (at least with this scaffold)
121
is substantially mediated by protein-protein interactions. Additional support for this assertion
122
was provided by EMSA supershift experiments in which the structure of the scaffold was
123
varied (Figure 1B). PcrA efficiently shifted the TEC regardless of the presence or length of
124
the upstream and downstream DNA in the scaffold. 125 PcrA binds tightly to a transcription elongation complex using its C-terminal Tudor
101
domain. Introduction
28 Crosslinking experiments revealed contacts between the 1B and 2B
77
subdomains of the helicase and the β flap tip of the RNAP β subunit, as well as the N-
78
terminal region of the RNAP β′ subunit (Epshtein et al., 2014). The extreme C-terminal
79
domain (CTD) of PcrA, which is disordered or was removed to aid crystallisation in the
80
available structures of this helicase, also plays a critical role in binding RNAP but its binding
81
site on RNAP is unknown (Gwynn et al., 2013). A structure of the isolated CTD shows that it
82
adopts a Tudor fold, which is connected to the main body of the helicase via a long,
83
apparently disordered, linker (Sanders et al., 2017). This is intriguing, because the canonical
84
TC-NER factor Mfd also employs a Tudor domain for interaction with RNAP (Deaconescu et
85
al., 2006). 86 In this work, we find that PcrA interacts tightly with a transcription elongation complex in
87
vitro in a manner dependent on the CTD. We show that (unlike Mfd) the CTD binds to a
88
lineage-specific insertion domain within the β subunit of RNAP, while the main body of the
89
helicase surrounds the RNA and DNA exit channels. The CTD engages with an exposed
90
beta-hairpin which defines a novel helicase interaction motif also found in other PcrA
91
partners including UvrB. We further show that PcrA efficiently unwinds DNA:RNA hybrids
92 in vitro and that blocking PcrA activity in vivo, by either overexpressing a dominant negative
93
mutant or by preventing PcrA-RNAP association, results in increased R-loop levels. On the
94
basis of these observations we develop a model for PcrA as an R-loop surveillance helicase,
95
where it acts to unwind DNA:RNA hybrids and minimise the potentially toxic effects of
96
transcription on DNA replication and repair (Epshtein et al., 2014; Matson, 1989; Wimberly
97
et al., 2013). 98 99 Results
100 125
Competition experiments showed that full length PcrA and free CTD competed for TEC
126 Competition experiments showed that full length PcrA and free CTD competed for TEC
126
association, and there was no evidence for a ternary complex formed between PcrA, the free
127
CTD and the TEC (Figure 1 – figure supplement 1D). The addition of unlabelled CTD
128
caused a decrease in the levels of fluorescently labelled PcrA bound to the TEC rather than a
129
further supershift. This strongly suggests that full length PcrA and the CTD bind to the same
130
(unknown) surface of RNAP and is consistent with the idea that the TEC recruits a single
131 PcrA molecule. Note that the ability of the free CTD to outcompete full length PcrA for
132
RNAP association is relevant for in vivo experiments presented below. 133
134
The C-terminal domain of PcrA interacts with the lineage-specific insertion domain 1
135
(SI1) of the β subunit of RNAP. Crosslinking of UvrD to RNAP has shown that the core
136
helicase interacts with the RNAP β-flap tip and the N-terminal region of the β′ subunit in
137
bacteria, but no information about the CTD-interaction domain was obtained (Epshtein et al.,
138
2014). Therefore, we pursued a complementary approach in the B. subtilis system, using
139
hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to study the
140
complex formed between the free CTD and RNAP. This technique allows one to measure the
141
rate of exchange of amide hydrogen atoms with deuterium atoms in the solvent. This rate is
142
affected by hydrogen bonding, surface accessibility, protein dynamics and changes in the
143
presence and absence of partner proteins and can therefore be used to probe potential binding
144
interfaces or conformational changes. 145 PcrA molecule. Note that the ability of the free CTD to outcompete full length PcrA for
132
RNAP association is relevant for in vivo experiments presented below. 133 The C-terminal domain of PcrA interacts with the lineage-specific insertion domain 1
135
(SI1) of the β subunit of RNAP. Crosslinking of UvrD to RNAP has shown that the core
136
helicase interacts with the RNAP β-flap tip and the N-terminal region of the β′ subunit in
137
bacteria, but no information about the CTD-interaction domain was obtained (Epshtein et al.,
138
2014). Therefore, we pursued a complementary approach in the B. Results
100 Comparison of the HDX at
164
different timepoints showed that protection of this region increased with time after exposure
165
to D2O, in agreement with a protein-protein interaction site. Fortunately, this region of
166
interest was well covered by the LC-MS/MS analysis with overlapping peptides, and the
167
smallest part of the polypeptide protected was the seven-residue sequence PETGEIL
168
(Figures 2A and 2B). Intriguingly, despite the ubiquity of UvrD/PcrA orthologues in the
169
bacterial domain and the strong conservation of the CTD sequence, the protected region in
170
RNAP belongs to a lineage-specific insertion domain (SI1, also referred to as βi5 (Lane and
171
Darst, 2010)) which differs significantly between organisms in terms of the length, sequence
172
and precise location within the primary structure. Nevertheless, a nine-residue sequence motif
173
(including the seven amino acids of interest) is present in most bacterial RpoB sequences
174
despite variation in its exact position. As an example, primary structure diagrams for the B. 175
subtilis and E. coli RpoB subunits are shown in Figure 2B with complete aligned sequences
176
for the β2 and SI1 regions shown in Figure 2 – figure supplement 2. Despite being quite
177
divergent compared to better-conserved parts of RpoB, the two SI1 domains contain a very
178
similar peptide motif (VDPETGEIL and IDESTGELI respectively). Although the motifs are
179
located in different parts of the SI1 domain primary structure they occupy a broadly similar
180
space within the global architecture of RNAP (Figure 2C). In E. coli the motif adopts a beta
181
hairpin structure, whereas this region is locally disordered in the B. subtilis TEC cryoEM
182
structure (Newing et al., 2020; Pei et al., 2020). The variability in the SI1 domain would
183
make it extremely challenging to appreciate the conservation of this motif across different
184
species from primary sequence alone. 185 Alignment of the beta-hairpin motif from B. subtilis-like SI1 domains demonstrated its high
186
conservation (shown in Weblogo format in Figure 2C). The consensus sequence obtained
187
from E. coli-like SI1 domains is very similar, consistent with the idea that they are in fact the
188
same motif (Figure 2C). The most conserved and striking feature of this putative helicase
189
interaction motif is a TGE triad which occupies the tip of a loop between two beta strands. 190
Indeed, the glutamic acid (E310 in B. Results
100 subtilis system, using
139
hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to study the
140
complex formed between the free CTD and RNAP. This technique allows one to measure the
141
rate of exchange of amide hydrogen atoms with deuterium atoms in the solvent. This rate is
142
affected by hydrogen bonding, surface accessibility, protein dynamics and changes in the
143
presence and absence of partner proteins and can therefore be used to probe potential binding
144
interfaces or conformational changes. 145 We obtained good sequence coverage and redundancy for both the CTD and the major
146
subunits of RNAP (Table 1). The experiment was first validated by analysing the protection
147
pattern on the CTD. As expected, data for the CTD in the CTD-RNAP complex showed
148
significant protection compared to the CTD alone (Figure 1C). To visualise the results, a
149
homology model of the Tudor domain of B. subtilis PcrA was created from the structure of
150
the closely related G. stearothermophilus protein (Sanders et al., 2017) (Figure 1D). The
151
areas of protection, which indicate the binding site location, overlap with residues that have
152
been shown to be important for the interaction between PcrA and RNAP through site-directed
153
mutagenesis studies (Sanders et al., 2017). Moreover, it is mostly localised on the face of the
154
Tudor domain that is typically expected to interact with partner proteins (Liu et al., 2015;
155
Westblade et al., 2010). This agreement with previous studies provided confidence that the
156
HDX-MS data was reporting correctly on the interface formed between PcrA and RNAP. 157 We next searched for protected peptides in the different RNAP subunits. It should be noted
158
that, given the large size of RNAP (374 kDa) compared to the tiny Tudor domain (9.3 kDa),
159
we did not expect to observe any protection for the large majority of the RNAP sequence if
160
the stoichiometry of interaction is 1:1. In accordance with this expectation, only a small
161
region of the β subunit of RNAP at approximately amino-acid position 300 showed
162
significant protection (Figure 2A, with HDX protection plots for the rest of RNAP β and the
163 other RNAP subunits in Figure 2 – figure supplement 1). Results
100 subtilis RpoB) is absolutely conserved across all
191
aligned RpoB sequences. To experimentally validate the motif, we purified the β subunit of
192
B. subtilis RNAP with and without substitutions at the E310 position. In vitro assays showed
193
that substitution of glutamic acid with either alanine or lysine at position 310 severely
194
disrupts pulldown of purified RpoB by PcrA (Figure 2D), confirming the importance of the
195
SI1 motif for the interaction. Lack of interaction with the E308 mutants was not due to global
196 mis-folding because both mutant proteins had equivalent CD spectra to wild type (Figure 2 –
197
figure supplement 3). 198 mis-folding because both mutant proteins had equivalent CD spectra to wild type (Figure 2 –
197
figure supplement 3). 198 The helicase interaction motif is found in several other PcrA interaction partners
199
including UvrB. In common with other SF1 helicases, PcrA/UvrD interacts with many
200
different partner proteins explaining its recruitment to different DNA transactions and the
201
resulting multi-functionality of this helicase (Gilhooly et al., 2013). To investigate whether
202
the helicase interaction motif we had identified in RpoB played a wider role in mediating
203
PcrA interactions we searched for it within the Bacillus subtilis proteome using PROSITE
204
with the query [VIL]-D-X-X-T-G-E-[VIL]-[VILT], where X is any amino acid and the
205
square brackets indicate ambiguity. This search retrieved seven proteins (only ~0.3 are
206
expected by chance), four of which (RpoB, UvrB, YxaL and YwhK) are known to interact
207
with PcrA (Figure 3A). Importantly, the structure of UvrB and high-confidence homology
208
models of YxaL and YwhK all show that, as is the case for the beta subunit of E. coli RNAP,
209
the conserved TGE triad is located at the tip of a surface loop flanked by β sheets (Figure
210
3B). There is currently no evidence for interaction between PcrA and the other three proteins
211
(QueA, RplX and YtzB) which may now be considered as putative PcrA interactors. When
212
this same motif search was applied more broadly across the entire SwissProt database it
213
returned only 607 sequences, of which 326 and 42 were bacterial RpoB and UvrB sequences
214
respectively, demonstrating its very high specificity for returning PcrA interactors. Results
100 215 YxaL and YwhK are β propeller proteins that have been identified as PcrA interactors using
216
yeast two-hybrid and biochemical assays (Noirot-Gros et al., 2002). The fragment of YxaL
217
found to interact with PcrA in the yeast two-hybrid assay is consistent with the location of the
218
newly identified motif. These two proteins are found in a very limited subset of bacterial
219
proteomes and their cellular function is obscure. Therefore, we next decided to focus on the
220
interaction of PcrA with UvrB; a ubiquitous Superfamily 2 helicase that is a key component
221
of the nucleotide excision repair apparatus (Kisker et al., 2015). Importantly, UvrB has
222
already been shown to bind to the C-terminal domain of E. coli UvrD (Manelyte et al., 2009)
223
and this interaction is, therefore, potentially analogous to that formed between the CTD and
224
RNAP β. To validate the predicted PcrA binding site on UvrB, we performed pulldown
225
experiments using his-tagged UvrB as bait and myc-tagged PcrA in B. subtilis extracts as
226
prey (Figure 3C). These experiments confirmed the expected interaction between PcrA and
227
wild type UvrB protein. Furthermore, and in agreement with our hypothesis, substitution of
228
the glutamic acid in the UvrB TGE motif (E233) severely disrupted the interaction with PcrA
229 while having no effect on the DNA-stimulated ATPase activity of UvrB (Figure 3 – figure
230
supplement 1). 231 while having no effect on the DNA-stimulated ATPase activity of UvrB (Figure 3 – figure
230
supplement 1). 231 Inspection of the domain architecture of UvrB reveals that the PcrA interaction motif is
232
located within domain 2, which also interacts with UvrA (Pakotiprapha et al., 2012; Truglio
233
et al., 2004) (Figure 3 – figure supplement 2A). However, because amino acid substitutions
234
reported to disrupt UvrA-UvrB interaction are not within the motif itself, binding of UvrA to
235
UvrB would not necessarily preclude binding of PcrA/UvrD. Interestingly, a high scoring
236
docking model for the interaction between the PcrA-CTD and UvrB using HADDOCK (Van
237
Zundert et al., 2016) suggests that the complex is stabilised by an electrostatic interaction
238
between UvrB E233 and the PcrA K727 residue, which is known to be critical for interaction
239
between PcrA and RNAP (Figure 3 – figure supplement 2B). Results
100 This led us to consider the possibility that the N-terminal
268
region of PcrA, including the helicase core, also contributes significantly to the interaction
269
with RNA polymerase. This view is consistent with crosslinking studies of UvrD-RNAP
270
which implicated the 1B and 2B subdomains of UvrD in interactions with the RNAP β-flap
271
tip and the N-terminal region of the β′ subunit (Epshtein et al., 2014). Note that these
272
structural features surround the RNA and DNA exit channels. 273 We did not pursue HDX experiments between PcrA-ΔCTD and RNAP because our previous
274
work had shown that this interaction was barely detected by pulldown/proteomics
275
experiments in vitro (Gwynn et al., 2013; Sanders et al., 2017) even with the favoured TEC
276
substrate (Kd > 1.5 µM; Figure 1A). Instead, to compare with data for the CTD-RNAP
277
complex, we studied interaction between full length PcrA and RNAP, obtaining good
278
coverage and redundancy values for the various constituent polypeptides (Table 1). PcrA
279
showed HDX protection in two main regions upon interaction with RNAP: the 2A domain
280
and the CTD (blue and magenta highlights respectively; Figure 4A and Figure 4 – figure
281
supplement 1A). Note that peptides from the long flexible linker between these two regions
282
were not detected above the confidence threshold and so we have no data for that part of the
283
protein. The protection pattern was visualised on a homology model of the active DNA
284
bound state of PcrA (Figure 4B) (Lee and Yang, 2006; Ordabayev et al., 2019; Velankar et
285
al., 1999), in which the CTD is disordered. Most of the protected regions outside of the CTD
286
prominently cluster in the 2A domain and on the side of the helicase which also binds to
287
DNA. The crosslinking positions identified in domains 1B and 2B in previous work are on
288
the same DNA-facing side of the helicase and are therefore broadly consistent with these data
289
(Epshtein et al., 2014). 290 The HDX protection afforded to the RNAP subunits in the presence of full length PcrA was
291
more extensive than in the presence of the CTD, and identified important regions in the two
292
largest subunits, β and β′ (butterfly plots for the rest of the subunits are shown in Figure 5 –
293
figure supplement 1). Results
100 The docked complex shows
240
how PcrA might be recruited to sites of damage after the damaged oligonucleotide has been
241
nicked, as DNA near the docking site is accessible. A conservation logo for the PcrA
242
interaction site on UvrB was created from reference proteomes (Figure 3 – figure
243
supplement 2C). The consensus sequence is highly similar to that obtained for the RNAP
244
interaction although, interestingly, the glutamate residue shows somewhat greater variability. 245 Our results predict that the interaction between the PcrA C-terminal Tudor domain and UvrB
246
is equivalent in molecular terms to its interaction with RNA polymerase. A corollary is that
247
UvrB and RNA polymerase might be engaged in a simple competition for the Tudor domain
248
of PcrA. To test this, we performed pulldown of RNA polymerase using PcrA as a bait and
249
titrated the cell extract (i.e. the prey proteins) with increasing concentrations of purified
250
recombinant UvrB (Figure 3D-E). As expected, introduction of UvrB significantly reduced
251
the amount of RNA polymerase that was recovered from the cell extract. As has been noted
252
previously, other RNAP interactors also bind to the RpoB subunit via Tudor domains,
253
including the transcription-repair coupling factor Mfd. Interestingly, using the same
254
pulldown assay, titration of free Mfd did not diminish the yield of RNAP (Figure 3D-E),
255
despite biotinylated Mfd pulling down RNAP subunits under similar conditions (Table 2). 256
This suggests that, although both proteins contain Tudor domains for interaction with RNAP,
257
PcrA and Mfd bind at different physical locations and without competing. This is in
258
agreement with recently-published observations for the E. coli system (Kawale and Burmann,
259
2020), as well as the lack of protection of the expected Mfd-interacting region of RpoB (the
260
β1 domain) that we observe in our HDX experiments (Figures 2A and S2B) (Smith and
261
Savery, 2005; Westblade et al., 2010). 262 263 The helicase core of RNAP interacts with RNAP near the RNA and DNA exit channels. 264
Despite the apparently critical role played by the CTD in RNAP interactions, previous work
265
in live E. coli and B. subtilis cells has found at most mild phenotypes associated with deletion
266
of the C-terminal domain of UvrD and PcrA, respectively (Manelyte et al., 2009; Merrikh et
267
al., 2015; Sanders et al., 2017). Results
100 In agreement with our data for the CTD-RNAP interaction, the SI1
294 region showed protection that we therefore assign to the interaction with the PcrA Tudor
295
domain (Figure 5A, green shadow and Figure 6B). The other protected region within the β
296
subunit corresponds to the β flap tip, as identified by crosslinking in the E. coli system
297
(Epshtein et al., 2014)( Figure 4 – figure supplement 1B). The β′ subunit also showed two
298
protected clusters in the N-terminal and central regions (Figure 5B and Figure 4 – figure
299
supplement 1C). The recent structure of the B. subtilis transcription elongation complex
300
allowed us to visualise the results (Newing et al., 2020) (Figure 5C) and reveals that
301
protected regions of RNAP surround the RNA and DNA exit channels. Unfortunately, the
302
zinc finger domain of the β′ subunit was not covered by our dataset but, given its close
303
proximity to the protected regions we have observed, as well as the crosslinking analysis of
304
the E. coli RNAP-UvrD complex (Epshtein et al., 2014), it is likely to be involved in the
305
interaction too. Taken together, our data suggested to us that the PcrA helicase might be
306
acting close to the RNA and DNA exit channels and are consistent with our earlier
307
conclusion that Mfd and PcrA bind to different parts of RNAP. 308 PcrA is able to unwind DNA-RNA hybrids by translocating on ssDNA. It is well
309
established that PcrA/UvrD is a 3′>5′ ssDNA translocase and helicase that binds
310
preferentially to 3′-ssDNA overhangs to initiate unwinding of flanking DNA duplexes. 311
However, UvrD has also been shown to unwind DNA-RNA hybrids efficiently (Matson,
312
1989) although we are not aware of a cellular function having been ascribed to this activity. 313
Moreover, no data is available for the enzyme’s activity on RNA duplexes. Therefore, given
314
the apparent recruitment of PcrA close to the RNA and DNA exit channels of RNAP, we next
315
investigated the relative helicase activity displayed by PcrA against all possible combinations
316
of a 3′-tailed duplex that can be formed by DNA and RNA oligonucleotides (Figure 6A). 317
PcrA was able to efficiently unwind the two substrates in which the 3′-ssDNA tail was
318
formed from DNA, including an all-DNA duplex and a DNA-RNA hybrid (Figure 6B). Results
100 In
319
contrast, the enzyme was unable to unwind either substrate containing a 3′-ssRNA tail (either
320
an RNA-DNA hybrid or an RNA duplex), even at elevated concentrations. Unwinding of
321
both the DNA duplex and the RNA-DNA hybrid was absolutely dependent on the presence of
322
the 3′-ssDNA tail (Figure 6 – figure supplement 1A-B), implying that it is initiated by
323
enzymes binding to ssDNA and moving in the 3′>5′ direction. This idea was further
324
corroborated by investigating the nucleic acid-dependence of the ATPase activity (Figure
325
6C). In the absence of DNA or RNA, the basal ATPase activity of PcrA was low (~1 s-1). 326
However, it was stimulated approximately 100-fold in the presence of poly(dT) single-
327 stranded DNA. In contrast, poly(U) single-stranded RNA had no effect on the observed
328
ATPase activity. Similar results were obtained with short DNA and RNA oligonucleotides
329
containing mixed base sequences (data not shown). These experiments demonstrate that PcrA
330
helicase can efficiently unwind DNA-RNA hybrids but can only do so by ATP-dependent
331
translocation on the DNA strand. Together with the HDX experiments localising PcrA to the
332
RNA and DNA exit channels, we hypothesised that PcrA might act to unwind R-loops
333
formed during transcription. 334 stranded DNA. In contrast, poly(U) single-stranded RNA had no effect on the observed
328
ATPase activity. Similar results were obtained with short DNA and RNA oligonucleotides
329
containing mixed base sequences (data not shown). These experiments demonstrate that PcrA
330
helicase can efficiently unwind DNA-RNA hybrids but can only do so by ATP-dependent
331
translocation on the DNA strand. Together with the HDX experiments localising PcrA to the
332
RNA and DNA exit channels, we hypothesised that PcrA might act to unwind R-loops
333
formed during transcription. 334 PcrA helicase suppresses R-loop levels in vivo. Both PcrA and UvrD have been shown to
335
interact with RNAP and to alleviate replication-transcription conflicts (in B. subtilis and E. 336
coli respectively) (Guy et al., 2009; Hawkins et al., 2019; Merrikh et al., 2015). However, the
337
precise role for this interaction remains unknown and study of the system is frustrated by the
338
essentiality of both PcrA and RNA polymerase, making phenotypic analysis more complex. Results
100 363 whether removal of mfd (that has been shown to counteract PcrA essentiality (Moreno-del
361
Alamo et al., 2020)) could relieve the mutant’s toxicity but found that the deletion had no
362
substantial effects on the growth defect (Figure 6 – figure supplement 2B). 363 To test our hypothesis that PcrA unwinds R-loops, we next measured DNA-RNA hybrid
364
levels in these PcrA overexpression strains using the S9.6 antibody (Boguslawski et al.,
365
1986). In this experiment, genomic DNA was purified, spotted onto a dot blot membrane and
366
probed for both DNA-RNA hybrids (S9.6) and total DNA (methylene blue staining) (Figure
367
6D). The relative hybrid to DNA level was then normalised to a control experiment in which
368
there was no gene in the ectopic expression cassette. Wild type PcrA overexpression did not
369
alter relative R-loop levels, whereas overexpression of the E224Q mutant led to a significant
370
increase (~2.7 fold). This increase is comparable to the effect of deleting known R-loop
371
suppression factors from bacterial cells such as rnhC or inhibiting rho using BCM in E. coli
372
(Raghunathan et al., 2018) (see also Figure 6G). 373 With the aim of testing whether the interaction of PcrA with RNAP is important for R-loop
374
regulation, we next overexpressed the CTD of WT PcrA or a CTD mutant (K727A) that does
375
not interact with RNAP (Sanders et al., 2017). Based upon in vitro TEC supershifts (Figure 1
376
– figure supplement 1D) and ex vivo pulldown assays (Figure 6F and Figure 6 – figure
377
supplement 3A), we knew that the free CTD inhibited the interaction between full length
378
PcrA and RNAP. Therefore, we reasoned that overexpression of the CTD in cells should
379
block endogenous wild type PcrA from being recruited by RNAP. The relative R-loop levels
380
in strains overexpressing the WT or mutant CTD were measured by dot-blot as before
381
(Figure 6G and Figure 6 – figure supplement 3B-C). Overexpression of the WT CTD
382
caused a significant increase in R-loop levels compared to WT cells or the empty expression
383
cassette control. This increase approached the levels associated with an rnhC deletion strain. 384
However, replacing the wild type CTD with a CTD mutant that fails to interact with RNAP
385
reduced R-loop levels to a value not significantly different to the strain with an empty
386
expression cassette. Results
100 339
To overcome this difficulty, we first exploited a dominant negative mutant of PcrA/UvrD in
340
which a glutamate in the Walker B motif is substituted to allow DNA and ATP binding, but
341
not hydrolysis and concomitant DNA translocation (Brosh and Matson, 1995). Studies in
342
many Additional Strand Catalytic E (ASCE)-class NTPases identify this residue as the
343
catalytic base which accepts a proton from water, thereby promoting nucleophilic attack at
344
the γ-phosphate and enabling ATP hydrolysis (Thomsen and Berger, 2008). Substitution of
345
this glutamate residue with glutamine prevents ATP hydrolysis, trapping the enzyme in an
346
ATP-bound or transition-like state (Hirano and Hirano, 2004; Soultanas et al., 1999). 347 Induction of PcrA-E224Q from an ectopic locus in otherwise wild type cells caused severe
348
growth defects which became most apparent as the cells entered exponential phase (Figure 6
349
– figure supplement 2A). This defect was not apparent when wild type PcrA was expressed
350
from the same locus and is therefore due to the E224Q mutation as opposed to the effects of
351
overexpression per se or the elevated cellular PcrA concentration that results. Interestingly,
352
delaying the induction of PcrA-E224Q until the onset of exponential phase (see Methods and
353
Figure 6 – figure supplement 2A for details) resulted in a much smaller effect, presumably
354
because a lower concentration of mutant PcrA had accumulated in early exponential phase. 355
This may suggest that the toxic effect of dysfunctional PcrA is mainly felt during rapid
356
growth. Removal of the CTD from the dominant negative mutant did not relieve the toxicity
357
we observed. This could indicate that PcrA-E224Q lacking the CTD can still interact weakly
358
with RNAP (as we have discussed above) or that the toxic effect we observe arises from
359
another role of this multifunctional enzyme that does not require the CTD. We also tested
360 whether removal of mfd (that has been shown to counteract PcrA essentiality (Moreno-del
361
Alamo et al., 2020)) could relieve the mutant’s toxicity but found that the deletion had no
362
substantial effects on the growth defect (Figure 6 – figure supplement 2B). Discussion The bacterial cell displays a remarkable ability to maintain genomic stability during rapid
395
growth when many different DNA and RNA transactions occur simultaneously on the same
396
chromosome. The UvrD-like family of helicases play a critical role at the interface of such
397
pathways, orchestrating DNA repair as damage arises and helping to minimise conflicts that
398
would otherwise occur between DNA replication and transcription (Dillingham, 2011). 399
Several reports have identified physical or functional interactions between PcrA/UvrD and
400
RNA polymerase which could underpin its emerging role in managing conflicts between
401
transcription and replication or repair processes. 402 We showed here that PcrA binds tightly to a pre-formed TEC in vitro to form a complex that
403
is stable during gel filtration, in contrast to complexes formed between free PcrA and RNAP. 404
The complex was unaffected by removal of the upstream or downstream DNA duplex from
405
the TEC, was still formed between the CTD (which lacks the helicase domains) and the TEC,
406
and was not formed when a truncated form of PcrA lacking the CTD was used. These
407
observations suggest that the increased stability associated with the PcrA:TEC complex is not
408
simply due to the helicase being able to engage with DNA/RNA that flanks the polymerase. 409
Instead, the RNA polymerase might adopt a conformation in the TEC that favours the
410
recruitment of PcrA via the CTD. 411 HDX-MS analysis confirmed that RNAP interacted with a face of the PcrA Tudor fold that is
412
frequently observed to form protein interactions in other systems (Musselman et al., 2012;
413
Ruthenburg et al., 2007). The site for this PcrA-CTD interaction on the RNA polymerase was
414
identified as a short peptide motif in the β subunit that sits within the SI1 domain. This is the
415
first protein-protein interaction reported for the enigmatic SI1 domain, which is a lineage-
416
specific insert in the β subunit whose function remains mysterious (Newing et al., 2020). This
417
interaction site is distinct from that bound by the TC-NER factor Mfd, which also engages
418
with the beta subunit via a Tudor domain but at the β1 domain. In agreement with this and
419
other data (Kawale and Burmann, 2020), we also found that PcrA and Mfd did not compete
420
for RNAP in a physical interaction assay. Results
100 This supports the idea that the activity of PcrA suppresses R-loop
387
formation and that this effect is dependent upon the ability of the CTD Tudor domain to
388
interact with RNAP and/or other partner proteins. Finally, to determine whether this function
389
of PcrA is conserved in other bacteria, we compared R-loop levels in WT and ΔuvrD E. coli
390
strains (where deletion of uvrD is not lethal). We observed that loss of UvrD caused a
391
significant increase in R-loops levels in the cell (Figure 6 – figure supplement 3D-E). 392 393 Discussion 421 The short peptide motif to which PcrA binds is highly conserved within Bacillus-like SI1
422
domains but does not obviously align with β subunit sequences from more distantly-related
423
organisms. Indeed, because PcrA/UvrD is ubiquitous and its CTD so highly-conserved, we
424
were initially surprised to find that the interaction with RNAP mapped to a highly variable
425 domain. This paradox was resolved using bioinformatics, which revealed that the same motif
426
is present widely in bacterial RNAPs, but that its positioning within the SI1 domain is
427
different depending on the lineage. Relative to other more universal features in RpoB
428
sequences, the interaction motif appears later in B. subtilis-like domains (which are
429
representative of Firmicutes) than it does in E. coli-like domains (which are representative of
430
Proteobacteria; see Figure 2 – figure supplement 2). Note however that we did find
431
examples of bacterial RpoB species for which we could find no clear helicase-interaction
432
motif present in the SI1 domain. Our sequence searches proved rewarding as they revealed
433
the presence of the same motif in other known PcrA-interacting proteins, including UvrB,
434
YxaL and YwhK. Moreover, this helicase interaction motif is also present in several poorly-
435
characterised proteins which might be previously unidentified interaction partners for PcrA. 436
Where a structure is available, the motif always adopts a beta-hairpin with the highly
437
conserved TGE triad protruding towards the solvent. The corollary of our observation that
438
RNAP and UvrB employ the same structural element to recruit PcrA is that they might
439
compete in the cell for the PcrA/UvrD helicase. This idea is supported directly by
440
competition pulldown assays, and is consistent with previous work in which the PcrA/UvrD
441
CTD was shown to be important for interaction with both RNAP and UvrB (Manelyte et al.,
442
2009; Sanders et al., 2017). However, the helicase interaction motif was absent from some
443
known PcrA partners including the mismatch repair factor MutL. This is in agreement with
444
recent work which showed that MutL binds and activates PcrA/UvrD via the 2B domain,
445
rather than the CTD (Ordabayev et al., 2018, 2019). 446 PcrA/UvrD, along with ppGpp, has been suggested to participate in an Mfd-independent
447
transcription-coupled DNA repair pathway (Epshtein et al., 2014; Pani and Nudler, 2017). Discussion Our HDX-MS
461
data also identified an interaction with the SI1 domain which we can assign to the PcrA CTD. 462
The exit channels for nucleic acids are distant from the SI1 domain, but the long linker
463
between the helicase core and the CTD is sufficiently long to accommodate both interaction
464
patches. Interestingly, the length of this linker varies in different PcrA orthologues in a
465
manner that reflects the distance between the RNAP exit channels and SI1 domains in each
466
system. The precise details of the PcrA-RNAP interaction will await a high-resolution
467
structure of the complex, but it is possible to build a speculative physical model which
468
satisfies both our own HDX data and the crosslinking study of the E. coli system. (Figure 7 –
469
figure supplement 1)
470 The involvement of the main body of the helicase in RNAP interactions may also help to
471
explain the phenotypes associated with CTD deletion. Given the apparently key role played
472
by the PcrA/UvrD CTD as a protein interaction hub, it is surprising that its complete removal
473
has little effect on nucleotide excision repair or replication-transcription conflicts in E. 474
coli(Merrikh et al., 2015; Sanders et al., 2017). Similarly, in B. subtilis where PcrA is
475
recruited to highly transcribed regions of the genome to facilitate replication through active
476
transcription units, the CTD seems to be dispensable (Merrikh et al., 2015). A possible
477
explanation lies in the fact that PcrA-RNAP association also involves the main body of the
478
helicase including the core motor domain 2A, whose activity is largely unchanged by
479
removal of the CTD. It is possible that residual weak interactions allow PcrA helicase core to
480
function effectively in the absence of the CTD in vivo. However, it should also be
481
acknowledged that stable interactions between the PcrA helicase core and RNAP cannot be
482
detected between purified proteins (this and previous work (Gwynn et al., 2013; Sanders et
483
al., 2017), and consequently we did not pursue HDX-MS experiments between these
484
constructs. Therefore, at least in vitro, the presence of the CTD appears to facilitate binding
485
of the core helicase domains near the RNA exit channels. Discussion In
448
this pathway, UvrD is thought to promote backtracking of RNAP stalled at bulky DNA
449
lesions and then recruits NER factors including UvrB to repair the damage. We find here that
450
RNAP and UvrB compete for PcrA interaction using the same conserved motif, meaning it is
451
unclear how UvrB would be recruited to the UvrD:RNAP complex. Moreover, the role of
452
UvrD in promoting strand-specific repair of DNA lesions remains controversial because a
453
genome-wide analysis of cyclobutane pyrimidine dimers showed no evidence for Mfd-
454
independent TC-NER and also that the levels of ppGpp do not change the repair rate in E. 455
coli (Adebali et al., 2017a, 2017b). Our data lead us to a new hypothesis to explain why
456
PcrA/UvrD interacts with RNA polymerase. 457 The HDX-MS data with full length PcrA showed that the core helicase domains interact with
458
multiple elements surrounding the RNA and DNA exit channels, for instance the β-flap tip
459
and the N-terminal part of the β′ subunit, as has been shown previously by crosslinking-MS
460
data for the complex between E. coli RNAP and UvrD (Epshtein et al., 2014). Our HDX-MS
461
data also identified an interaction with the SI1 domain which we can assign to the PcrA CTD. 462
The exit channels for nucleic acids are distant from the SI1 domain, but the long linker
463
between the helicase core and the CTD is sufficiently long to accommodate both interaction
464
patches. Interestingly, the length of this linker varies in different PcrA orthologues in a
465
manner that reflects the distance between the RNAP exit channels and SI1 domains in each
466
system. The precise details of the PcrA-RNAP interaction will await a high-resolution
467
structure of the complex, but it is possible to build a speculative physical model which
468
satisfies both our own HDX data and the crosslinking study of the E. coli system. (Figure 7 –
469
figure supplement 1)
470 The HDX-MS data with full length PcrA showed that the core helicase domains interact with
458
multiple elements surrounding the RNA and DNA exit channels, for instance the β-flap tip
459
and the N-terminal part of the β′ subunit, as has been shown previously by crosslinking-MS
460
data for the complex between E. coli RNAP and UvrD (Epshtein et al., 2014). Discussion Moreover, our observation of
486
increased DNA-RNA hybrids in cells in which the free CTD is overexpressed provides a
487
direct link between the RNAP binding function of the CTD and R-loop metabolism. 488 The location of the helicase core close to the RNA exit channel led us to consider roles for
489
PcrA in manipulating the transcript. Due to the enzyme’s inability to translocate on RNA or
490 to unwind duplex RNAs, we hypothesised that PcrA might act to unwind DNA/RNA hybrids
491
that are formed upstream of the transcription bubble. This idea was supported by experiments
492
in Bacillus subtilis showing that R-loop concentration in cells was increased by 2-3 fold by
493
overexpressing dominant negative PcrA or free CTD (which blocks PcrA-RNAP interaction). 494
This strongly suggests that an activity of wild type PcrA, which is dependent upon
495
interactions made by its CTD, is important for suppressing R-loops. Although out work does
496
not establish the mechanism by which PcrA decreases R-loop levels in cells, we hypothesise
497
that it does so by unwinding RNA-DNA hybrids that form behind the transcription elongation
498
complex (Figure 7). The well-documented 3′>5′ polarity of PcrA/UvrD helicase (Bird et al.,
499
1998), together with its inability to move along RNA strands, suggests two possible working
500
models for the unwinding of R-loops. In the first “co-directional” model, the helicase
501
translocates on the template strand and in the same direction as the RNA polymerase,
502
unwinding R-loops as they form. Another possibility (the “backtracking model”) is that the
503
helicase translocates on the non-template strand of the R-loop and in the opposite direction to
504
transcription, indirectly unwinding the DNA/RNA hybrid by making the polymerase
505
backtrack (Epshtein et al., 2014). In this scenario the lid domain of RNAP, rather than the
506
helicase itself, would act as the “ploughshare” to separate the two strands. We favour the first
507
model because our HDX protection data places the 2A domain of PcrA (which is at the front
508
of the moving helicase) in direct contact with the RNA exit region, suggesting that the
509
helicase translocates towards the RNA polymerase. Indeed, the close contact between PcrA
510
and RNAP suggested by our data could mean that R-loops are unwound as they are formed at
511
the exit channels, rather than being targeted after they have extended behind the TEC. Discussion Finally, a very recent study
537
involving depletion of PcrA in rnhB, rnhC and dinG backgrounds led to the conclusion that
538
PcrA alleviates replication-transcription conflicts, with biochemical analysis supporting the
539
idea (broadly similar to that presented here) that the helicase activity of PcrA acts to unwind
540
and remove R-loops in concert with R-loop nucleases(Moreno-Del Álamo et al., 2021). 541 Further experiments are now required to test the idea that PcrA unwinds co-transcriptional R-
523
loops more directly, but the model is appealing for several reasons. A role for PcrA in
524
alleviating R-loops can explain its function in alleviating replication:transcription conflicts as
525
well as why the helicase is enriched at highly transcribed rRNA and tRNA genes that have
526
been shown to be prone to R-loop formation in yeast and human cells (Boubakri et al., 2010;
527
Chan et al., 2014; Chen et al., 2017; Guy et al., 2009; Merrikh et al., 2015). Indeed, recent
528
work in E. coli has shown that uvrD is synthetically lethal with rnhA, in agreement with a
529
role for this helicase in unwinding R-loops that may be distinctive from those dealt with by
530
other R-loop suppression factors (Wolak et al., 2020). An R-loop suppression function might
531
also explain why loss of PcrA leads to hyper-recombination and the formation of RecFOR-
532
and RecA-dependent toxic recombination intermediates (Arthur and Lloyd, 1980; Moreno-
533
del Alamo et al., 2020; Petit and Ehrlich, 2002; Veaute et al., 2005). The displaced ssDNA
534
within an unresolved R-loop may act as a “frustrated” substrate for ssDNA gap repair,
535
resulting in RecA recruitment and strand exchange but failing to be processed correctly into
536
repaired products because of the presence of the R-loop and TEC. Finally, a very recent study
537
involving depletion of PcrA in rnhB, rnhC and dinG backgrounds led to the conclusion that
538
PcrA alleviates replication-transcription conflicts, with biochemical analysis supporting the
539
idea (broadly similar to that presented here) that the helicase activity of PcrA acts to unwind
540
and remove R-loops in concert with R-loop nucleases(Moreno-Del Álamo et al., 2021). 541 Discussion 512
However, we also note that previous observations of UvrD-dependent backtracking of RNAP
513
are more consistent with the second backtracking model (Epshtein et al., 2014; Sanders et
514
al., 2017). In these models, we propose that the role of the CTD is simply to target the
515
helicase activity to its physiological substrate in vivo (i.e. to increase the local concentration
516
of PcrA near R-loops), rather than to catalytically activate the DNA motor protein in response
517
to its engagement with RNAP. This view is consistent with our observation that free CTD can
518
block R-loop suppression through dominant negative effects on wild type PcrA, and also with
519
previously published work in which we have observed only moderate effects of adding
520
RNAP and/or removing the CTD on the ATPase and helicase activities of PcrA (Gwynn et
521
al 2013; Velankar et al 1999)
522 Further experiments are now required to test the idea that PcrA unwinds co-transcriptional R-
523
loops more directly, but the model is appealing for several reasons. A role for PcrA in
524
alleviating R-loops can explain its function in alleviating replication:transcription conflicts as
525
well as why the helicase is enriched at highly transcribed rRNA and tRNA genes that have
526
been shown to be prone to R-loop formation in yeast and human cells (Boubakri et al., 2010;
527
Chan et al., 2014; Chen et al., 2017; Guy et al., 2009; Merrikh et al., 2015). Indeed, recent
528
work in E. coli has shown that uvrD is synthetically lethal with rnhA, in agreement with a
529
role for this helicase in unwinding R-loops that may be distinctive from those dealt with by
530
other R-loop suppression factors (Wolak et al., 2020). An R-loop suppression function might
531
also explain why loss of PcrA leads to hyper-recombination and the formation of RecFOR-
532
and RecA-dependent toxic recombination intermediates (Arthur and Lloyd, 1980; Moreno-
533
del Alamo et al., 2020; Petit and Ehrlich, 2002; Veaute et al., 2005). The displaced ssDNA
534
within an unresolved R-loop may act as a “frustrated” substrate for ssDNA gap repair,
535
resulting in RecA recruitment and strand exchange but failing to be processed correctly into
536
repaired products because of the presence of the R-loop and TEC. Materials and Methods
543 Strain construction. All strains used in this work are listed in Table 3. The plasmids and
544
primers used to create them are described in Tables 4 and 5, respectively. Standard
545
techniques were used for strain construction (Harwood and Cutting, 1990). B. subtilis was
546
transformed using the LM-MD method or an optimised two-step starvation procedure
547
(Anagnostopoulos and Spizizen, 1961; Burby and Simmons, 2017) Integrations within the
548
ectopic locus were verified by PCR and/or sequencing. 549 Protein expression and purification. His-tagged Bacillus subtilis RNA polymerase was
550
purified as described previously from the MH5636 strain (Gwynn et al., 2013; Qi and Hulett,
551
1998). Wild-type and mutant B. subtilis PcrA and biotinylated PcrA were purified as
552
described (Gwynn et al., 2013). Plasmids expressing PcrA ΔCTD (residues 1-653) and PcrA
553
V448C were generated by site-directed mutagenesis of the wild type construct using the
554 Quikchange II XL kit (Agilent Technologies). In the last gel filtration chromatography of
555
PcrA V448C, DTT was omitted to allow efficient fluorescent labelling. Fluorescent PcrA was
556
prepared by mixing the V448C mutant with Cy3-maleimide (Cytiva) in a 1:2 molar ratio and
557
incubating overnight at 4 ºC. The reaction was quenched by the addition of 5 mM DTT. The
558
free dye was separated from the labelled protein by loading the mixture on a Superdex 200
559
5/150 GL. Fluorescent labelling efficiency was calculated spectrophotometrically following
560
the manufacturer’s instructions. A plasmid expressing the PcrA-CTD was generated by PCR
561
amplification from the wild type plasmid followed by sub-cloning into the pET47b plasmid. 562
B. subtilis PcrA-CTD was then purified in the same way as Geobacillus stearothermophilus
563
PcrA-CTD but the gel-filtration chromatography was substituted for MonoS (Cytiva) ion-
564
exchange chromatography (Sanders et al., 2017). 565 Quikchange II XL kit (Agilent Technologies). In the last gel filtration chromatography of
555
PcrA V448C, DTT was omitted to allow efficient fluorescent labelling. Fluorescent PcrA was
556
prepared by mixing the V448C mutant with Cy3-maleimide (Cytiva) in a 1:2 molar ratio and
557
incubating overnight at 4 ºC. The reaction was quenched by the addition of 5 mM DTT. The
558
free dye was separated from the labelled protein by loading the mixture on a Superdex 200
559
5/150 GL. Fluorescent labelling efficiency was calculated spectrophotometrically following
560
the manufacturer’s instructions. Materials and Methods
543 The
586 uncleaved sample was purified in the same way but without the second HisTrap
587
chromatography step. 588 uncleaved sample was purified in the same way but without the second HisTrap
587
chromatography step. 588 uncleaved sample was purified in the same way but without the second HisTrap
587
chromatography step. 588 His-tagged B. subtilis RNAP β subunit was expressed and purified in the same manner as
589
UvrB. The uncleaved sample was then further purified using a heparin column equilibrated in
590
20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT followed by elution with a NaCl gradient. The
591
protein was dialysed against 20 mM Tris-HCl pH 7.9, 300 mM NaCl, 2 mM EDTA, 1 mM
592
DTT and 10% glycerol overnight at 4 ºC. 593 His-tagged B. subtilis RNAP β subunit was expressed and purified in the same manner as
589
UvrB. The uncleaved sample was then further purified using a heparin column equilibrated in
590
20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT followed by elution with a NaCl gradient. The
591
protein was dialysed against 20 mM Tris-HCl pH 7.9, 300 mM NaCl, 2 mM EDTA, 1 mM
592
DTT and 10% glycerol overnight at 4 ºC. 593 Analytical size-exclusion chromatography. Transcription elongation complexes (TEC)
594
were formed as described previously (Sidorenkov et al., 1998) using scaffold 1 (Table 6) and
595
the RNA sequence: AUCGAGAGG (IBA life sciences). Briefly, the RNA oligonucleotide
596
was incubated with the template strand (TS) for 5 minutes at 45 °C in buffer T (50 mM Tris
597
pH 7.9, 150 mM NaCl, 10 MgCl2 and 1 mM DTT) and cooled to room temperature at a rate
598
of 1 °C/min. Then, RNAP was added and the solution was incubated at room temperature for
599
10 minutes. Finally, non-template strand (NTS) was added and incubated at 37°C for 10
600
minutes. The final concentration of each component was: 118 nM TS, 236 nM RNA, 2.36
601
μM NTS and 66 nM RNAP. To form the PcrA-TEC complex, the TEC was concentrated 10-
602
fold using Amicon Ultra-0.5 3 kDa NMWL centrifugal filters and then incubated with 4.6
603
µM PcrA for 10 min at room temperature. Materials and Methods
543 A plasmid expressing the PcrA-CTD was generated by PCR
561
amplification from the wild type plasmid followed by sub-cloning into the pET47b plasmid. 562
B. subtilis PcrA-CTD was then purified in the same way as Geobacillus stearothermophilus
563
PcrA-CTD but the gel-filtration chromatography was substituted for MonoS (Cytiva) ion-
564
exchange chromatography (Sanders et al., 2017). 565 B. subtilis uvrB, mfd and rpoB were amplified by colony PCR from B. subtilis 168 single
566
colonies and cloned into pET47b, pET22b and pET28a, respectively (cloning
567
oligonucleotides available in the Supplementary Methods and Table 5). Mutations to
568
generate plasmids for expression of UvrB E233A, RpoB E310A and RpoB E310K were
569
made by site-directed mutagenesis of the wild type constructs using the Quikchcange II XL
570
kit (Agilent). B. subtilis Mfd was purified in the same way as E. coli Mfd, as described
571
previously (Chambers et al., 2003). His-tagged B. subtillis UvrB was expressed in BL21
572
(DE3) cells. Following induction at mid-log phase with 1 mM IPTG, cells were grown
573
overnight at 18 ºC. Cell pellets were resuspended in 20mM Tris–HCl pH 7.9, 300mM NaCl,
574
20mM imidazole, 0.1 mM DTT supplemented with EDTA-free cOmplete protease inhibitor
575
cocktail (Roche) and lysed by sonication. The soluble fraction was loaded on a 5 ml HisTrap
576
column (Cytiva) in the same buffer and eluted with an imidazole gradient. A portion of the
577
material was supplemented with 3C protease and the two separate samples were then dialysed
578
against 20 mM Tris-HCl pH 7.9, 300 mM NaCl, 20 mM imidazole and 0.1 mM DTT
579
overnight at 4 ºC. For the sample that had been cleaved with 3C, the protein was loaded again
580
onto a HisTrap column to remove the cleaved tag and the protease (which is itself his-
581
tagged). The flow through was diluted into 20 mM Tris-HCl, 1 mM DTT to reduce the salt
582
concentration before loading onto a MonoQ column (Cytiva) equilibrated in 20 mM Tris-
583
HCl, 100 mM NaCl, 1 mM DTT. The protein was eluted with a NaCl gradient and fractions
584
containing UvrB were pooled and dialysed against storage buffer (20 mM Tris-HCl pH 7.5,
585
200 mM NaCl, 2 mM EDTA, 1 mM DTT and 10% glycerol) overnight at 4 ºC. Materials and Methods
543 The wash and dilution buffer (20 mM
619
Tris pH 7.5, 150 mM NaCl, 0.1% Triton X-100) was supplemented with1 mM EDTA for
620
experiments with the streptavidin beads and 20 mM imidazole for Ni-NTA beads. For the in
621
vitro pulldown, purified β subunit was used at 2 µM concentration. For experiments that
622
contained competitors 1.5 µM PcrA or PcrA-CTD, 1.5 µM Mfd and 3 µM UvrB were used. 623
In vitro pulldowns were analysed by Coomassie staining the SDS-PAGE gel. Ex vivo
624
pulldowns were analysed by Western blotting using a monoclonal anti-RNAP β subunit
625
antibody (Abcam; ab202891) or a monoclonal anti-myc antibody (Proteintech; 67447-1-Ig). 626
In the latter experiments, a portion of the gel was excised before transfer to the blotting
627
membrane to confirm uniform bait levels by Coomassie staining, except where this was not
628
possible with UvrB due to its similar molecular weight to PcrA. Images were quantified using
629
ImageQuant (Cytiva). 630 Hydrogen-deuterium exchange coupled to mass spectrometry HDX-MS experiments
631
were performed using an automated robot (LEAP Technologies) coupled to an M-Class
632
Acquity UPLC, HDX manager (Waters Ltd.), and Synapt G2S-i mass spectrometer. For the
633
CTD-RNAP experiments, protein samples were prepared containing either or both 5 µM
634
RNAP and 10 µM CTD. For the PcrA-RNAP experiments samples were prepared using 0.5
635
μM RNAP and/or 2 μM PcrA. 30 μl of the protein solution in sample buffer (10 mM
636
potassium phosphate buffer pH 7.4) was added to 135 μl deuterated buffer (10 mM potassium
637
phosphate buffer in deuterated water, pD 7.4) and incubated at 4ºC for 0.5, 2, 5 and 10
638
minutes. After labelling, the reaction was terminated by the addition of 50 μl of sample to
639
100 μl quench buffer (10 mM potassium phosphate, 2 M guanidine- HCl, pH 2.2) resulting in
640
a final pH of approximately 2.5. 50 μl of quenched sample were loaded onto an immobilised
641
ethylene-bridged hybrid (BEH) pepsin column (Waters Ltd.). The resulting peptides were
642
passed through a VanGuard BEH C18 pre- column and a C18 column (Waters Ltd.) and
643
separated by gradient elution of 0–40% MeCN (0.1% v/v formic acid) in H2O (0.3% v/v
644
formic acid). For the CTD-RNAP experiments, quench and pepsin wash buffers were
645
supplemented with 0.05% (w/v) DDM. Materials and Methods
543 Samples were loaded onto a Superdex 200
604
Increase 5/150 GL (Cytiva) connected to an AKTA FPLC instrument (Cytiva) in buffer T (50
605
mM Tris pH 7.9, 150 mM NaCl, 10 MgCl2 and 1 mM DTT) at room temperature. Samples
606
were directly injected in the injection loop prior to the chromatography. Where necessary,
607
samples were collected to analyse their content by SDS-PAGE. 608 Electrophoretic mobility shift assays TECs were assembled as above but using different
609
DNA:RNA scaffolds containing fluorescently-labelled oligonucleotides (Table 6), the buffer
610
supplemented with 2.5% (v/v) Ficoll and analysed for their ability to bind to PcrA. Wild type
611
or fluorescent PcrA at the concentration indicated was added to the TEC at room temperature
612
for 10 minutes before loading the samples onto a 4.5 % polyacrylamide TBE-PAGE gel. The
613
gels were imaged using a Typhoon FLA 9500 (Cytiva). 614 Analysis of protein:protein interactions using pulldown assays B. subtilis cell extracts
615
were prepared for ex vivo pulldowns as described previously (Gwynn et al., 2013) using
616
strains 1A1 or IU79, the latter supplemented with 1% xylose to induce the expression of myc-
617
tagged PcrA. 1 µM biotin-tagged proteins were used as baits on streptavidin beads, whereas 2
618 µM his-tagged proteins were used with Ni-NTA beads. The wash and dilution buffer (20 mM
619
Tris pH 7.5, 150 mM NaCl, 0.1% Triton X-100) was supplemented with1 mM EDTA for
620
experiments with the streptavidin beads and 20 mM imidazole for Ni-NTA beads. For the in
621
vitro pulldown, purified β subunit was used at 2 µM concentration. For experiments that
622
contained competitors 1.5 µM PcrA or PcrA-CTD, 1.5 µM Mfd and 3 µM UvrB were used. 623
In vitro pulldowns were analysed by Coomassie staining the SDS-PAGE gel. Ex vivo
624
pulldowns were analysed by Western blotting using a monoclonal anti-RNAP β subunit
625
antibody (Abcam; ab202891) or a monoclonal anti-myc antibody (Proteintech; 67447-1-Ig). 626
In the latter experiments, a portion of the gel was excised before transfer to the blotting
627
membrane to confirm uniform bait levels by Coomassie staining, except where this was not
628
possible with UvrB due to its similar molecular weight to PcrA. Images were quantified using
629
ImageQuant (Cytiva). 630 µM his-tagged proteins were used with Ni-NTA beads. Materials and Methods
543 646 The UPLC was interfaced to the mass spectrometer via electrospray ionisation. HDMSE and
647
dynamic range extension modes (Data Independent Analysis couple with IMS separation)
648
were used to separate peptides by IMS prior to fragmentation in the transfer cell. Data were
649
analysed using PLGS and DynamX software provided with the mass spectrometer. The
650
restrictions applied for peptide identification in DynamX were the following: minimum
651 intensity 1000, minimum products per amino acid 0.3, maximum sequence length 25,
652
maximum ppm error 6, replication file threshold 4. The difference plots were generated using
653
the in-house developed algorithm Paved (Cornwell et al., 2018), Deuteros (Lau et al., 2019)
654
and the data represented in Graphpad Prism 7.0. Difference plots were then mapped onto
655
structures in Chimera (Pettersen et al., 2004) and PyMOL using available structures and/or
656
homology models generated by Swiss-model (Waterhouse et al., 2018). Four technical
657
repeats were performed for each HDX-MS experiment, each using single protein samples
658
pooled from independent preparations. The data have been deposited with the PRIDE
659
database (Accession number PXD025332). 660 Bioinformatic analysis Protein orthologues were searched for in the reference proteomes of
661
the Protein Information Resource using the B. subtilis protein sequence as a query (Chen et
662
al., 2011). Obtained sequences were manually curated to remove sequences that were
663
substantially smaller than the B. subtilis sequence or that contained large gaps. The
664
conservation logo was created using WebLogo (Crooks et al., 2004) and sequence alignment
665
figure created with ESPript 3.0 (Robert and Gouet, 2014). B. subtilis proteins’ homology
666
models were created using SWISS-MODEL with the indicated PDB files (Waterhouse et al.,
667
2018). Protein-protein interaction docking analysis was performed using Haddock 2.2 and the
668
best structures from the clusters with the lowest HADDOCK score were compared (Van
669
Zundert et al., 2016). 670 CD spectroscopy CD spectra were collected using a JASCO J-810 spectropolarimeter fitted
671
with a Peltier temperature control (Jasco UK). 0.5 mg/ml protein samples were buffer
672
exchanged into phosphate buffered saline (PBS; 8.2 mM NaH2PO4, 1.8 mM KH2PO4, 137
673
mM NaCl and 2.7 mM KCl (pH 7.4)) by 16 hr dialysis at 4˚C using a membrane with a
674
MWCO of 10 kDa. Materials and Methods
543 Using 0.25 mg/ml of protein in a 0.1 cm quartz cuvette, data were
675
acquired across a 190-206 nm absorbance scan with a scan rate of 100 nm/min at 20ºC. Raw
676
data was normalised to molar ellipticity (MRE (deg.cm2.dmol-1)) using calculation of the
677
concentration of peptide bonds and the cell path length. A buffer only baseline was subtracted
678
from all datasets. 679 Helicase assays Helicase assays were carried out essentially as described previously with the
680
following changes (Gwynn et al., 2013). The reactions were performed at 20 ºC in 50 mM
681
Tris pH 7.5, 50 mM NaCl, 2 mM MgCl2 and 1 mM DTT and started by the addition of the
682
protein. The list of DNA and RNA substrates used is shown in Table 7. The products were
683 run on 12% acrylamide TBE gels, imaged using a Typhoon phosphorimager and quantified
684
using ImageQuant software. 685 run on 12% acrylamide TBE gels, imaged using a Typhoon phosphorimager and quantified
684
using ImageQuant software. 685 ATPase assay The ATPase activities of PcrA and UvrB were measured using an enzyme
686
linked assay in which ATP hydrolysis is coupled to NADH oxidation (Kiianitsa et al., 2003). 687
The assay was carried out at 25 ºC using 1 nM PcrA, 50 nM UvrB and 5 µM nucleic acids (as
688
indicated). For measurements of PcrA ATPase activity the reaction buffer contained 50 mM
689
Tris pH 7.5, 50 mM NaCl, 5 mM MgCl2 and 5 mM DTT. The method used for UvrB was as
690
described previously (Webster et al., 2012). 691 ATPase assay The ATPase activities of PcrA and UvrB were measured using an enzyme
686
linked assay in which ATP hydrolysis is coupled to NADH oxidation (Kiianitsa et al., 2003). 687
The assay was carried out at 25 ºC using 1 nM PcrA, 50 nM UvrB and 5 µM nucleic acids (as
688
indicated). For measurements of PcrA ATPase activity the reaction buffer contained 50 mM
689
Tris pH 7.5, 50 mM NaCl, 5 mM MgCl2 and 5 mM DTT. The method used for UvrB was as
690
described previously (Webster et al., 2012). 691 Growth curves Single colonies were grown overnight in LB at 30 ºC. Each strain was
692
diluted to an OD600 of 0.01 in LB supplemented with 1 mM IPTG and then grown at 37 ºC. Materials and Methods
543 The gel was cut into two parts
717
prior to transfer, and the upper portion was Coomassie stained to use as loading control and
718
the lower portion was transferred to a PVDF membrane by electroblotting. Myc-tagged PcrA
719
was detected using a monoclonal anti-myc antibody (Proteintech) followed by an anti-mouse-
720
HRP goat antibody (Santa Cruz Biotechnology). 721 Immunoblotting B. subtilis cells were grown until stationary phase in LB at 37 ºC, harvested
715
by centrifugation and the pellet was resuspended in SSC prior to sonication. OD600 was used
716
to normalize the loading of samples in the SDS-PAGE gel. The gel was cut into two parts
717
prior to transfer, and the upper portion was Coomassie stained to use as loading control and
718
the lower portion was transferred to a PVDF membrane by electroblotting. Myc-tagged PcrA
719
was detected using a monoclonal anti-myc antibody (Proteintech) followed by an anti-mouse-
720
HRP goat antibody (Santa Cruz Biotechnology). 721 Mfd Pulldown-Tandem mass tagging mass spectrometry (TMT-MS). B. subtilis lysates
722
were prepared and pulldown performed using the 1A1 strain as described above. Two
723
conditions were tested: 1 µM biotinylated Mfd and no-bait control. Samples were then
724
analysed by TMT-MS to determine which prey proteins were enriched in the pulldown
725
compared to control. 726 Pulled-down samples were reduced (10mM TCEP, 55°C for 1h), alkylated (18.75mM
727
iodoacetamide, room temperature for 30min.) and then digested from the beads with trypsin
728
(2.5µg trypsin; 37°C, overnight). The resulting peptides were then labelled with TMT ten-
729
plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific,
730
Loughborough, LE11 5RG, UK) and the labelled samples pooled and desalted using a
731
SepPak cartridge according to the manufacturer’s instructions (Waters, Milford,
732
Massachusetts, USA). Eluate from the SepPak cartridge was evaporated to dryness and
733
resuspended in buffer A (20 mM ammonium hydroxide, pH 10) prior to fractionation by high
734
pH reversed-phase chromatography using an Ultimate 3000 liquid chromatography system
735
(Thermo Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column
736
(130Å, 3.5 µm, 2.1 mm X 150 mm, Waters, UK) in buffer A and peptides eluted with an
737
increasing gradient of buffer B (20 mM Ammonium Hydroxide in acetonitrile, pH 10) from
738
0-95% over 60 minutes. Materials and Methods
543 693
Absorbance measurements were taken at the indicated times, averaged and plotted using
694
Graphpad Prism. 695 Growth curves Single colonies were grown overnight in LB at 30 ºC. Each strain was
692
diluted to an OD600 of 0.01 in LB supplemented with 1 mM IPTG and then grown at 37 ºC. 693
Absorbance measurements were taken at the indicated times, averaged and plotted using
694
Graphpad Prism. 695 RNA/DNA hybrid dot blot Single colonies were grown overnight in LB at 30 ºC. Cultures
696
were diluted to an OD600 of 0.05 into fresh LB medium supplemented with 1 mM IPTG for
697
both B. subtilis morning and overnight inductions. For the FL-PcrA overexpression
698
experiment, overnight cultures were induced the next morning at the time that they were
699
diluted. For the CTD experiments, induction was started while inoculating the single colonies
700
for the overnight growth. Cultures were grown until an OD600 1.1-1.2 and genomic DNA was
701
purified using the DNeasy Blood and Tissue (Qiagen) or the GenElute Bacterial Genomic
702
DNA Kit (Sigma-Aldrich) following manufacturer’s instructions. gDNA was quantified by
703
Nanodrop and a fraction of DNA was treated with 5 U RNase H (NEB) for 1h at 37 ºC. 704
gDNA serial dilutions and the RNase treated gDNA were spotted on a positively charged
705
Hybond-N+ nylon membrane (Amersham) using a dot-blot apparatus. The DNA was probed
706
with the S9.6 antibody (1:1000 dilution, Millipore) in 1% BSA/TBST overnight at 4 ºC after
707
UV-crosslinking (0.12 J/cm2) and blocking the membrane with 5% milk/TBST for 1h at RT. 708
An anti-mouse HRP antibody (1:10000 dilution, Santa Cruz Biotechnology) was used as
709
secondary antibody. Images were acquired with Odyssey Fc (Li-COR Biosciences). DNA
710
loading was calculated by staining with 0.05% methylene blue in 0.5 M sodium acetate buffer
711
(pH 5.2) after washing the membrane with 5% acetic acid as described previously (Ko et al.,
712
2010; Raghunathan et al., 2018). Images were quantified using ImageQuant (Cytiva). Error
713
bars show the SEM of at least 3 independent experiments. 714 Immunoblotting B. subtilis cells were grown until stationary phase in LB at 37 ºC, harvested
715
by centrifugation and the pellet was resuspended in SSC prior to sonication. OD600 was used
716
to normalize the loading of samples in the SDS-PAGE gel. Materials and Methods
543 761 All spectra were acquired using an Orbitrap Fusion Tribrid mass spectrometer controlled by
752
Xcalibur 2.1 software (Thermo Scientific) and operated in data-dependent acquisition mode
753
using an SPS-MS3 workflow. FTMS1 spectra were collected at a resolution of 120 000, with
754
an automatic gain control (AGC) target of 200 000 and a max injection time of 50ms. 755 All spectra were acquired using an Orbitrap Fusion Tribrid mass spectrometer controlled by
752
Xcalibur 2.1 software (Thermo Scientific) and operated in data-dependent acquisition mode
753
using an SPS-MS3 workflow. FTMS1 spectra were collected at a resolution of 120 000, with
754
an automatic gain control (AGC) target of 200 000 and a max injection time of 50ms. 755
Precursors were filtered with an intensity threshold of 5000, according to charge state (to
756
include charge states 2-7) and with monoisotopic peak determination set to peptide. 757
Previously interrogated precursors were excluded using a dynamic window (60s +/-10ppm). 758
The MS2 precursors were isolated with a quadrupole isolation window of 1.2m/z. ITMS2
759
spectra were collected with an AGC target of 10 000, max injection time of 70ms and CID
760
collision energy of 35%. 761 For FTMS3 analysis, the Orbitrap was operated at 50 000 resolution with an AGC target of
762
50 000 and a max injection time of 105ms. Precursors were fragmented by high energy
763
collision dissociation (HCD) at a normalised collision energy of 60% to ensure maximal
764
TMT reporter ion yield. Synchronous Precursor Selection (SPS) was enabled to include up to
765
5 MS2 fragment ions in the FTMS3 scan. 766 The raw data files were processed and quantified using Proteome Discoverer software v2.1
767
(Thermo Scientific) and searched against the UniProt Bacillus subtilis (strain 168) database
768
(downloaded December 2018: 4284 entries) using the SEQUEST HT algorithm. Peptide
769
precursor mass tolerance was set at 10ppm, and MS/MS tolerance was set at 0.6Da. Search
770
criteria included oxidation of methionine (+15.995Da), acetylation of the protein N-terminus
771
(+42.011Da) and Methionine loss plus acetylation of the protein N-terminus (-89.03Da) as
772
variable modifications and carbamidomethylation of cysteine (+57.021Da) and the addition
773
of the TMT mass tag (+229.163Da) to peptide N-termini and lysine as fixed modifications. 774
Searches were performed with full tryptic digestion and a maximum of 2 missed cleavages
775
were allowed. Materials and Methods
543 The resulting fractions (4 in total) were evaporated to dryness and
739
resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion
740
Tribrid mass spectrometer (Thermo Scientific). 741 High pH RP fractions were further fractionated using an Ultimate 3000 nano-LC system in
742
line with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). In brief, peptides
743
in 1% (vol/vol) formic acid were injected onto an Acclaim PepMap C18 nano-trap column
744
(Thermo Scientific). After washing with 0.5% (vol/vol) acetonitrile 0.1% (vol/vol) formic
745
acid, peptides were resolved on a 250 mm × 75 μm Acclaim PepMap C18 reverse phase
746 analytical column (Thermo Scientific) over a 150 min organic gradient with a flow rate of
747
300 nl min−1. Solvent A was 0.1% formic acid and Solvent B was aqueous 80% acetonitrile
748
in 0.1% formic acid. Peptides were ionized by nano-electrospray ionization at 2.0kV using a
749
stainless-steel emitter with an internal diameter of 30 μm (Thermo Scientific) and a capillary
750
temperature of 275°C. 751 analytical column (Thermo Scientific) over a 150 min organic gradient with a flow rate of
747
300 nl min−1. Solvent A was 0.1% formic acid and Solvent B was aqueous 80% acetonitrile
748
in 0.1% formic acid. Peptides were ionized by nano-electrospray ionization at 2.0kV using a
749
stainless-steel emitter with an internal diameter of 30 μm (Thermo Scientific) and a capillary
750
temperature of 275°C. 751 All spectra were acquired using an Orbitrap Fusion Tribrid mass spectrometer controlled by
752
Xcalibur 2.1 software (Thermo Scientific) and operated in data-dependent acquisition mode
753
using an SPS-MS3 workflow. FTMS1 spectra were collected at a resolution of 120 000, with
754
an automatic gain control (AGC) target of 200 000 and a max injection time of 50ms. 755
Precursors were filtered with an intensity threshold of 5000, according to charge state (to
756
include charge states 2-7) and with monoisotopic peak determination set to peptide. 757
Previously interrogated precursors were excluded using a dynamic window (60s +/-10ppm). 758
The MS2 precursors were isolated with a quadrupole isolation window of 1.2m/z. ITMS2
759
spectra were collected with an AGC target of 10 000, max injection time of 70ms and CID
760
collision energy of 35%. Materials and Methods
543 The reverse database search option was enabled and all data was filtered to
776
satisfy a false discovery rate (FDR) of 5%. Only the proteins with a high FDR confidence and
777 more than one unique peptide were accepted as hits. Fold change was calculated using the
778
beads-only control. 779 more than one unique peptide were accepted as hits. Fold change was calculated using the
778
beads-only control. 779 780
Acknowledgements
781
This work was funded by the European Union via the DNAREPAIRMAN innovative training
782
network (Urrutia-Irazabal, Savery and Dillingham) and by a BBSRC ALERT grant (Ault and
783
Sobott; BB/M012573/1). We are grateful to Abigail Smith and Gwendolyn Brouwer for
784
helpful discussions, to Harry Thompson for technical assistance with CD measurements, to
785
Kate Heesom and Phil Lewis for technical assistance with TMT-MS, to Peter McGlynn,
786
Heath Murray, Marie-Agnès Petit, Houra Merrikh and Christopher Merrikh for advice and
787
the sharing of plasmids and strains used in this work, and to Peter Lewis for generously
788
sharing the co-ordinates of the B. subtilis transcription elongation complex ahead of public
789
release. 790
791
Figure Legends
792
Figure 1. Interactions between PcrA and a transcription elongation complex are
793
mediated by protein-protein interactions involving the PcrA-CTD (A) EMSA supershift
794
assays to monitor association of PcrA with a TEC. The PcrA-CTD is necessary and sufficient
795
for stable formation of the PcrA-RNAP complex. WT PcrA and ΔCTD PcrA were titrated
796
from 0.25µM to 1.5 µM. The PcrA-CTD was titrated from 0.5 µM to 3 µM. (B) EMSA
797
supershift assay showing that binding of PcrA is not dependent on the presence of upstream
798
or downstream DNA in the TEC. The star indicates the position of the fluorescent label at the
799
5′ end of the template strand of the scaffold. PcrA was used at 1 µM. The oligonucleotides
800
used to assemble the scaffolds are shown in Table 6. (C) Relative HDX measured for the
801
PcrA-CTD in the CTD-RNAP complex compared to CTD alone. The black line shows the
802
differential relative uptake and the pink and blue shadowing, the SEM of the CTD and CTD-
803
RNAP conditions, respectively. The four offset traces show different exchange times. Materials and Methods
543 804
Negative uptake values over the CTD baseline with non-overlapping shadowing show
805
protected amino acid regions on the CTD when it is in complex with RNAP. Note the key
806
regions of the CTD (aa ~690-705 and aa ~720-Ct; shaded yellow) are protected upon binding
807
RNAP. (D) Left - homology model of the PcrA-CTD showing regions protected from HDX
808 Acknowledgements
781 This work was funded by the European Union via the DNAREPAIRMAN innovative training
782
network (Urrutia-Irazabal, Savery and Dillingham) and by a BBSRC ALERT grant (Ault and
783
Sobott; BB/M012573/1). We are grateful to Abigail Smith and Gwendolyn Brouwer for
784
helpful discussions, to Harry Thompson for technical assistance with CD measurements, to
785
Kate Heesom and Phil Lewis for technical assistance with TMT-MS, to Peter McGlynn,
786
Heath Murray, Marie-Agnès Petit, Houra Merrikh and Christopher Merrikh for advice and
787
the sharing of plasmids and strains used in this work, and to Peter Lewis for generously
788
sharing the co-ordinates of the B. subtilis transcription elongation complex ahead of public
789
release. 790 Figure 1. Interactions between PcrA and a transcription elongation complex are
793
mediated by protein-protein interactions involving the PcrA-CTD (A) EMSA supershift
794
assays to monitor association of PcrA with a TEC. The PcrA-CTD is necessary and sufficient
795
for stable formation of the PcrA-RNAP complex. WT PcrA and ΔCTD PcrA were titrated
796
from 0.25µM to 1.5 µM. The PcrA-CTD was titrated from 0.5 µM to 3 µM. (B) EMSA
797
supershift assay showing that binding of PcrA is not dependent on the presence of upstream
798
or downstream DNA in the TEC. The star indicates the position of the fluorescent label at the
799
5′ end of the template strand of the scaffold. PcrA was used at 1 µM. The oligonucleotides
800
used to assemble the scaffolds are shown in Table 6. (C) Relative HDX measured for the
801
PcrA-CTD in the CTD-RNAP complex compared to CTD alone. The black line shows the
802
differential relative uptake and the pink and blue shadowing, the SEM of the CTD and CTD-
803
RNAP conditions, respectively. The four offset traces show different exchange times. 804
Negative uptake values over the CTD baseline with non-overlapping shadowing show
805
protected amino acid regions on the CTD when it is in complex with RNAP. Note the key
806
regions of the CTD (aa ~690-705 and aa ~720-Ct; shaded yellow) are protected upon binding
807
RNAP. (D) Left - homology model of the PcrA-CTD showing regions protected from HDX
808 in the complex with RNAP (dark blue). Right - amino acids known to be important for
809
interaction with RNAP (purple residues). 810 in the complex with RNAP (dark blue). Acknowledgements
781 Right - amino acids known to be important for
809
interaction with RNAP (purple residues). 810 Figure 1 – Figure Supplement 1. Interactions between PcrA and a transcription
811
elongation complex are mediated by protein-protein interactions involving the PcrA-
812
CTD. (A) PcrA interacts physically with the TEC during size exclusion chromatography. 813
Chromatographs are shown for free PcrA (green), the TEC alone (blue) and the TEC:PcrA
814
mixture (magenta) at a 1:7 ratio. (B) SDS-PAGE gel showing samples taken from the size
815
exclusion runs for TEC only (blue) and the TEC:PcrA mixture (magenta) at the position
816
indicated by the arrow in (A). Note the presence of PcrA at an apparently very high
817
molecular weight when the TEC is present, providing evidence for a physical interaction. (C)
818
PcrA interacts physically with the TEC in native polyacrylamide gels. The gel shows a
819
EMSA “supershift” assay in which a fluorescently labelled transcription bubble scaffold is
820
shifted by PcrA alone, RNAP alone (i.e. the TEC) and a mixture of RNAP and PcrA. PcrA
821
was used at 1 µM concentration. Note that a unique supershifted band is formed in the final
822
lane providing evidence for a PcrA-TEC complex. The free scaffold runs off the bottom of
823
this gel due to the long electrophoresis time required to separate the shifted bands. (D) The
824
CTD competes with FL PcrA for binding to the TEC in native polyacrylamide gels. The
825
upper panel shows the TEC signal (the template strand is Cy5- labelled) and the lower panel,
826
the FL PcrA (V448C mutant labelled with Cy3). The ΔCTD construct is not able to supershift
827
the TEC. When CTD is added to the TEC-PcrA complex, the band shifts downwards to the
828
expected position of the CTD-TEC complex. FL PcrA and CTD were used at 3 µM
829
concentration. The CTD titration is the same titration gel shown in Figure 1A right panel. The
830
dashed line shows the position of the CTD-TEC. 831 Figure 2. The PcrA-CTD binds to a conserved motif in the SI1 domain of RNAP (A)
832 g
( )
Relative HDX measured for a region of the RNAP β subunit (residue numbers on x axis)
833
within the CTD-RNAP complex (blue) compared to RNAP alone (red). A small region of
834
RpoB (at amino-acid positions around ~300) becomes significantly protected by interaction
835
with the PcrA CTD as the exchange time becomes longer. (B) The protected region maps to a
836
conserved motif in the SI1 domain of B. subtilis RpoB. This region is organised differently in
837
E. coli RpoB, but the same conserved amino acid motif appears in a slightly different position
838
in the structure (black arrow). (C) Structure of the B.subtilis (upper panel) and E. coli (lower
839
panel) β2 (red) - SI1(green) domains indicating the beta-loop structure containing a putative
840
interaction motif at the tip (black arrows). This sequence is well-conserved in bacterial RNA
841 polymerases and the consensus sequence is shown in weblogo format beneath each structure. 842
(D) In vitro pulldown of RpoB using PcrA as a bait (see Methods for details). Mutation of the
843
conserved glutamate (E301) in the putative helicase interaction motif dramatically reduces
844
RpoB pulldown. 845 polymerases and the consensus sequence is shown in weblogo format beneath each structure. 842
(D) In vitro pulldown of RpoB using PcrA as a bait (see Methods for details). Mutation of the
843
conserved glutamate (E301) in the putative helicase interaction motif dramatically reduces
844
RpoB pulldown. 845 Figure 2 – Figure Supplement 1. HDX protection plots for the remaining RNAP
846
subunits in the CTD-RNAP experiment. (A) Complete data for the β subunit shown in
847
Paved format as used for Figure 2A. The green box indicates the protected area. (B-F)
848
DYNAMX HDX butterfly plots for the β, β’, α, δ and ε subunits of RNAP. Negative values
849
represent protected regions and positive values represent regions that are exposed upon
850
binding of the CTD. The green box in panel B indicates the region of β that is protected by
851
the PcrA CTD. Note that there are no significant protection signals anywhere else in the
852
entire RNAP complex. For difference plots, four replicates were performed for each of the
853
four independent colour-coded time points (orange dots 0.5 min, red dots 2 min, blue dots 5
854
min, black dots 10 min). Figure 2. The PcrA-CTD binds to a conserved motif in the SI1 domain of RNAP (A)
832 Grey shading indicates the standard deviation of all charge states
855
and replicates per peptide. 856 Figure 2 – Figure Supplement 2. The CTD-interacting motif is located in different
857
regions of the SI1 domain among landmark organisms. A multiple sequence alignment for
858
the β2 (red line) and SI1 (green line) domains of the RNAP β subunit for the organisms
859
indicated. Pink shading indicates the putative helicase interaction motif which is positioned
860
differently in E. coli-like compared to B. subtilis-like RNAP. Note that the motif was not
861
clearly identified in all bacterial RNAPs. 862 Figure 2 – Figure Supplement 3. Mutations to E301 do not alter the overall structure of
863
the β subunit of RNAP. CD spectra for the proteins indicated were obtained at 0.25 mg/ml
864
as described in the Materials and Methods. The spectra are all similar and show a high α
865
helical content as expected based on the cryo-EM structure (predicted spectrum shown as red
866
line), suggesting that the wild type and mutant proteins are globally folded. 867
Figure 3. Many PcrA partner proteins contain the helicase interaction motif (A) Putative
868
helicase interaction motif (blue) in known PcrA interaction partners from B. subtilis. A beta
869
hairpin structure (blue) is formed by the interaction motif in each of the proteins. Two of the
870
structures are homology models as indicated. (B) Sequences of putative helicase interaction
871
motifs in four known PcrA partner proteins. (C) Pulldown of Myc-tagged PcrA from B. 872
subtilis cell extracts using UvrB as bait (for details see the Methods). Mutation of the
873 Figure 2 – Figure Supplement 3. Mutations to E301 do not alter the overall structure of
863
the β subunit of RNAP. CD spectra for the proteins indicated were obtained at 0.25 mg/ml
864
as described in the Materials and Methods. The spectra are all similar and show a high α
865
helical content as expected based on the cryo-EM structure (predicted spectrum shown as red
866
line), suggesting that the wild type and mutant proteins are globally folded. 867 Figure 3. Many PcrA partner proteins contain the helicase interaction motif (A) Putative
868
helicase interaction motif (blue) in known PcrA interaction partners from B. subtilis. A beta
869
hairpin structure (blue) is formed by the interaction motif in each of the proteins. Figure 2. The PcrA-CTD binds to a conserved motif in the SI1 domain of RNAP (A)
832 subtilis cell extracts using biotinylated PcrA as bait. Where
877
indicated the prey was supplemented with purified UvrB or Mfd. Free UvrB, but not Mfd,
878
competes for the interaction site formed between PcrA and RNA polymerase. Error bars
879
show the SEM of three independent experiments. Two-tailed Student’s t test determined
880
statistical significance (*p value < 0.05). 881 Figure 3 – Figure Supplement 1. The E233A mutation does not affect the ATPase
882
activity of UvrB. ssDNA stimulated steady-state ATPase activity of WT and E233A UvrB
883
was measured as described in the Methods. The reported values are the mean turnover
884
number and standard deviation for at least 5 independent experiments. 885 Figure 3 – Figure Supplement 1. The E233A mutation does not affect the ATPase
882
activity of UvrB. ssDNA stimulated steady-state ATPase activity of WT and E233A UvrB
883
was measured as described in the Methods. The reported values are the mean turnover
884
number and standard deviation for at least 5 independent experiments. 885 Figure 3 – Figure Supplement 2. The CTD interacts with UvrB domain 2 and close to
886
the damaged DNA site. (A) Domain organisation of UvrB showing the interaction sites for
887
UvrA, UvrC and the location of the PcrA/UvrD helicase interaction motif identified in this
888
study. (B) Docking model generated by HADDOCK (Van Zundert et al., 2016) showing
889
DNA-bound UvrB (PDB: 6o8e; colours according to panel A) and the PcrA CTD (pink). The
890
DNA strands are shown in orange and grey. (C) Sequence conservation logos for the helicase
891
interaction motif in UvrB and the PcrA-CTD. Note that the glutamic acid is somewhat more
892
variable than in the logo generated for the RNAP β subunit, although the interaction motifs
893
are otherwise highly similar. 894 Figure 4. The PcrA helicase core is protected by interaction with RNA polymerase (A)
895
Relative HDX measured for full length PcrA (residue numbers on x axis) in the PcrA-RNAP
896
complex (blue) compared to PcrA alone (red). The magenta rectangle highlights strong
897
protection of the CTD afforded by interaction with RNAP as expected based on results
898
presented earlier in this manuscript. The blue rectangle highlights a second region of strong
899
protection within the 2A domain of PcrA. (B) The PcrA helicase core (homology model from
900
PDB: 3PJR) showing domain organisation. Figure 2. The PcrA-CTD binds to a conserved motif in the SI1 domain of RNAP (A)
832 Two of the
870
structures are homology models as indicated. (B) Sequences of putative helicase interaction
871
motifs in four known PcrA partner proteins. (C) Pulldown of Myc-tagged PcrA from B. 872
subtilis cell extracts using UvrB as bait (for details see the Methods). Mutation of the
873 Figure 3. Many PcrA partner proteins contain the helicase interaction motif (A) Putative
868
helicase interaction motif (blue) in known PcrA interaction partners from B. subtilis. A beta
869
hairpin structure (blue) is formed by the interaction motif in each of the proteins. Two of the
870
structures are homology models as indicated. (B) Sequences of putative helicase interaction
871
motifs in four known PcrA partner proteins. (C) Pulldown of Myc-tagged PcrA from B. 872
subtilis cell extracts using UvrB as bait (for details see the Methods). Mutation of the
873 conserved glutamate (E233A) in the putative helicase interaction motif dramatically reduces
874
PcrA pulldown. PcrA was detected using an anti-Myc antibody (upper gel). Equivalent
875
loading of WT and mutant UvrB was confirmed by Coomassie staining (lower gel). (D) and
876
(E) Pulldown of RNAP from B. subtilis cell extracts using biotinylated PcrA as bait. Where
877
indicated the prey was supplemented with purified UvrB or Mfd. Free UvrB, but not Mfd,
878
competes for the interaction site formed between PcrA and RNA polymerase. Error bars
879
show the SEM of three independent experiments. Two-tailed Student’s t test determined
880
statistical significance (*p value < 0.05). 881 conserved glutamate (E233A) in the putative helicase interaction motif dramatically reduces
874
PcrA pulldown. PcrA was detected using an anti-Myc antibody (upper gel). Equivalent
875
loading of WT and mutant UvrB was confirmed by Coomassie staining (lower gel). (D) and
876
(E) Pulldown of RNAP from B. subtilis cell extracts using biotinylated PcrA as bait. Where
877
indicated the prey was supplemented with purified UvrB or Mfd. Free UvrB, but not Mfd,
878
competes for the interaction site formed between PcrA and RNA polymerase. Error bars
879
show the SEM of three independent experiments. Two-tailed Student’s t test determined
880
statistical significance (*p value < 0.05). 881 conserved glutamate (E233A) in the putative helicase interaction motif dramatically reduces
874
PcrA pulldown. PcrA was detected using an anti-Myc antibody (upper gel). Equivalent
875
loading of WT and mutant UvrB was confirmed by Coomassie staining (lower gel). (D) and
876
(E) Pulldown of RNAP from B. igure 5. The PcrA helicase core binds to RNAP close to the RNA and DNA exit Figure 5. The PcrA helicase core binds to RNAP close to the RNA and DNA exit
911
channel (A) Differential protection plot for the β subunit of RNAP showing the differential
912
relative uptake at 10 minutes of exposure to deuterium for a PcrA-RNAP complex (blue)
913
compared to RNAP alone (red). Green and yellow rectangles highlight regions of RpoB that
914
are significantly protected in the complex state. (B) Differential protection plot for the β′
915
subunit of RNAP showing the differential relative uptake at 10 minutes of exposure to
916
deuterium for a PcrA-RNAP complex compared to RNAP alone. Purple and pink rectangles
917
highlight regions of RpoC that are significantly protected in the complex state. (C) Three
918
views of the B. subtilis TEC (PDB: 6WVJ) showing regions protected by interaction with
919
PcrA. DNA is shown in yellow and black. RNA is green. Coloured circles correspond to the
920
protected regions highlighted in panels A and B. The green circle highlights the helicase
921
interaction motif already identified as the site of binding of the PcrA-CTD. Note that the
922
other major protection sites surround the DNA/RNA exit channels. The crosslinking sites
923
(orange) are from reference (Epshtein et al., 2014). 924 Figure 5 – Figure Supplement 1. HDX protection data for the remaining RNAP
925 Figure 2. The PcrA-CTD binds to a conserved motif in the SI1 domain of RNAP (A)
832 The DNA substrate is shown in black and orange
901
and the CTD (which is disordered in this structure) is indicated as a purple circle. (C) The
902
same structure showing the mapping of the HDX-protection data (bottom; blue indicates
903
protection in the complex, red indicates exposure and green indicates a lack of data). Note
904 that the HDX-protection data maps largely to one face of the helicase within domains 2A and,
905
to a lesser extent, 2B. 906 that the HDX-protection data maps largely to one face of the helicase within domains 2A and,
905
to a lesser extent, 2B. 906 Figure 4 – Figure Supplement 1. Deuterium uptake dynamics for regions of PcrA and
907
RNAP that are significantly protected in the PcrA-RNAP complex. The plots show all
908
four timepoints for the protected regions in (A) PcrA, (B-C) the RNAP β subunit, with the
909
spiral representing the β-flap tip region and (D-E) the RNAP β’ subunit. 910 Figure 4 – Figure Supplement 1. Deuterium uptake dynamics for regions of PcrA and
907
RNAP that are significantly protected in the PcrA-RNAP complex. The plots show all
908
four timepoints for the protected regions in (A) PcrA, (B-C) the RNAP β subunit, with the
909
spiral representing the β-flap tip region and (D-E) the RNAP β’ subunit. 910 Figure 5 – Figure Supplement 1. HDX protection data for the remaining RNAP
925 Figure 5 – Figure Supplement 1. HDX protection data for the remaining RNAP
925
subunits in the PcrA-RNAP interaction experiment. DYNAMX HDX butterfly plots for
926
the (A) α and (B) ε subunits. Negative values represent protected regions and positive values,
927
regions that are exposed upon binding of PcrA. For difference plots, four replicates were
928
performed for each of the four independent colour-coded time points shown. Grey shading
929
indicates the standard deviation of all charge states and replicates per peptide. 930 subunits in the PcrA-RNAP interaction experiment. DYNAMX HDX butterfly plots for
926
the (A) α and (B) ε subunits. Negative values represent protected regions and positive values,
927
regions that are exposed upon binding of PcrA. For difference plots, four replicates were
928
performed for each of the four independent colour-coded time points shown. Grey shading
929
indicates the standard deviation of all charge states and replicates per peptide. 930 Figure 6. PcrA unwinds DNA-RNA hybrids in vitro and supresses R-loops in vivo (A)
931
DNA and RNA substrates used for helicase assays. Thick lines represent DNA strands and
932
thin lines, RNA strands. The oligonucleotides used to form these substrates are shown in
933
Table 7. (B) Quantification of unwinding as a function of PcrA concentration for the 3′-tailed
934
substrates shown in panel A. The substrate is only efficiently unwound if the longer of the
935
two nucleic acids strands is DNA. Error bars show the standard deviation of at least 3
936 Figure 6. PcrA unwinds DNA-RNA hybrids in vitro and supresses R-loops in vivo (A)
931
DNA and RNA substrates used for helicase assays. Thick lines represent DNA strands and
932
thin lines, RNA strands. The oligonucleotides used to form these substrates are shown in
933
Table 7. (B) Quantification of unwinding as a function of PcrA concentration for the 3′-tailed
934
substrates shown in panel A. The substrate is only efficiently unwound if the longer of the
935
two nucleic acids strands is DNA. Error bars show the standard deviation of at least 3
936 independent experiments. (C) The ATPase activity of PcrA is strongly stimulated by single-
937
stranded DNA but not single-stranded RNA. Error bars show the standard deviation of at
938
least 3 independent experiments. (D) Anti R-loop antibody (S9.6) dot blot for nucleic acid
939
samples purified from three strains of B. subtilis. Figure 5 – Figure Supplement 1. HDX protection data for the remaining RNAP
925 These strains contain an integrated
940
expression cassette for either wild type PcrA or a dominant negative form of PcrA (E224Q). 941
The control strain (EV) contains an integrated but empty expression cassette. The S9.6 signal
942
is normalized using methylene blue as a stain for all DNA. Note the high S9.6 signal for the
943
strain expressing PcrA E224Q. (E) Quantification of four independent repeats of the
944
experiment shown in (c). Error bars show the SEM. Expression of a dominant negative form
945
of PcrA increases R-loop content (relative to DNA) in B. subtilis by ~2.5 fold. (F)
946
Quantification of pulldown experiments of RNAP from B. subtilis cell extracts using
947
biotinylated PcrA as bait and supplemented with purified PcrA WT or CTD. Addition of the
948
CTD competes with WT PcrA to bind to RNAP. Error bars show the SEM of three
949
independent repeats. (G) Relative R-loop levels in strains of B. subtilis expressing free CTD,
950
a CTD mutant that interacts weakly with RNAP, or with a control expression cassette. A
951
ΔrnhC strain is shown as a control for elevated R-loop levels. Error bars show the SEM of at
952
least 3 independent experiments. In all panels the statistical significance was determined
953
using two-tailed Student’s t test (*p value < 0.05, **p value < 0.01, ***p value < 0.001,
954
****p value < 0.0001). 955 independent experiments. (C) The ATPase activity of PcrA is strongly stimulated by single-
937
stranded DNA but not single-stranded RNA. Error bars show the standard deviation of at
938
least 3 independent experiments. (D) Anti R-loop antibody (S9.6) dot blot for nucleic acid
939
samples purified from three strains of B. subtilis. These strains contain an integrated
940
expression cassette for either wild type PcrA or a dominant negative form of PcrA (E224Q). 941
The control strain (EV) contains an integrated but empty expression cassette. The S9.6 signal
942
is normalized using methylene blue as a stain for all DNA. Note the high S9.6 signal for the
943
strain expressing PcrA E224Q. (E) Quantification of four independent repeats of the
944
experiment shown in (c). Error bars show the SEM. Expression of a dominant negative form
945
of PcrA increases R-loop content (relative to DNA) in B. subtilis by ~2.5 fold. (F)
946
Quantification of pulldown experiments of RNAP from B. Figure 5 – Figure Supplement 1. HDX protection data for the remaining RNAP
925 subtilis cell extracts using
947
biotinylated PcrA as bait and supplemented with purified PcrA WT or CTD. Addition of the
948
CTD competes with WT PcrA to bind to RNAP. Error bars show the SEM of three
949
independent repeats. (G) Relative R-loop levels in strains of B. subtilis expressing free CTD,
950
a CTD mutant that interacts weakly with RNAP, or with a control expression cassette. A
951
ΔrnhC strain is shown as a control for elevated R-loop levels. Error bars show the SEM of at
952
least 3 independent experiments. In all panels the statistical significance was determined
953
using two-tailed Student’s t test (*p value < 0.05, **p value < 0.01, ***p value < 0.001,
954
****p value < 0.0001). 955 Figure 6 – Figure Supplement 1. PcrA requires a 3′-ssDNA tail to unwind DNA
956
duplexes. (A) Representative helicase assays using 3′-tailed or duplex substrates consisting
957
of annealed DNA (black) and RNA (red) oligonucleotides. The sequences of the
958
oligonucleotides are in Table 7). (B) Quantification of unwinding as a function of PcrA
959
concentration for 3′-tailed and fully duplex blunt-ended substrates (indicated by the prefix b). 960
For the 3′-tailed substrates, the substrate is only efficiently unwound if the longer of the two
961
nucleic acids strands is DNA. The fully duplex substrates are not unwound efficiently. Part of
962
this data is reproduced from Figure 6 for comparison. Error bars show the standard deviation
963
of at least 3 independent experiments. 964 Figure 6 – Figure Supplement 2. Overexpression of PcrA E224Q causes growth defects
965
in WT and Δmfd B. subtilis. Growth curves for (A) WT strains or (B) Δmfd strains
966
overexpressing wild type PcrA or E224Q at 37ºC. The overexpression was induced with 1
967
mM IPTG at the indicated times. The overexpression cassette was integrated in the
968 Figure 6 – Figure Supplement 2. Overexpression of PcrA E224Q causes growth defects
965
in WT and Δmfd B. subtilis. Growth curves for (A) WT strains or (B) Δmfd strains
966
overexpressing wild type PcrA or E224Q at 37ºC. The overexpression was induced with 1
967
mM IPTG at the indicated times. The overexpression cassette was integrated in the
968 codirectional orientation. Three biological replicates were averaged at each time point and
969
error bars represent the standard error of the mean. 970 Figure 6 – Figure Supplement 3. Figure 7. Hypothetical models for PcrA-dependent R-loop suppression during
983 Figure 7. Hypothetical models for PcrA-dependent R-loop suppression during
983
transcription. R-loops form during transcription but lead to genomic instability and are
984
therefore targeted for removal by PcrA. In the co-directional model, PcrA helicase (green)
985
interacts with an R-loop associated RNAP (blue) via the PcrA-CTD (magenta). It then
986
engages the template DNA strand before translocating in the 3′>5′ direction and directly
987
unwinding the DNA:RNA hybrid. In this model, R-loops may be unwound as they are
988
formed if the helicase is in close contact with the TEC. In the backtracking model, PcrA
989
binds to the displaced (non-template) strand behind RNAP and pulls it backwards, thereby
990
unwinding the R-loop indirectly (see main text for further details and discussion). 991 Figure 7. Hypothetical models for PcrA-dependent R-loop suppression during
983
transcription. R-loops form during transcription but lead to genomic instability and are
984
therefore targeted for removal by PcrA. In the co-directional model, PcrA helicase (green)
985
interacts with an R-loop associated RNAP (blue) via the PcrA-CTD (magenta). It then
986
engages the template DNA strand before translocating in the 3′>5′ direction and directly
987
unwinding the DNA:RNA hybrid. In this model, R-loops may be unwound as they are
988
formed if the helicase is in close contact with the TEC. In the backtracking model, PcrA
989
binds to the displaced (non-template) strand behind RNAP and pulls it backwards, thereby
990
unwinding the R-loop indirectly (see main text for further details and discussion). 991 Figure 7 – Figure Supplement 1. Working model for the interaction between PcrA and a
992
TEC. The figure shows the PcrA core (homology model from PDB: 3PJR) coloured by
993
domain organisation (red, blue, green and yellow) as in Figure 4B. This is joined to the PcrA-
994
CTD (template PDB: 5DMA, purple ribbons) via an extended linker (manually-modeled;
995
purple spheres). The B. subtilis TEC (PDB: 6WVJ) with the addition of the β-flap tip (PDB:
996
6FLQ) and extended nucleic acids (PDB: 6ALF) is shown as a surface representation. Light
997
blue and dark grey regions on the TEC show protected regions and regions for which there is
998
no data, respectively, from the HDX-MS experiment. Orange residues show the crosslinks
999
identified in the E. coli XL-MS experiment (Epshtein et al., 2014). The template strand is
1000 Figure 7 – Figure Supplement 1. Figure 5 – Figure Supplement 1. HDX protection data for the remaining RNAP
925 Overexpression of the PcrA CTD in B. subtilis and
971
deletion of uvrD in E. coli increase R-loop levels in the cell. (A) Representative anti-β
972
subunit immunoblot (upper panel) and Coomassie-stained gel (lower panel) of a pulldown
973
from B. subtilis extracts using biotinylated PcrA as bait. The gel shows that both free PcrA
974
(full length) and free CTD can compete with the bait (tagged PcrA) to bind to RNAP. Where
975
indicated, 1.5 µM untagged PcrA or CTD were added to the cell lysate. Quantification of
976
these data is shown in Figure 6F. (B) and (C) Representative dot blots of B. subtilis CTD
977
overexpression strains (as indicated) using the S9.6 antibody. Methylene blue was used as
978
loading control. Quantification of these data is shown in Figure 6G. (D) and (E),
979 Representative RNA/DNA hybrid dot blot of genomic DNA from the indicated E. coli strains
980
and quantification. Error bars show the SEM of 4 independent experiments. In all panels the
981
statistical significance was determined using two-tailed Student’s t test (*p value < 0.05). 982 Representative RNA/DNA hybrid dot blot of genomic DNA from the indicated E. coli strains
980
and quantification. Error bars show the SEM of 4 independent experiments. In all panels the
981
statistical significance was determined using two-tailed Student’s t test (*p value < 0.05). 982 black, the non-template strand is yellow and the RNA is green. This simple model is intended
1001
to show that it is possible to satisfy the large majority of the protected regions we identify on
1002
RNAP by docking a single PcrA monomer. Importantly, note that the PcrA linker region is of
1003
an appropriate length to allow simultaneous binding of the PcrA helicase core to the
1004
DNA/RNA exit channels and the PcrA-CTD to the distant SI1 domain.
1005 Figure 7. Hypothetical models for PcrA-dependent R-loop suppression during
983 Working model for the interaction between PcrA and a
992
TEC. The figure shows the PcrA core (homology model from PDB: 3PJR) coloured by
993
domain organisation (red, blue, green and yellow) as in Figure 4B. This is joined to the PcrA-
994
CTD (template PDB: 5DMA, purple ribbons) via an extended linker (manually-modeled;
995
purple spheres). The B. subtilis TEC (PDB: 6WVJ) with the addition of the β-flap tip (PDB:
996
6FLQ) and extended nucleic acids (PDB: 6ALF) is shown as a surface representation. Light
997
blue and dark grey regions on the TEC show protected regions and regions for which there is
998
no data, respectively, from the HDX-MS experiment. Orange residues show the crosslinks
999
identified in the E. coli XL-MS experiment (Epshtein et al., 2014). The template strand is
1000 black, the non-template strand is yellow and the RNA is green. This simple model is intended
1001
to show that it is possible to satisfy the large majority of the protected regions we identify on
1002
RNAP by docking a single PcrA monomer. Importantly, note that the PcrA linker region is of
1003
an appropriate length to allow simultaneous binding of the PcrA helicase core to the
1004
DNA/RNA exit channels and the PcrA-CTD to the distant SI1 domain. 1005 black, the non-template strand is yellow and the RNA is green. This simple model is intended
1001
to show that it is possible to satisfy the large majority of the protected regions we identify on
1002
RNAP by docking a single PcrA monomer. Importantly, note that the PcrA linker region is of
1003
an appropriate length to allow simultaneous binding of the PcrA helicase core to the
1004
DNA/RNA exit channels and the PcrA-CTD to the distant SI1 domain. 1005 1006 Table 1. Sequence coverage and redundancy for the proteins analysed by HDX-MS. 007
Protein
CTD-RNAP complex
PcrA-RNAP complex
Sequence
coverage (%)
Sequence
redundancy
Sequence
coverage (%)
Sequence
redundancy
CTD/
PcrA
87.2
3.61
78.5
2.38
α
93.9
3.48
89.2
2.59
β
83.7
2.84
84.9
2.24
β’
88.7
2.58
80.3
2.05
δ
75.7
1.79
17.3
1.53
ε
100
4.26
91.3
2.38
ω
-
-
-
-
σA
42.6
1.29
-
-
008 1007 Table 1. Sequence coverage and redundancy for the proteins analysed by HDX-MS. Table 2. Mfd pulls down RNAP subunits from Bacillus subtilis cell extracts. Figure 7. Hypothetical models for PcrA-dependent R-loop suppression during
983 Proteins
1010
enriched in the biotinylated Mfd bait condition compared to the no-bait control pulldown (see
1011
Methods for details). Subunits of RNAP that are enriched in the Mfd pulldown are indicated
1012
in bold text. Accession refers to the UniProt accession code, GN refers to gene name and FC,
1013
to fold change. 1014 Table 2. Mfd pulls down RNAP subunits from Bacillus subtilis cell extracts. Proteins
1010
enriched in the biotinylated Mfd bait condition compared to the no-bait control pulldown (see
1011
Methods for details). Subunits of RNAP that are enriched in the Mfd pulldown are indicated
1012
in bold text. Accession refers to the UniProt accession code, GN refers to gene name and FC,
1013
to fold change. 1014 Accession
Description
LogFC
P37474
Transcription-repair-coupling factor GN=mfd
7.887
O34863
UvrABC system protein A GN=uvrA
6.127
O34628
Uncharacterized protein YvlB GN=yvlB
5.779
Q795Q5
Uncharacterized membrane protein YttA GN=yttA
3.735
O34942
ATP-dependent DNA helicase RecG GN=recG
3.304
Q06796
50S ribosomal protein L11 GN=rplK
3.269
P20429
DNA-directed RNA polymerase subunit alpha
GN=rpoA
2.899
O32006
Resolvase homolog YokA GN=yokA
2.898
O07542
UPF0342 protein YheA GN=yheA
2.800
O35011
DNA-directed RNA polymerase subunit omega
GN=rpoZ
2.780
P39592
Uncharacterized HTH-type transcriptional regulator YwbI
GN=ywbI
2.667
Q08792
Uncharacterized HTH-type transcriptional regulator YcxD
GN=ycxD
2.654
O34949
Uncharacterized HTH-type transcriptional regulator YkoM
GN=ykoM
2.600
O34381
HTH-type transcriptional regulator PksA GN=pksA
2.512 1015 Table 3. List of B. subtilis and E. coli strains used in this work. 1016 Strain
Genotype
Reference
Plasmid to generate the strain
Parent strain
MH5636
rpoC-His10::cat trpC2
(Qi and
Hulett, 1998) -
JH642
1a1
trpC2
(Koo et al.,
2017)
-
-
IU79
lacA::Pxyl-myc-PcrA::mls insb pMAP39 in pcrA
This work
pMAP39(Petit et al., 1998) and
pBS2EXylRPxylA(V2)- myc-PcrA
1a1
IU6
amyE::Phyperspank::spec HO trpC2
This work
pDRIII (HO)
1a1
IU35
amyE::Phyperspank::spec CD trpC2
This work
pDRIII (CD)
1a1
IU41
amyE::Phyperspank-mycPcrA::spec CD trpC2
This work
pDRIII(CD)-mycPcrA
1a1
IU56
amyE::Phyperspank-mycPcrA-E224Q::spec CD trpC2
This work
pDRIII(CD)-mycPcrA-E224Q
1a1
BKK00550 Δmfd:kan trpC2
(Koo et al.,
2017)
-
1a1
IU60
amyE::Phyperspank::spec Δmfd:kan trpC2
This work
pDRIII (CD)
BKK00550
IU61
amyE::Phyperspank-myc-PcrA::spec Δmfd:kan trpC2
This work
pDRIII(CD)-mycPcrA
BKK00550
IU62
amyE::Phyperspank-myc-PcrA-E224Q::spec Δmfd:kan trpC2
This work
pDRIII(CD)-mycPcrA-E224Q
BKK00550
IU65
amyE::Phyperspank-myc-PcrAΔCTD-E224Q::spec trpC2
This work
pDRIII(CD)-mycPcrAΔCTD-E224Q
1a1
IU66
amyE::Phyperspank-myc-PcrAΔCTD-E224Q::spec Δmfd:kan trpC2 This work
pDRIII(CD)-mycPcrAΔCTD-E224Q
BKK00550
BKK28620 trpC2 ΔrnhC::kan
(Koo et al.,
2017)
-
1a1
IU3
amyE::Phyperspank::spec(HO) trpC2
This work
pDRIII(HO)
BKK28620
IU5
amyE::Phyperspank-myc-CTD::spec (HO) trpC2
This work
pDRIII(HO)-mycCTD
BKK28620
IU9
amyE::Phyperspank-myc-CTD-K727A::spec (HO) trpC2
This work
pDRIII(HO)-mycCTD-K727A
BKK28620
TB28
E. Figure 7. Hypothetical models for PcrA-dependent R-loop suppression during
983 Coli ΔlacIZYA
(Bernhardt
and De Boer,
2004)
-
MG1655
N6632
E. Coli ΔuvrD::dhfr
(Guy et al.,
2009)
-
MG1655 1018 Table 4. Plasmids used in this work 1019 Table 4. Plasmids used in this work Plasmid name
Vector
Insert(s)
Reference
pET22b-PcrA
pET22b
PcrA
(Gwynn et al.,
2013)
pET22b-bioPcrA
pET22b
BioPcrA
(Gwynn et al.,
2013)
pET22b-PcrAV448C
pET22b
PcrA V448C
This work
pET22b-PcrAΔCTD
pET22b
PcrAΔCTD
This work
pET47b-CTD
pET47b
CTD
This work
pET28a-rpoB
pET28a
RNAP β subunit
This work
pET28a-rpoB-E310A
pET28a
RNAP β subunit E310A
This work
pET28a-rpoB-E310K
pET28a
RNAP β subunit E310K
This work
pET47b-UvrB
pET47b
UvrB
This work
pET47b-UvrB-E233A
pET47b
UvrB E233A
This work
pET22b-Mfd
pET22b
Mfd
This work
pBS2EXyl-mycPcrA
pBS2EXylRPxylA (V2)
pBS0EPliaI
mycPcrA
(Popp et al.,
2017)
pDRIII(HO)
pDRIII(HO)
-
(Fisher et al.,
2017)
pDRIII(CD)
pDRIII(CD)
-
This work
pDRIII(CD)-mycPcrA
pDRIII(CD)
mycPcrA
This work
pDRIII(HO)-mycPcrA
pDRIII(HO)
mycPcrA
This work
pDRIII(CD)-mycPcrA-E224Q
pDRIII(CD)
PcrA-E224Q
This work
pDRIII(CD)-mycPcrAΔCTD-E224Q pDRIII(CD)
PcrAΔCTD-E224Q
This work
pDRIII(HO)-mycCTD
pDRIII(HO)
mycCTD
This work
pDRIII(HO)-mycCTD-K727A
pDRIII(HO)
mycCTD-K727A
This work
pMAP39
pBSspec+
(nt 201 to 699 of pcrA) at
HincII
(Petit et al.,
1998) 1022 Table 5. Oligonucleotides used in this work for cloning (all sequences written 5′>3′)
Name
Sequence
Purpose
PcrA_V448C_F
AAGCGATTCAGCAGTGTGATTTTATCG
SDM
PcrA_V448C_R
CGATAAAATCACACTGCTGAATCGCTT
SDM
His-
PcrA_CTD_XmaI_F
GACTCCCGGGAAAGAAACAAGAGCGACGTC
PcrA CTD (664-739) subcloning
in pET47b
BsuPcrA_BamHI_R
GATCGGATCCTTACTGCTTTTCAATAGGAGCAAATG
PcrA CTD (664-739) subcloning
in pET47b
PcrA_E224Q_F
CATCCACGTTGATCAGTATCAGGATACGAAC
SDM
PcrA_E224Q_R
GTTCGTATCCTGATACTGATCAACGTGGATG
SDM
BSuPcrA_deltaCTD_F
CCTAAATGAGAAATAAGAAACAAG
SDM
BSuPcrA_deltaCTD_R
CTTGTTTCTTATTTCTCATTTAGG
SDM
bsurpob_ndeI_F
CAGGTGCATATGTTGACAGGTCAACTAGTTCAGTATG
RpoB subcloning in pET28a
bsurpob_xhoI_R
TTAATTCTCGAGTTATTCTTTTGTTACTACATCGCGTTC
RpoB subcloning in pET28a
rpoB_E301A_F
GATCCTGAAACAGGAGCAATCCTTGCTGAAAAAG
SDM
rpoB_E301A_R
CTTTTTCAGCAAGGATTGCTCCTGTTTCAGGATC
SDM
rpoB_E310K_F
GATCCTGAAACAGGAAAAATCCTTGCTGAAAAAG
SDM
rpoB_E310K_R
CTTTTTCAGCAAGGATTTTTCCTGTTTCAGGATC
SDM
BSuUvrB_SmaI_F
AGCAGCCCGGGGTGAAAGATCGCTTTGAGTTAGTCTCGAAATATC
UvrB subcloning in pET47b
BsuUvrB_XhoI_R
GCATCTCGAGTCATCCTTCCGCTTTTAGCTCTAAAAGTAAATC
UvrB subcloning in pET47b
BsuUvrB_E233A_F
GCTGACAGGAGCAATTCTCGGCGAC
SDM
BsuUvrB_E233A_R
GTCGCCGAGAATTGCTCCTGTCAGC
SDM
bMfd_5′NdeI_F
TTAATCATATGGACAACATTCAAACCTTT
Mfd subcloning in pET22b
bMfd_XhoI_3′_R
ATTAACTCGAGTTACGTTGATGAAATGGTTTG
Mfd subcloning in pET22b
bMfd_mutA2586G_F
CCTGACGCGAAGGTAGCGTATGCGCATGGGAAAATG
SDM
bMfd_mutA2586G_R
CATTTTCCCATGCGCATACGCTACCTTCGCGTCAGG
SDM
bMfdmutT2361C_F
CGCGTACGCTGCACATGTCTATGCTTG
SDM
bMfd_mutT2361C_R
CAAGCATAGACATGTGCAGCGTACGCG
SDM
pDRIII-inver-upsR-
BlpI
TACTTAGCTAAGCCTAACTCACATTAATTGCGTTGCG
Invert MCS in pDRIII
pDRIII-inver-downsF-
BamHI
TAATTTGGATCCCTAAGCAGAAGGCCATCCTG
Invert MCS in pDRIII
CTD_HindIII_F
ATCGTAAGCTTAAAGAAACAAGAGCGACGTC
CTD subcloning in pDRIII
CTD_SphI_R
GCTTTGCATGCTTACTGCTTTTCAATAGGAGCAAATG
CTD and PcrA subcloning in
pDRIII myc-
PcrA_5′SalI_RBS_F
CGTTGTCGACAGGAGGTATACATATGGAGCAAAAG
PcrA subcloning in pDRIII
PcrA_K727A_F
CTGTCGGCGTGGCACGCCTGTTAGCAG
SDM
PcrA_K727A_R
CTGCTAACAGGCGTGCCACGCCGACAG
SDM 1024 Table 6. Oligonucleotides used in this work for assembling the TEC (all sequences 5′>3′). Figure 7. Hypothetical models for PcrA-dependent R-loop suppression during
983 1025
Name
Strand
Modification
Sequence (5′-3′)
RNA I
-
-
AUCGAGAGG
Standard scaffold
TS
5′ Cy5
TGTCACTTCGCCGTGTCCCTCTCGATGGCTGTAAG TATACT
NTS
AGTATACTTACAGCCATCGAGAGGGACACGGCGAAGTG ACA
Downstream gap in TS
TS
5′ Cy5
CGTGTCCCTCTCGATGGCTGTAAGTATACT
NTS
AGTATACTTACAGCCATCGAGAGGGACACGGCGAAGTGACA
Upstream gap in NTS
TS
5′ Cy5
TGTCACTTCGCCGTGTCCCTCTCGATGGCTGTAAGTATAC
NTS
AGCCATCGAGAGGGACACGGCGAAGTGACA
Short scaffold
TS
5′ Cy5
CGTGTCCCTCTCGATGGCT
NTS
AGCCATCGAGAGGGACACG
No duplex downstream
TS
5′ Cy5
CGTGTCCCTCTCGATGGCTGTAAGTATACT
NTS
AGTATACTTACAGCCATCGAGAGGGACACG
No duplex upstream
TS
5′ Cy5
TGTCACTTCGCCGTGTCCCTCTCGATGGCT
NTS
AGCCATCGAGAGGGACACGGCGAAGTGACA
1026 6. Oligonucleotides used in this work for assembling the TEC (all sequences 5′>3′). 1025 1026 Table 7. Oligonucleotides used in this work for helicase assays (all sequences 5′>3′). 027
Oligo
name
Type
Sequence (5′-3′)
IU_1
DNA
GGGAGCCGGTCTGCGTCTGGTGTACTCTTCTGCTTTCTCG
IU_1R
RNA
GGGAGCCGGUCUGCGUCUGGUGUACUCUUCUGCUUUCUCG
IU_2
DNA
CCAGACGCAGACCGGCTCCC
IU_2R
RNA
CCAGACGCAGACCGGCUCCC
IU_3
DNA
GGGAGCCGGTCTGCGTCTGG
IU_3R
RNA
GGGAGCCGGUCUGCGUCUGG
028 1027 1028 References
1029 Adebali, O., Chiou, Y.-Y., Hu, J., Sancar, A., and Selby, C.P. (2017a). Genome-wide
1030
transcription-coupled repair in Escherichia coli is mediated by the Mfd translocase. Proc. 1031
Natl. Acad. Sci. 114, E2116–E2125. 1032 Adebali, O., Sancar, A., and Selby, C.P. (2017b). Mfd translocase is necessary and sufficient
1033
for transcription-coupled repair in Escherichia coli. J. Biol. Chem. jbc.C117.818807. 1034 Aguilera, A., and García-Muse, T. (2012). R Loops: From Transcription Byproducts to
1035
Threats to Genome Stability. Mol. Cell 46, 115–124. 1036 Anagnostopoulos, C., and Spizizen, J. (1961). Requirements for Transformation in Bacillus
1037
Subtilis. J. Bacteriol. 81, 741–746. 1038 Arthur, H.M., and Lloyd, R.G. (1980). Hyper-recombination in uvrD mutants of Escherichia
1039
coli K-12. MGG Mol. Gen. Genet. 180, 185–191. 1040 Bernhardt, T.G., and De Boer, P.A.J. (2004). Screening for synthetic lethal mutants in
1041
Escherichia coli and identification of EnvC (YibP) as a periplasmic septal ring factor with
1042
murein hydrolase activity Mol Microbiol 52 1255 1269
1043 Bernhardt, T.G., and De Boer, P.A.J. (2004). Screening for synthetic lethal mutants in
1041
Escherichia coli and identification of EnvC (YibP) as a periplasmic septal ring factor with
1042 Bernhardt, T.G., and De Boer, P.A.J. (2004). Screening for synthetic lethal mutants in
1041 Escherichia coli and identification of EnvC (YibP) as a periplasmic septal ring factor with
1042
murein hydrolase activity. Mol. Microbiol. 52, 1255–1269. 1043 Bird, L.E., Brannigan, J.A., Subramanya, H.S., and Wigley, D.B. (1998). Characterisation of
1044
Bacillus stearothermophilus PcrA helicase: Evidence against an active rolling mechanism. 1045
Nucleic Acids Res. 26, 2686–2693. 1046 Boguslawski, S.J., Smith, D.E., Michalak, M.A., Mickelson, K.E., Yehle, C.O., Patterson,
1047
W.L., and Carrico, R.J. (1986). Characterization of monoclonal antibody to DNA · RNA and
1048
its application to immunodetection of hybrids. J. Immunol. Methods 89, 123–130. 1049 Boubakri, H., De Septenville, A.L., Viguera, E., and Michel, B. (2010). The helicases DinG,
1050
Rep and UvrD cooperate to promote replication across transcription units in vivo. EMBO J. 1051
29, 145–157. 1052 Brosh, R.M., and Matson, S.W. (1995). Mutations in motif II of Escherichia coli DNA
1053 helicase II render the enzyme nonfunctional in both mismatch repair and excision repair with
1054
differential effects on the unwinding reaction. J. Bacteriol. 177, 5612–5621. 1055 Bruand, C., and Ehrlich, S.D. (2000). UvrD-dependent replication of rolling-circle plasmids
1056
in Escherichia coli. Mol. Microbiol. 35, 204–210. 1057 (
)
p
p
g
p
in Escherichia coli. Mol. Microbiol. References
1029 35, 204–210. 1057 Burby, P.E., and Simmons, L.A. (2017). MutS2 promotes homologous recombination in
1058 Burby, P.E., and Simmons, L.A. (2017). MutS2 promotes homologous recombination in
1058
Bacillus subtilis. J. Bacteriol. 199, 1–13. 1059 Bacillus subtilis. J. Bacteriol. 199, 1–13. 1059 Chambers, A.L., Smith, A.J., and Savery, N.J. (2003). A DNA translocation motif in the
1060
bacterial transcription-repair coupling factor, Mfd. Nucleic Acids Res. 31, 6409–6418. 1061 Chan, Y.A., Aristizabal, M.J., Lu, P.Y.T., Luo, Z., Hamza, A., Kobor, M.S., Stirling, P.C.,
1062 and Hieter, P. (2014). Genome-Wide Profiling of Yeast DNA:RNA Hybrid Prone Sites with
1063
DRIP-Chip. PLoS Genet. 10. 1064 Chen, C., Natale, D.A., Finn, R.D., Huang, H., Zhang, J., Wu, C.H., and Mazumder, R. 1065 (2011). Representative Proteomes: A Stable, Scalable and Unbiased proteome set for
1066
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English
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Residual angulation of distal tibial diaphyseal fractures in children younger than ten years
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Journal of orthopaedic surgery and research
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© 2014 Jung 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/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. * Correspondence: jrkeem@jbnu.ac.kr
2Department of Orthopaedics Surgery, Chonbuk National University Medical
School, 567 Baekje-ro, Dukjin-gu, Jeonju 561-756, South Korea
Full list of author information is available at the end of the article Sung Taek Jung1, Hyuk Park2, Ju-Hyung Lee3 and Jung Ryul Kim2* Sung Taek Jung1, Hyuk Park2, Ju-Hyung Lee3 and Jung Ryul Kim2* Sung Taek Jung1, Hyuk Park2, Ju-Hyung Lee3 and Jung Ryul Kim2* Abstract Background: The purpose of this study was to evaluate the factors that influence residual angulation after treating
pediatric distal tibial diaphyseal fractures. Methods: We retrospectively reviewed the records of 75 children under the age of ten who were treated at two
referral centers for distal tibial diaphyseal fractures. The mean patient age was 6.8 ± 2.3 years, and the mean
follow-up duration was 4.1 ± 1.3 years (range, 3 to 6 years). Early postoperative and late follow-up radiographs were
used to measure angulation. Results: Twenty-four patients had valgus angulations >5° at the final follow-up. There was no varus, or anteroposterior
residual angulations >5°. There was more residual valgus angulation when the postoperative angulation was >5°
(p = 0.006) and when intramedullary nail and external fixators were applied for treatment (p = 0.004). Multivariate
logistic regression analysis showed that postoperative angulation (adjusted odds ratio (OR) 4.33, 95% confidence
interval (CI) 1.07–17.53) and treatment methods (intramedullary nail: adjusted OR 7.33, 95% CI 1.31–41.07; external fixator:
adjusted OR 11.35, 95% CI 1.91–67.40 compared with the cast group) were associated with residual deformity. Conclusions: Valgus angulation after pediatric distal tibial fractures persisted in this study sample. Accurate reduction
should be performed to prevent residual deformity. Conclusions: Valgus angulation after pediatric distal tibial fractures persisted in this study sample. Accurate reduction
should be performed to prevent residual deformity. Keywords: Residual deformity, Distal tibia, Diaphysis, Remodeling Jung et al. Journal of Orthopaedic Surgery and Research 2014, 9:84
http://www.josr-online.com/content/9/1/84 Jung et al. Journal of Orthopaedic Surgery and Research 2014, 9:84
http://www.josr-online.com/content/9/1/84 Jung et al. Journal of Orthopaedic Surgery and Research 2014, 9:84
http://www.josr-online.com/content/9/1/84 Jung et al. Journal of Orthopaedic Surgery and Research 2014, 9:84
http://www.josr-online.com/content/9/1/84 Background
h
l remodeling process in such injuries is complicated be-
cause of the activity of the muscles in the anterior and
lateral compartments of the lower leg and the decreased
growth potential of the distal tibia compared to that of the
proximal tibia [4-6]. Cozen investigated valgus deformities
after proximal tibial fractures in children [7]. In skeletally
immature patients, there is femur overgrowth after mid-
diaphyseal fracture; similarly, in patients under ten years
old, there may be increased valgus angulation of the tibia
after injury [8]. A possible cause of this valgus deformity
after tibia injury is overgrowth of the tibia with tethering
by the fibula. The normal process of bone remodeling in the diaphysis
and metaphysis of a growing child may realign initially
malunited fragments. This dynamic remodeling makes
the anatomic reduction less significant in a child than it
is in a comparable injury in an adult. Children’s bones
remodel in response to the normal stresses of body weight,
muscle action, joint reaction forces, and intrinsic control
mechanisms including the periosteum [1-3]. If there is any
residual angulation after bone union, there is spontaneous
correction. The physis responds to such malalignment by
differential growth, which aligns the shaft perpendicular to
the major joint reaction forces [4]. Recently, sufficient fracture stabilization and external
fixation for severe fracture can be achieved with intra-
medullary fixation in skeletally immature patients
[9-12]. However, despite high union rates, flexible intra-
medullary fixation and external fixation have significant
complications [13]. The most common complication
observed is malunion of at least 10° in either the sagittal
or coronal plane [13]. However, prior studies have not In general, axial malalignments will remodel in children
after forearm or femoral shaft fractures. However, tibial
angular deformities, especially distal tibial fractures, are
less favorable injuries with regard to remodeling. The * Correspondence: jrkeem@jbnu.ac.kr
2Department of Orthopaedics Surgery, Chonbuk National University Medical
School, 567 Baekje-ro, Dukjin-gu, Jeonju 561-756, South Korea
Full list of author information is available at the end of the article Page 2 of 7 Jung et al. Journal of Orthopaedic Surgery and Research 2014, 9:84
http://www.josr-online.com/content/9/1/84 Table 1 Patient demographics and classification
Parameters
Values
Period
2001. 6–2008. Methods Written informed consent was obtained from the patient’s
guardian/parent/next of kin for the publication of this
report and any accompanying images. This study was
approved by the institutional review board of Chonbuk
National University, Republic of Korea. All patients
under ten years old who sustained distal tibial fractures
between 2001 and 2008 and were treated at one of two
tertiary referral centers were included. On anteroposterior
plain radiographs, the total tibial length (from the center
of the tibial spine to the center of the tibial plafond) was
divided into three equal parts (proximal 1/3, middle 1/3,
distal 1/3). All patients with distal 1/3 closed fractures and
open fractures (grades I, II, IIIA according to the system
reported by Gustilo [14]) and who had been followed for
more than 3 years were included in this study. Patients
were excluded if they presented with undisplaced frac-
tures, grades IIIB and IIIC open fractures, fractures
associated with physeal injuries, or pathologic fractures. Patients were also excluded if there was a lack of adequate
radiographs, or if they were lost to follow-up prior to
3 years after treatment. for immobilization. If the reduction was not stable, either
intramedullary nails (ESIN, Titanium Elastic Intramedullary
Nail, Synthesis, USA) or external fixation (EF, AO ASIF
External Fixator, Synthesis, USA) was performed. We did
not perform fibular fracture fixation. All patients wore a long
leg splint after ESIN for 4 weeks. Weight-bearing ability was
determined on the basis of fracture stability. Patients who
had ≥50% cortical contact in the transverse plane were
allowed to bear weight with crutches as tolerated. During EF,
fluoroscopic guidance was used to place four bicortical self-
drilling fixator pins in the tibia, with two proximal and two
distal to the fracture site. The EF were removed when there
was radiographic evidence of bridging calluses on three of
four cortices, and the patient had no pain at the fracture site
when bearing weight on the external fixator. Most patients
were managed with a below-the-knee plaster posterior splint
and remained non-weight-bearing until the first postopera-
tive visit at 2 weeks. The splint was then removed, and the
patient was allowed to bear weight as tolerated. A total of 104 fractures in 98 patients were identified,
and 29 of these fractures were excluded. Background
h
l 8
Cases
75 fracture (75 cases)
Sex (M:F)
45 (60):30 (40)
Age (years)
6.8 ± 2.3
Follow-up duration (years)
4.1 ± 1.3
Fracture configuration
Transverse
25 (33.3)
Oblique
40 (53.3)
Spiral
10 (13.3)
Soft tissue injury
Closed
49 (65.3)
Open type I
15 (20.0)
Open type II
6 (8.0)
Open type IIIA
5 (6.6)
Fibular fracture
Intact
24 (32.0)
Fracture
51 (68.0)
Treatment method
Cast
29 (38.7)
Intramedullary nail
25 (33.3)
External fixation
21 (28.0)
Data are presented as a number (percentage) or average ± standard deviation. investigated residual deformities or the factors that may
influence them after distal tibial diaphyseal fractures. Therefore, the purpose of this retrospective study was
to evaluate residual angulation and the factors affecting
residual angulation after pediatric distal tibial fractures. Methods Of those excluded,
nine fractures were undisplaced fractures, seven were
Gustilo grade IIIB or IIIC fractures, three were associated
with physeal injuries, and one was pathologic due to osteo-
fibrous dysplasia. Five patients were lost to follow-up and
four did not have adequate radiographs. The study includes
the remaining 75 fractures from 75 patients. Demographic data is summarized in Table 1 and includes
age at the time of injury, sex, treatment method, and
whether the fracture was open or closed (grades I, II, IIIA). Radiographic data included fracture types (transverse,
oblique, or spiral), presence of associated fibular fracture,
and distal tibiofibular synostosis at follow-up. Radiographic
angulation was measured by comparing the immediate
postoperative anteroposterior and lateral radiographs to the
follow-up radiographs. Residual angulation was defined as
more than 5° on the most recent anteroposterior or lateral
plane radiographs. All measurements were made by two
independent evaluators (P.H. and L.J.H). Results The mean patient age was 6.8 ± 2.3 years (range, 4 to
10 years) at the time of injury, with 45 boys and 30 girls
included. There were more left-sided (54) than right-sided
(21) fractures. The mean follow-up period was 4.1 ± 1.3 years
(range, 3 to 6 years). There were 25 transverse, 40 oblique, and 10 spiral
fractures. Fifty-one fractures were associated with sim-
ultaneous fibular fractures. Twenty-six (34.6%) were open
fractures, with fifteen grade I, six grade II, and five grade
IIIA. Twenty-nine patients were managed with a long leg
cast, 25 patients with ESIN, and 21 patients with EF. The average time to union for all fractures was 13.7 weeks
(range, 6 to 22 weeks). The average time to union for closed
and open fractures was 11.5 weeks and 15.2 weeks, re-
spectively. The average time to union was 8.8 weeks
(range, 6 to 10 weeks) for the cast group, 10.9 weeks
(range, 7 to 15 weeks) for the ESIN group, and 16.3 weeks
(range, 8 to 22 weeks) for the EF group. There were no
nonunions. Two cases (8%) were complicated by infection;
however, both were resolved after repeated irrigation,
debridement, and intravenous antibiotics. At the final
follow-up visit, one patient with an open grade IIIA frac-
ture had a 1.5-cm overgrowth involving the injured tibia. Clinical properties such as the radiographic results of
the residual deformities are shown in Table 3 and were an-
alyzed by Fisher’s exact test. There were no statistically
significant differences in age (p = 0.313) or sex (p = 0.614)
between the two groups. The fracture configuration was
divided into two types (transverse vs. oblique, and spiral). There was no statistically significant difference in fracture
configuration (p = 0.793). The subgroup analysis between
oblique and spiral fracture types also did not reveal a The overall improvement of postoperative angulation
was 8.3° at the last follow-up visit. All residual deformities
were valgus deformities (Figures 1 and 2). At the follow-up Figure 1 Serials radiographs of a nine-year-old boy who sustained a right distal tibiofibular fracture. (a) Anteroposterior radiograph at
trauma. (b) Immediate postoperative radiograph. Treatment involved external fixation of the tibia. The postoperative angulation was 10° valgus. (c) A standing orthoroetgenogram after 5 years shows persistent valgus angulation. Figure 1 Serials radiographs of a nine-year-old boy who sustained a right distal tibiofibular fracture. (a) Anteroposterior radiograph at
trauma. Statistical analysis
d Patient demographics were summarized using means
and standard deviations for continuous variables and
proportions for categorical variables. The study sub-
jects were stratified into two groups based on the pres-
ence of valgus deformity. For comparison between the
two groups, we changed age to a categorical variable
(greater or less than 6 years). Statistical differences be-
tween the two groups were analyzed using Fisher’s exact
test. Variables with a p value lower than 0.2 including A senior pediatric orthopedic surgeon (S.T.J. or J.R.K.)
determined the surgical method, and all patients were
treated by one of these two surgeons. Closed fracture
reduction was performed under general anesthesia
with fluoroscopic assistance in the operating room. If
the closed reduction was acceptable, a cast was placed Page 3 of 7 Jung et al. Journal of Orthopaedic Surgery and Research 2014, 9:84
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http://www.josr-online.com/content/9/1/84 visit, 24 fractures had >5° residual valgus angulations. There
were no varus anteroposterior angulations >5°. The mean
changes in the valgus and varus angles immediately after
surgery and at the final follow-up are shown in Table 2. In
the valgus group, the mean angulation increased from the
postoperative measure to that of the final follow-up. In
contrast, the mean immediate postoperative angulation in
varus group was shown in almost neutral angulation at
final follow-up (average 0.43°, range; −0.6 to 3.9). Five pa-
tients who originally had varus angulations postoperatively
had valgus angulations at the final follow-up. Another six
patients who had varus angulations after surgery still had
such angulations at the final follow-up. Although there
was no significant difference in angulation change during
follow-up between the valgus and varus groups, most of
the varus angulations were remodeled and were almost
neutral angulations at the final follow-up; however, most
postoperative valgus angulations were either persisted or
progressed. Three patients who had valgus deformities >10°
after 3 years of follow-up underwent corrective osteotomy
(Figure 3). Eight patients exhibited tibiofibular synostosis at
the fracture site, and every patient developed valgus de-
formities of the ankle (Figure 4). age and sex were retained in the final logistic regression
model to determine the factor(s) related with deformity. All data were analyzed using SPSS statistics software
version 20.0 (SPSS Inc., Chicago, IL, USA). Results (b) Immediate postoperative radiograph. Treatment involved external fixation of the tibia. The postoperative angulation was 10° valgus. (c) A standing orthoroetgenogram after 5 years shows persistent valgus angulation. Jung et al. Journal of Orthopaedic Surgery and Research 2014, 9:84
Page 4 of 7
http://www.josr-online.com/content/9/1/84 Page 4 of 7 Jung et al. Journal of Orthopaedic Surgery and Research 2014, 9:84
http://www.josr-online.com/content/9/1/84 Figure 2 Serials radiographs of an eight-year-old boy who sustained a left distal tibiofibular fracture. (a) Anteroposterior radiograph at
trauma. (b) Immediate postoperative radiograph. Treatment involved flexible intramedullary nailing. Postoperative angulation was 8° valgus. (c) A
standing orthoroetgenogram after 5 years shows persistent valgus angulation. Figure 2 Serials radiographs of an eight-year-old boy who sustained a left distal tibiofibular fracture. (a) Anteroposterior radiograph at
trauma. (b) Immediate postoperative radiograph. Treatment involved flexible intramedullary nailing. Postoperative angulation was 8° valgus. (c) A
standing orthoroetgenogram after 5 years shows persistent valgus angulation. significant difference (p = 0.702). Open types IIIB and
IIIC fractures were excluded in this study. Thus, we
compared closed fractures and open type IIIA frac-
tures. Having an open fracture was not significantly as-
sociated with having a residual deformity (p = 0.797). There was no statistically significant difference in the
presence of a fibular fracture (p = 0.793). According to
the treatment methods, residual valgus deformities
were observed in three (12.5%) patients in the cast
group, ten (41.7%) in the ESIN group, and eleven
(45.8%) in the EF group (p = 0.004). The Fisher’s exact
test showed that the initial angulation (p = 0.002),
treatment method (p = 0.004), and immediate postop-
erative angulation (p = 0.006) were all associated with
having a residual deformity. Discussion Remodeling after tibial shaft fractures is greatly influenced
by the direction of angulation [15,16]. Angulation de-
formities are more often varus deformities than they are
valgus. Regardless, remodeling of the angulated tibial
shaft fracture is often incomplete [17-20]. Specifically,
distal tibia fractures are less likely to remodel success-
fully because of the activity of the surrounding muscles
in the anterior and lateral compartments of the lower
leg. In addition, the distal tibia has a lower growth
potential than that of the proximal tibia. Deformities to
the distal tibia tend to be in the coronal plane, perpen-
dicular to the axis of the ankle joint [6]. The develop-
ment of the valgus deformity under consideration has
been attributed to a growth discrepancy between the
tibia and fibula, with the fibula exerting a tethering
effect. There is an asymmetric stimulation of the physis
when the tibia grows faster than the fibula [7]. The
deformity is predominantly characterized by a lateral
angulation of the tibia with the apex of the deformity at
the fracture site. This anatomy implies that the tibial
overgrowth results from the stimulation of its proximal
growth plate; the fibula does not overgrow and therefore
the deformity forms and progresses at the fracture site
in the tibial metaphysis [8]. Swaan and Oppers found
that in 86 children (with girls 1–8 years old and boys 1
to 10 years old) treated for tibial fractures, there was
moderate spontaneous correction of the residual angu-
lations after bone union [16]. In girls (ages 9 to 12 years)
and boys (ages 11 to 12 years), approximately 50% of
the angulations were corrected. No more than 25% of
the deformities were corrected in children over 13 years
of age. Hansen et al. reported that of 102 pediatric tibial
fractures, 25 had malunions ranging from 4° to 19° [5]. At Results of multiple logistic regression analysis are shown
in Table 4. The backward addition method was used for
modeling. Age and sex were included in the final model. Multiple logistic regression analysis showed that both
initial angulations >5° (adjusted odds ratio (OR) 3.80,
95% confidence interval (CI) 1.12–12.87) and the treatment
method compared to casting (intramedullary nail: adjusted
OR 7.33, 95% CI 1.31–41.07; external fixation: adjusted
OR 11.35, 95% CI 1.91–67.40) were associated with re-
sidual deformities. Discussion Table 2 Angulation changes during follow-up
Postoperative
(°; range)
Last follow-up
(°; range)
Mean change
(°; range)
Valgus group
3.49 (0.1 to 8.8)
5.47 (0.1 to 16)
2.95 (0 to 10.7)
Varus group
−3.58 (−5.9 to −2)
0.43 (−0.6 to 3.9)
4.01 (0.8 to 6.6)
p value
0.276
+ valgus angle, −varus angle. Table 2 Angulation changes during follow-up Page 5 of 7 Jung et al. Journal of Orthopaedic Surgery and Research 2014, 9:84
http://www.josr-online.com/content/9/1/84 Figure 3 Serials radiographs of a seven-year-old girl who sustained a left distal tibiofibular fracture. (a) Anteroposterior radiograph at
trauma. (b) Immediate postoperative radiograph. Treatment involved external fixation of the tibia without fibular fixation. (c) Standing
anteroposterior radiographs after 1 year shows 20° valgus angulation. (d) Standing anteroposterior radiograph after 3 years of follow-up shows
15° valgus angulation and a 15-mm tibial overgrowth. (e) Gross image of valgus angulation around the ankle joint. (f) A postoperative radiograph
3 years after corrective osteotomy and shortening. (g) A radiograph 3 years after corrective osteotomy shows an excellent correction. Figure 3 Serials radiographs of a seven-year-old girl who sustained a left distal tibiofibular fracture. (a) Anteroposterior radiograph at
trauma. (b) Immediate postoperative radiograph. Treatment involved external fixation of the tibia without fibular fixation. (c) Standing
anteroposterior radiographs after 1 year shows 20° valgus angulation. (d) Standing anteroposterior radiograph after 3 years of follow-up shows
15° valgus angulation and a 15-mm tibial overgrowth. (e) Gross image of valgus angulation around the ankle joint. (f) A postoperative radiograph
3 years after corrective osteotomy and shortening. (g) A radiograph 3 years after corrective osteotomy shows an excellent correction. deformities at follow-up [21]. Although fractures with
varus malalignments (of 5° to 13°) completely corrected at
the physis, most valgus deformities (of 5° to 7°) were
not fully corrected [22]. In the present study, 24 of 75
fractures in children under the age of ten were uncorrected. All residual deformities were valgus deformities. Valgus the follow-up visit, residual angular malunions ranged
from 3° to 19°. Every patient had a complete correction. Spontaneous correction accounted for approximately
13.5% of the total deformity. Benneck and Steinert
reported that 26 of 28 children with varus or valgus
deformities at union had significant residual angular Figure 4 Radiograph of a ten-year-old girl who sustained a left distal tibiofibular fracture. (a) Anteroposterior radiograph at trauma. Discussion (b) A radiograph after 3 years of follow-up shows tibiofibular synostosis and an ankle valgus deformity associated with fibular shortening. Figure 4 Radiograph of a ten-year-old girl who sustained a left distal tibiofibular fracture. (a) Anteroposterior radiograph at trauma. (b) A radiograph after 3 years of follow-up shows tibiofibular synostosis and an ankle valgus deformity associated with fibular shortening. Jung et al. Journal of Orthopaedic Surgery and Research 2014, 9:84
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http://www.josr-online.com/content/9/1/84 Page 6 of 7 Table 3 Comparisons between the normal and valgus deformity groups
Residual angulation
<5 (n = 51)
≥5 (n = 24)
p value
Age
≤6 years
22
(43.1)
7
(29.2)
0.313
>6 years
29
(56.9)
17
(70.8)
Sex
Male
32
(62.7)
13
(54.2)
0.614
Female
19
(37.3)
11
(45.8)
Fracture type
Transverse
16
(31.4)
9
(37.5)
0.793
Oblique and spiral
35
(68.6)
15
(62.5)
Open fracture
No
34
(66.7)
15
(62.5)
0.797
Yes
17
(33.3)
9
(37.5)
Fibular fracture
No
18
(35.3)
6
(25.0)
0.793
Yes
33
(64.7)
18
(75.0)
Operation methods
Cast
26
(51.0)
3
(12.5)
0.004a
Intramedullary nail
15
(29.4)
10
(41.7)
External fixation
10
(19.6)
11
(45.8)
Postoperative angulation
<5
45
(88.2)
14
(58.3)
0.006
≥5
6
(11.8)
10
(41.7)
Data are presented as a number (percentage) and were analyzed by Fisher’s exact test. aCast vs. intramedullary nail and external fixation. Table 3 Comparisons between the normal and valgus deformity groups
Residual angulation
<5 (n = 51) Table 3 Comparisons between the normal and valgus deformity groups Data are presented as a number (percentage) and were analyzed by Fisher’s exact test. aCast vs. intramedullary nail and external fixation. follow-up visit. Second, we did not fix fibular fractures. Although there were accurate postoperative reductions
achieved with surgery, the residual valgus angulations
developed during the follow-up period. In the present
study, valgus angulations progressed over the follow-up
period, while varus angulations improved. We suggest that
a small amount of varus reduction may be better than is
an acceptable valgus range. Further research is needed to
determine whether the valgus deformity progression can
be prevented by stabilizing the fibular fracture and main-
taining the fibular length. deformities require more precise reduction because they
are perpendicular to the axis of the ankle joint. Discussion During the first 18 months after a fracture in a child,
there may be spontaneous tibial remodeling [6]. The
remodeling capacity is determined by the child’s age
and the degree of angulation [5,17]. In this study, there
were no statistically significant age differences. How-
ever, every child has a different growth rate, which
makes it difficult to analyze the relationship between
age and angular deformity. Severely displaced fractures and factures that were
unstable after closed reduction were treated with an
intramedullary nail or external fixator. Residual valgus
deformities were significantly prevalent in patients treated
with an intramedullary nail and external fixator. We sus-
pected that there are two reasons that the surgical groups
had a higher prevalence of residual valgus deformities. First, the postoperative angulations persisted until the final Posttraumatic tibiofibular synostosis is a rare compli-
cation of pediatric distal tibiofibular fractures [23,24]. Tibial tethering was noted in every patient. The normal
growth pattern of distal fibular migration relative to the
tibia was reversed. This reversed growth pattern resulted
in a shorter distance between the proximal tibial physis
and the fibula. Additionally, there was proximal migra-
tion of the distal fibular physis relative to the distal
tibia. Shortening of the lateral malleolus leads to greater
valgus alignment of the ankle [15,24,25]. In this study, eight
patients who had tibiofibular synostosis in comminuted
fractures of the distal tibia and fibula associated with
the interosseous membrane also developed a valgus ankle
deformity. In patients with tibiofibular synostosis, the val-
gus deformity of the distal tibia persisted throughout the
entire follow-up period. Table 4 Multivariate logistic regression analysis for risk
factors associated with deformity
N
Adjusted
OR
95% CI
p value
Postoperative
angulation
<5
59
1
≥5
16
4.33
1.07–17.53
0.040
Operation
methods
Cast
29
1
Intramedullary nail
25
7.33
1.31–41.07
0.023
External fixation
21
11.35
1.91–67.40
0.008
OR odds ratio, CI confidence interval. The OR was adjusted for age and sex. Table 4 Multivariate logistic regression analysis for risk
factors associated with deformity Competing interests Competing interests
The authors declare that they have no competing interests. 20. Bohn WW, Durbin RA: Ipsilateral fractures of the femur and tibia in
children and adolescents. J Bone Joint Surg Am 1991, 73–3:429–439. 21. Steinert V, Bennek J: Tibial fractures in children. Zentralbl Chir 1966,
91–38:1387–1392. Table 4 Multivariate logistic regression analysis for risk
factors associated with deformity This study has several limitations to consider. First, we
retrospectively analyzed a relatively small sample of
patients over a short follow-up period. Second, the
treatment method was dependent upon various clinical
findings. Therefore, the treatment method may not have Page 7 of 7 Jung et al. Journal of Orthopaedic Surgery and Research 2014, 9:84
http://www.josr-online.com/content/9/1/84 been an independent predictor of valgus deformity, but
may have represented patients’ overall health. been an independent predictor of valgus deformity, but
may have represented patients’ overall health. 15. Buckley SL, Smith G, Sponseller PD, Thompson JD, Griffin PP: Open fractures
of the tibia in children. J Bone Joint Surg Am 1990, 72–10:1462–1469. 16. Swaan JW, Oppers VM: Crural fractures in children. A study of the
incidence of changes of the axial position and of enhanced longitudinal
growth of the tibia after the healing of crural fractures. Arch Chir Neerl
1971, 23–4:259–272. Author details
1 1Department of Orthopaedics Surgery, Chonnam National University Medical
School, 160, Baekseo-ro, Gwangju 501-746, South Korea. 2Department of
Orthopaedics Surgery, Chonbuk National University Medical School, 567
Baekje-ro, Dukjin-gu, Jeonju 561-756, South Korea. 3Department of Preventive
Medicine, Chonbuk National University Medical School, 567 Baekje-ro,
Dukjin-gu, Jeonju 561-756, South Korea. 1Department of Orthopaedics Surgery, Chonnam National University Medical
School, 160, Baekseo-ro, Gwangju 501-746, South Korea. 2Department of
Orthopaedics Surgery, Chonbuk National University Medical School, 567
3 ,
,
,
gj
,
p
Orthopaedics Surgery, Chonbuk National University Medical School, 567
Baekje-ro, Dukjin-gu, Jeonju 561-756, South Korea. 3Department of Preventive
Medicine, Chonbuk National University Medical School, 567 Baekje-ro,
Dukjin-gu, Jeonju 561-756, South Korea. Baekje-ro, Dukjin-gu, Jeonju 561-756, South Korea. 3Department of Preventive
Medicine, Chonbuk National University Medical School, 567 Baekje-ro,
Dukjin-gu, Jeonju 561-756, South Korea. Authors’ contributions STJ and HP participated in the data collection and analysis and wrote the
manuscript. JHL contributed to the conception and design of the study, the
data collection, the statistical analysis, and the manuscript drafting. JRK
conceived the idea for this study, prepared the protocol, and collected and
analyzed the data. All authors read and approved the final manuscript. 22. Beaty JH, Kasser JR: Fractures in Children. 7th edition. Philadelphia: Lippincott
Williams & Wilkins; 2010:948–956. 23. Munjal K, Kishan S, Sabharwal S: Posttraumatic pediatric distal tibiofibular
synostosis: a case report. Foot Ankle Int 2004, 25–6:429–433. 24. Frick SL, Shoemaker S, Mubarak SJ: Altered fibular growth patterns after
tibiofibular synostosis in children. J Bone Joint Surg Am 2001, 83-A-2:247–254. Acknowledgements
Thi
d 25. Wiltse LL: Valgus deformity of the ankle: a sequel to acquired or congenital
abnormalities of the fibula. J Bone Joint Surg Am 1972, 54–3:595–606. This study was supported by a grant of the Korean Health Technology R&D
Project, Ministry of Health & Welfare, Republic of Korea (A121141). doi:10.1186/s13018-014-0084-5
Cite this article as: Jung et al.: Residual angulation of distal tibial
diaphyseal fractures in children younger than ten years. Journal of
Orthopaedic Surgery and Research 2014 9:84. Conclusions Spontaneous bone remodeling after distal tibial diaphy-
seal fractures in children is often incomplete. The goal
of treating these fractures is to obtain accurate reduction
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forearm fractures in children. Clin Orthop Relat Res 1988, 237:190–195. 3. Vittas D, Larsen E, Torp-Pedersen S: Angular remodeling of midshaft
forearm fractures in children. Clin Orthop Relat Res 1991, 265:261–264. 4. Gasco J, de Pablos J: Bone remodeling in malunited fractures in children
Is it reliable? J Pediatr Orthop B 1997, 6–2:126–132. 5. Hansen BA, Greiff J, Bergmann F: Fractures of the tibia in children. Acta Orthop Scand 1976, 47–4:448–453. p
6. Dwyer AJ, John B, Krishen M, Hora R: Remodeling of tibial fractures in
children younger than 12 years. Orthopedics 2007, 30–5:393–396. 6. Dwyer AJ, John B, Krishen M, Hora R: Remodeling of tibial fractures in
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• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
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Submit your manuscript at
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• Convenient online submission
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• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of: 10. Siegmeth A, Wruhs O, Vecsei V: External fixation of lower limb fractures in
children. Eur J Pediatr Surg 1998, 8–1:35–41. 10. Siegmeth A, Wruhs O, Vecsei V: External fixation of lower limb fractures in
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treatment of tibial fractures in children: are elastic stable intramedullary
nails an improvement over external fixation? J Bone Joint Surg Am 2005,
87–8:1761–1768. 13. Goodwin RC, Gaynor T, Mahar A, Oka R, Lalonde FD: Intramedullary flexible
nail fixation of unstable pediatric tibial diaphyseal fractures. J Pediatr
Orthop 2005, 25–5:570–576. 14. Gustilo RB: Current concepts in the management of open fractures. Instr
Course Lect 1987, 36:359–366. 14. Gustilo RB: Current concepts in the management of open fractures. Instr
Course Lect 1987, 36:359–366.
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English
| null |
Preparation (HgBa2Ca2Cu2.85La0.15O8+δ) compound and Study of the impact of partial substitution of mercury with (In2O3)on the structural characteristics
|
Mağallaẗ al-Kūfaẗ li-l-fiziyā'/Journal of Kufa - physics
| 2,023
|
cc-by
| 4,226
|
K e y w o r d s :
Partial substitution
the (SSR) method
(XRD) analysis
degree of crystallinity
crystallite size and strain JOURNAL OF KUFA−PHYSICS journal.uokufa.edu.iq/index.php/jkp/index | ISSN: 2077–5830 Preparation of (HgBa2Ca2Cu2.85La0.15O8+δ) superconducting compound
and Study the impact of partial substitution of mercury with (In2O3) on
the structural characteristics * Ekhlas Hassan1
Haider MJ. Haider2
1Material Department, Faculty of Engineering, University of Kufa, Najaf, IRAQ
2Physics Department, College of Education for Girls, University of Kufa, Najaf, IRAQ . *Corresponding Author E-mail: eklaseh.alkhazraji@uokufa.edu.iq 1. INTRODUCTION It has been discovered that there is a
distinction between substances and chemical
elements when it comes to electrical resistance
or conductivity. Since the early of 1960s,
superconducting materials have demonstrated
distinctive and inherent properties, making them
crucial for electronic device applications that
call
for
cooling
at
extremely
low
temperatures[1-3]. Because
of
their
high
transition
temperatures,
mercury-based
superconducting
capsules
like
HgBa2CuO4+δ(Tc=940k),
HgBa2CaCu2O6+δ(Tc=1270k),
and
HgBa2Ca2Cu3O8+δ(Tc=1340k) are considered to
be among the high-temperature superconducting
materials [4] By processing it in a furnace with It has been discovered that there is a
distinction between substances and chemical
elements when it comes to electrical resistance
or conductivity. Since the early of 1960s,
superconducting materials have demonstrated
distinctive and inherent properties, making them
crucial for electronic device applications that
call
for
cooling
at
extremely
low
temperatures[1-3]. Because
of
their
high
transition
temperatures,
mercury-based
superconducting
capsules
like According to several studies, favorable
alternatives boost the phase development and
superconducting characteristics of (Hg-1223). The partial substitution of some higher valence
elements, such as Pb, Y, or Re, or other
elements, might enhance the critical current
density and phase development of Mercury -
1223[5]. They have not used the Hg-Base family as
frequently as other HTSC materials. This is
because HTSC phases containing mercury are
extremely difficult to synthesize and extremely
susceptible to contamination by moisture and
carbon dioxide. Samples of Mercury-Base are
known to degrade fast after production. To
increase the stability of the HTSC Mercury-
Base stage, a lot of work has been done
recently[3]. HgBa2CaCu2O6+δ(Tc=1270k), HgBa2Ca2Cu3O8+δ(Tc=1340k) are considered to
be among the high-temperature superconducting
materials [4]. By processing it in a furnace with
high pressure, the critical transition temperature
has risen to 160 K[5]. Extensive research on superconductivity
has been performed with encouraging outcomes
since the discovery of the first high-temperature
superconductors based on mercury (Hg-1201)
material with a high Tc. Many of the major
problems
with
superconductivity
remain
involve superconducting materials, despite the
field's enormous achievements. The difficulty of
shaping these materials into wires or circuits
such that they may transport high currents that
are not permitted in typical conductors is one of
these difficulties and has been a key problem
ever since superconductivity was discovered[6-
8]. One of the systems in the HBCCO family of
superconducting mercury compounds is this In the current study, (SSR) method was
used to create polycrystalline compounds of
(Hg1-xInxBa2Ca2Cu2.85La0.15O8+δ) where (x=0,
0.05, 0.1, 0.15, 0.20, 0.25). A R T I C L E I N F
A B S T R A C T In the current work, we focused on the impact of Indium oxide
partial substitution at the (Hg) site on the structural characteristics of the
(HgBa2Ca2Cu2.85La0.15O8+δ) compound with (0,0.05,0.1,0.15,0.2 and
0.25). Samples were prepared by the (SSR) method. The combined
powder was crushed with a pressure of 7 tons per square centimeter to
create a disc with a thickness of (0.2 cm) and a diameter of (1.5 cm). The samples sintered for 24 hours at a rate that was changed from
ambient temperature to 820 °C. For the samples which were doped with
(In=0.05) in comparison to other samples, the highest value of the c-axis
lattice parameter and a pseudo-tetragonal structure were discovered
using (XRD) analysis. It was discovered that altering the In
concentrations in all of the results of our sample changes the volume
fraction VPh(1223), mass density dm, and c/a. It was also determined
that the change in lattice parameters, degree of crystallinity, crystal size,
and strain (in different ways) resulted from a change in the indium oxide
concentrations for all samples. Article history:
Received: 30 DEC, 2022
Revised: 03 FEB, 2023
Accepted: 05 FEB, 2023
Available Online: 03 JUN, 2023 K e y w o r d s :
Partial substitution
the (SSR) method
(XRD) analysis
degree of crystallinity
crystallite size and strain DOI: https://doi.org/10.31257/2018/JKP/2023/v15.i01.10917 DOI: https://doi.org/10.31257/2018/JKP/2023/v15.i01.1091 تحضير المركة(HgBa2Ca2Cu2.85La0.15O8+δ)
ودراسة تأثير اإلستبذال الجزئي للزئبق
الخواص التركيبية اخالص حسن1 ال
ــــ ـــ خ
الص
ة ـــ فٍ انعًم انحبنٍ ،إجرَُب
دراست حىل تأثُر االستبذال انجزئٍ ألكسُذ اإلَدذَىو فدٍ يىقد
(Hg)
عهً انخصدبئ انتركُبُدت نهًركد(HgBa2Ca2Cu2.85La0.15O8+δ) بُسد إسدتبذال
(
.0...00..10..100..20..20
)
. تددى تحرددُر انعُُددبث بطرَقددت تفبعددم انحبنددت انصددهبت(SSR) 22 JOURNAL OF KUFA–PHYSICS | Vol. 15, No. 1 (2023) Ekhlas Hassan, Haider MJ. Haider .تى كبس يسحىق
انًرك برغط7
( ًطدٍ نكدم سدُتًُتر يربد نتكدىٍَ قدر بسد..2
)سدى
( وقطدر1.0
سدى). نبددذث انعُُدبث نًدذة22
ًسددبعت بًعدذل تغُددر يدٍ درجدت انحددرارة انغرفدت إندد
02. درجت يئىَت. (ببنُسبت نهعُُبث انتدٍ تحتدىٌ َسدبت اإلسدتبذالIn =0.05
) يقبرَدت ببنعُُدبث
األخري ، تى اكتشبف أعهً قًُت نًعب يم شبُكت انًحىرc
ُوتركtetragonal
ببستخذاو تحهُم
(
XRD
ٍ). تدددى اكتشدددبف أٌ تغُُدددر انتراكُدددز فدددٍ جًُددد َتدددبئل انعُُدددت َغُدددر انكسدددر انحجًددد
VPh(1223)
وكثبفت انكتهُت
dm
وc /a
. تدى اسدتخذاو تحهُدمXRD
، نتحذَدذ يعدبي ث انشدبُكت
)ودرجدت انتبهددىر وحجددى انبهدرة ، واالَفعددبل (بطددرق يختهفدت
حُددإ إَهددب تتغُدر يدد تغُددر تراكُددز
أكسُذ اإلَذَىو نجًُ انعُُبث. one,
the
high-critical
temperature
superconducting material is produced by a
Mercury oxide airplane [9]. 1. INTRODUCTION We will examine the
structural characteristics of compounds and
investigate the impact of partial Indium
substitution on crystallinity degree, crystallite
size, and strain (using multiple methods). 2. EXPERIMENTAL DETAILS Using
the
(SSR)
method,
the
superconducting
compound
(Hg1-
xInxBa2Ca2Cu2.85La0.15O8+δ) with (x=0, 0.05, 23 JOURNAL OF KUFA–PHYSICS | Vol. 15, No. 1 (2023) Ekhlas Hassan, Haider MJ. Haider 0.1, 0.15, 0.20, 0.25), was prepared by using
suitable weights of powders of (HgO, BaO,
CaO, CuO, La2O3, In2O3 ) (with a purity of
more than 99.9%). With sensitivity order (10-4)
g, a sensitive balance was used to estimate the
reactants. To
homogenize
the
reactants,
isopropanol (C2H3O5) was added and the
reactants were mixed in a gate mortar for two
hours
to
obtain
fineness
and
optimal
homogeneity of the powders, then the resulting
powders were dried at a temperature of 200 oC
for a period of (1hr) in a drying oven. The
samples were compressed into discs with a
thickness of ( 0.2cm) and a diameter of (1.5 cm)
using a hydraulic press under pressure (7
tons/cm2) for one minute. To produce a
coherent material and to assure the process, the
samples were sintered inside a sintering furnace
for 24 hours at a temperature of 820 degrees
Celsius. The temperature was progressively
raised (i.e. at the rate of 10 oC/min). The
samples were gradually cooled within the
furnace at the same heating rate after optimal
atoms diffusion. To determine the samples'
structural characteristics, the samples were
examined at within the diffraction angle range
(10-80). The constants (a, 3. RESULTS AND DISCUSSION 1 (2023) Ekhlas Hassan, Haider MJ. Haider X
Cryesti
lanity
(%)
crystallite size (D) (nm)
Scher
rer
meth
od
modifi
ed
Scherre
r
method
Willia
mson
-Hall
method
Size
-strain
plot
method
Halder
-Wagner
Langford
method
0
54.39
30.91
5
37.234
62.739
30.743
30.788
0.05
63.51
28.33
7
33.882
45.460
27.240
30.257
0.1
60.84
33.17
5
35.906
35.643
37.272
41.356
0.15
56.42
37.94
2
45.178
83.026
23.264
25.853
0.2
58.93
28.98
1
26.788
21.904
19.045
21.168
0.25
55.76
32.62
4
35.648
63.895
34.320
38.153
Table
3:
Strain
of
(Hg1-
xInxBa2Ca2Cu2.85La0.15O8+δ) compound using
different Methods. method, the Williamson-Hall method, and the
Size-strain plot approach. crystallite size (D) (nm) Table 1: Demonstrates the generated samples'
phase ratios, lattice coefficients, (c/a), and
density. X
a=b (A0)
c (A0)
c/a
V (A0)3
Dm (gm/cm3)
Vph (H)
Vph (L)
Tc (K)
P
0
3.892
15.865
4.076
240.36
5
6.122
77.3
27.7
120.4
0.1266
0.05
3.841
16.065
4.182
237.04
0
6.208
80
20
132.6
0.16
0.1
3.895
15.863
4.072
240.68
8
6.114
78.3
21.7
121.6
0.1283
0.15
3.895
15.874
4.074
240.90
0
6.109
72.7
27.3
118.8
0.1245
0.2
3.836
15.865
4.135
233.54
6
6.301
77.3
22.7
120.5
0.1267
0.25
3.889
15.589
4.008
235.81
8
6.240
72.7
27.3
116.2
0.1213
Figure
1:
X-ray
diffraction
for
(Hg1-
xInxBa2Ca2Cu2.85La0.15O8+δ) bulk polycrystalline
samples where (x=0, 0.05, 0.1, 0.15, 0.20, 0.25). Table 2: Crystallite size and degree of
crystallinity of (Hg1-xInxBa2Ca2Cu2.85La0.15O8+δ)
compound using different Methods. Table 1: Demonstrates the generated samples'
phase ratios, lattice coefficients, (c/a), and
density. X
a=b (A0)
c (A0)
c/a
V (A0)3
Dm (gm/cm3)
Vph (H)
Vph (L)
Tc (K)
P
0
3.892
15.865
4.076
240.36
5
6.122
77.3
27.7
120.4
0.1266
0.05
3.841
16.065
4.182
237.04
0
6.208
80
20
132.6
0.16
0.1
3.895
15.863
4.072
240.68
8
6.114
78.3
21.7
121.6
0.1283
0.15
3.895
15.874
4.074
240.90
0
6.109
72.7
27.3
118.8
0.1245
0.2
3.836
15.865
4.135
233.54
6
6.301
77.3
22.7
120.5
0.1267
0.25
3.889
15.589
4.008
235.81
8
6.240
72.7
27.3
116.2
0.1213 X
strain(ƹ)
Williamson-
Hall method
Size-strain
plot method
Halder-Wagner
Langford method
0
0.00234
0.001016883
0.00128688
0.05
1.39E-03
0.000139781
0.000907313
0.1
3.99E-04
0.000545328
0.003539712
0.15
2.10E-03
0.001302404
0.008453875
0.2
5.16E-04
0.001378669
0.008948899
0.25
2.39E-03
7.09132E-05
0.000460296 3. RESULTS AND DISCUSSION The
produced
samples
of
(Hg1-
xInxBa2Ca2Cu2.85La0.15O8+δ) were seen to have
various Indium concentrations(x=0, 0.05, 0.1,
0.15, 0.20, 0.25) that were given a general
structural analysis using the (XRD) method. All
of the samples were polycrystalline and
correlate to tetragonal, according to the (XRD)
data gathered from a variety of samples. In
Fig.1, the representative (XRD) pattern is
displayed. The spectra revealed that all samples
were composed mostly of some impurity phases
in addition to the low -Tc phase(1212) and the
high-Tc phase (1223). The arranging defects
throughout the c-axis may be the cause of the
emergence of more than two phases [13]. According to a comparison of the XRD
patterns', the relative intensities for samples
with ln=0.05, 0.15, 0.2, and 0.25 with the same
reflections' relative intensity in the sample with
ln=0.0, all samples have reflection intensities of
the High-Tc phase reflections (peaks H) and
Low-Tc phase reflections (peaks L), with the
(peaks H) changing and the (peaks L) is
changing with changing ln. b, and c) were determined theoretically
using Miller indices and d-values of the
observed XRD pattern reflections. Additionally,
the cell unit density was determined, and the
percentages of the phases created in the samples
were calculated using the equation below [10,
11]: Table 1 shows the lattice constants ( a, b,
and c), mass density dm, and (V phase) which
were found through the results of (XRD). The conduction channel in Hg-base is holes
in the Cu-O2 layers, which are strengthened by
the Hg-O layer, as a result of the replacement of
ln for Hg owing to the different ionic radii. Hg-
1223's charge transfer from the Hg layer to the
Cu
layer
is
adjusted
by
the
c-axis'
deformation[13]. ( )
∑
∑ ∑ ∑ ( ) ( )
∑
∑ ∑ ∑ ( ) and the concentrations of hole per Cu
ion(P) was calculated by means of [12]: and the concentrations of hole per Cu
ion(P) was calculated by means of [12]: Crystallite size, strain, and degree of
crystallinity are calculated using XRD analysis. Different techniques were used to determine the
crystal size, including the Halder-Wagner
Langford
method,
the
modified
Scherrer ( ) [(
⁄
) ( )
⁄
]
⁄ Investigation of the impact of partial
substitution of concentrations on the degree of
crystallinity, and crystallite size, strain was
conducted using various techniques. 24 JOURNAL OF KUFA–PHYSICS | Vol. 15, No. 3.1 Scherrer Method Eq.2 refers to The Scherrer equation, which
is mostly based on data from X-ray diffraction,
which is a formula that links the average crystal
size in materials to a peak expansion in the
diffraction pattern. The scientist (Paul Shearer)
gave it its name. The average crystallite size (D)
in the crystal's vertical direction was calculated
using the following Scherrer formula [14, 15]. Figure
1:
X-ray
diffraction
for
(Hg1-
xInxBa2Ca2Cu2.85La0.15O8+δ) bulk polycrystalline
samples where (x=0, 0.05, 0.1, 0.15, 0.20, 0.25). Table 2: Crystallite size and degree of
crystallinity of (Hg1-xInxBa2Ca2Cu2.85La0.15O8+δ)
compound using different Methods. where K=0.9 (constant), The wavelength of where K=0.9 (constant), The wavelength of where K=0.9 (constant), The wavelength of
an X-ray is (λ =0.15406 nm), and = highest
peak intensity at full width in Radian (FWHM). Utilizing the Origin program, the crystallite size
may be calculated. Fig.2 shows a graph of
(cosθ)
as
a
function
of
(1/ßhkl)
for
(Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ)
bulk
polycrystalline using the Origin software. The
average crystallite size can be calculated from
the Eq.2 which was found as (30.9154064) nm. where K=0.9 (constant), The wavelength of
an X-ray is (λ =0.15406 nm), and = highest
peak intensity at full width in Radian (FWHM). Utilizing the Origin program, the crystallite size
may be calculated. Fig.2 shows a graph of
(cosθ)
as
a
function
of
(1/ßhkl)
for
(Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ)
bulk Figure
1:
X-ray
diffraction
for
(Hg1-
xInxBa2Ca2Cu2.85La0.15O8+δ) bulk polycrystalline
samples where (x=0, 0.05, 0.1, 0.15, 0.20, 0.25). Table 2: Crystallite size and degree of
crystallinity of (Hg1-xInxBa2Ca2Cu2.85La0.15O8+δ)
compound using different Methods. polycrystalline using the Origin software. The
average crystallite size can be calculated from
the Eq.2 which was found as (30.9154064) nm. 25 JOURNAL OF KUFA–PHYSICS | Vol. 15, No. 1 (2023) Ekhlas Hassan, Haider MJ. Haider Similarly,
the
crystallite
size
of
(Hg1-
xInxBa2Ca2Cu2.85La0.15O8+δ)
was
calculated
when (X =0,0.1,0.15,0.2 and 0.25), and their
values are presented in the Table 2. Figure 3: Graph of ( ) as a function of
(
)for(Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ)
bulk polycrystalline presented to The Modified
Scherrer crystallite size. 0
50
100
150
200
250
300
0.82
0.84
0.86
0.88
0.90
0.92
0.94
0.96
0.98
1.00
cosθ
1/β
x=0.05
Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ
Figure 2: Graph of cosθ as a function of 1/ßhkl
for
(Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ)
bulk
polycrystalline presented to Scherrer crystallite
size. 0
50
100
150
200
250
300
0.82
0.84
0.86
0.88
0.90
0.92
0.94
0.96
0.98
1.00
cosθ
1/β
x=0.05
Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ Figure 3: Graph of ( ) as a function of Figure 3: Graph of ( ) as a function of
(
)for(Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ)
bulk polycrystalline presented to The Modified
Scherrer crystallite size. 3.2 Williamson-Hall (W-H) method Figure 5: Graph of [ ]
as a function of [
]for
(Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ)
bulk
polycrystalline presented to Size-strain plot
(SSP). of 3.3 The Size-strain plot (SSP) method The
(SSP)
approach
provides
better
isotropic expansion performance because it
focuses on low angle reflections, where the
resolution is higher than at higher angles. This
is because the peaks in the XRD data at low
angles usually overlap due to overlapping
phases. Consequently, The (SSP) method,
which examines peak profiles, ignores the
lattice microstructures and only considers how
crystalline size affects XRD peak broadening. Figure 5: Graph of [ ]
as a function of [
]for
(Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ)
bulk
polycrystalline presented to Size-strain plot
(SSP). Figure 5: Graph of [ ]
as a function of [
]for
(Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ)
bulk
polycrystalline presented to Size-strain plot
(SSP). 3.2 Williamson-Hall (W-H) method This is a way to determine the crystallite
size and strain of crystals. The size causes the
physical line broadening of the XRD peak. 1/β Figure 2: Graph of cosθ as a function of 1/ßhkl
for
(Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ)
bulk
polycrystalline presented to Scherrer crystallite
size. Peak widening is only taken into account
by the (W-H) system as a function of a
diffraction angle (2θ). This is thought to have
been a combination of size- and strain-induced
widening. The crystal volume was also calculated
using the Modified Scherrer equation, which is
calculated by taking (Ln) to both sides of Eq.3,
so we get the following equation[16]: Consequently,
the
total
broadening
resulting from size and strain at a certain peak
with the hkl value may be represented as
follows: ( ) (
) (
) ( ) ( ) ( ) ( )
The peak broadening caused by strain may
be demonstarted as This equation represents a straight-line
equation
between ( )
on
a
Y-axis
and (
) on an X-axis using the Origin
software, then The peak broadening caused by strain may
be demonstarted as ( )
Where ƹ (slop) represents the strain [15, 17, 18]. ( )
Where ƹ (slop) represents the strain [15, 17, 18]. (
)
( From Fig. 3 and Eq.4 the crystallite
size(D)
can
be
calculated,
where
the
corresponding values are shown in Table 2. When rearranging Eq.8 we get: Which represents straight line equation
between on a Y-axis and on
an X-axis using the Origin software, then 26 JOURNAL OF KUFA–PHYSICS | Vol. 15, No. 1 (2023) Ekhlas Hassan, Haider MJ. Haider [
] on an X-axis using
the Origin software, then
√
(
) (
)
From Fig.4 and Eq.9 can be calculated
crystallite size(D), that values are shown in
Table2. [
] on an X-axis using
the Origin software, then (
) [
] on an X-axis using
the Origin software, then [
] on an X-axis using
the Origin software, then [
] on an X-axis using
the Origin software, then From Fig.4 and Eq.9 can be calculated
crystallite size(D), that values are shown in
Table2. √ Figure 4: Graph of as a function
of
for
(Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ)
bulk
polycrystalline presented to Williamson-Hall
method. (
) From Fig.5 and Eq.12 crystalline size(D)
can be calculated, where it values are presented
in Table 2. 4. Conclusion We have investigated the impact of the
synchronic substitution at the Hg site in the
HgO layer, which lacks oxygen of (Hg1-
xInxBa2Ca2Cu2.85La0.15O8+δ)
cuprate
superconductor which has been prepared under
optimum conditions. In comparison to samples
without any In content, (XRD) examination
revealed a pseudo-tetragonal structure with an
increase in the c-axis lattice constant. As-grown
samples' transition temperatures are discovered
to be sensitive to In concentrations. We have investigated the impact of the
synchronic substitution at the Hg site in the
HgO layer, which lacks oxygen of (Hg1-
xInxBa2Ca2Cu2.85La0.15O8+δ)
cuprate Figure 6: Graph of (
) a function of (
)
for
(Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ)
bulk
polycrystalline presented to Halder-Wagner
Langford method. superconductor which has been prepared under
optimum conditions. In comparison to samples
without any In content, (XRD) examination
revealed a pseudo-tetragonal structure with an
increase in the c-axis lattice constant. As-grown
samples' transition temperatures are discovered
to be sensitive to In concentrations. 3.4 Halder-Wagner Langford method The advantage of using this strategy is that
peaks in the low-frequency range are focused
on, the deviation peaks overlapping with the
much
lower,
mid-angle
regions,
Peak
broadening is considered to be a symmetrical
Voigt function in the (HW) method. The following formula is used to do SSP
measurement[14, 19, 20]: [ ]
[
] [ ]
[
] The connection between crystallite size and
lattice strain is now provided by the Halder-
Wagner process and is as follows[20-22]: (
)
(
) (
) Which represents straight line equation
between[ ] on a Y-axis and 27 27 JOURNAL OF KUFA–PHYSICS | Vol. 15, No. 1 (2023) Ekhlas Hassan, Haider MJ. Haider The aforementioned equation is a straight-
line equation, hence we will now create a graph
of(
)vs. (
)
, as shown in Fig.6, where
(
) on the X- axis and(
)
on the Y-axis. The aforementioned equation is a straight-
line equation, hence we will now create a graph illustration of the link between crystallinity
levels and indium concentration is shown in
Fig7. It should be noticed that when the indium
concentration increased, the crystallization
values changed. of(
)vs. (
)
, as shown in Fig.6, where
(
) on the X- axis and(
)
on the Y-axis. Figure 7: Degree of Crystallinity as a
function
of
In
concentration
for
(Hg1-
xInxBa2Ca2Cu2.85La0.15O8+δ)
bulk
polycrystalline samples. This straight line's slope provides the value of
(1/crystallite size) among them, we find the (D)
shown in Table 2 and the Intercept represent
(
)
From this, we can find the strain (ƹ)which
is shown in Table 3. Figure 6: Graph of (
) a function of (
)
for
(Hg0.95In0.05Ba2Ca2Cu2.85La0.15O8+δ)
bulk
polycrystalline presented to Halder-Wagner
Langford method. Figure 7: Degree of Crystallinity as a
function
of
In
concentration
for
(Hg1-
xInxBa2Ca2Cu2.85La0.15O8+δ)
bulk
polycrystalline samples. Figure 7: Degree of Crystallinity as a
function
of
In
concentration
for
(Hg1-
xInxBa2Ca2Cu2.85La0.15O8+δ)
bulk
polycrystalline samples. 3.5 The degree of Crystallinity Typically, crystallinity is expressed as a
percentage of the volume of the crystalline
substance. However, even among perfectly
crystalline materials, the level of structural
perfection can vary. The following equation
may be used to calculate the crystallinity using
X-ray crystallography[14, 15]: where the optimal value ( Tc=132.6 k)
occurs when focus (x = 0.05) coincides with the
network constant's maximum value (c=16.065). The degree of Crystallinity calculations further
demonstrates that the best ratio (63.51%) was at
a concentration (x = 0.05), confirming that is
the optimum substitution ratio in comparison to
the other substituents. Acknowledgment: We appreciate the cooperation of the
Department of Physics at the College of
Education for Girls at the University of Kufa
and the cooperation of the superconducting
laboratory official at the College of Education,
Ibn Al-Haitham at the University of Baghdad. [7]
M.R. Jobayr, A.H.A. Al Razak, S.H. Mahdi,
R.N. Fadhil,
Study
optoelectronic properties for polymer
composite
thick
film,
in:
AIP
Conference
Proceedings,
AIP
Publishing LLC, 2018, pp. 030021. ( ) ( ) The (W-H) system only considers peak
widening as a function of a diffraction angle
(2θ), which is anticipated to have been a mix of The findings that shown in Table 2 were
obtained using the aforementioned equation. An 28 JOURNAL OF KUFA–PHYSICS | Vol. 15, No. 1 (2023) Ekhlas Hassan, Haider MJ. Haider Conference
Proceedings,
AIP
Publishing LLC, 2019, pp. 020033. size-induced broadening and strain-induced
broadening. [5]
M.K. Kamil, M.A. Hasan, K.A. Jasim,
A.H. Shaban, Synthesis of HgSr2-
xYxCa2Cu3O8+ δ Superconducting
Compound, in: Key Engineering
Materials, Trans Tech Publ, 2021, pp. 42-47. [5] As opposed to this, the (SSP) approach,
which analyzes peak profiles, simply takes into
account the impact of crystalline size on XRD
peak broadening and says nothing about the
lattice microstructures. In the (HW) approach,
peak broadening is thought to be a symmetrical
Voigt function. [6]
A. Arya, A. Sharma, Structural,
electrical properties and dielectric
relaxations
in
Na+-ion-conducting
solid polymer electrolyte, Journal of
Physics: Condensed Matter, 30 (2018)
165402. 5. REFERENCES [1]
A. Sacco, Electrochemical impedance
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Frequency
dependent
dielectric
properties
of
Al/maleic
anhydride (MA)/p-Si structures, arXiv
preprint arXiv:1604.07789, (2015). [2]
Z. Alborzi, V. Daadmehr, Isovalent
substitution effects of arsenic on
structural and electrical properties of
iron-based
superconductor
NdFeAsO0. 8F0. 2,
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of
Superconductivity
and
Novel
Magnetism, 33 (2020) 387-396. [9]
C. Chu, L. Deng, B. Lv, Hole-doped
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high
temperature
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Physica
C:
Superconductivity
and
its
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partial substitution of copper with
nickel oxide on the Structural and
electrical properties of HgBa2 Ca2
Cu3xNix
O8+
δ
superconducting
compound, in: Journal of Physics:
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2018, pp. 012071. [3]
M.R. Jobayr, S. Mahdi, E. Salman, K. Jasim,
Effect
of
antimony
on
characteristics of HgBa 2 CaCu 2-x Sb
x O 8+ δ superconducting, Journal of
Ovonic Research, 18 (2022). [4]
H.M. Haider, K.M. Wadi, H.A. Mahdi,
K.A. Jasim, A.H. Shaban, Studying
the partial substitution of barium
with cadmium oxide and its effect on
the
electrical
and
structural
properties of HgBa2Ca2Cu3O8+ δ
superconducting compound, in: AIP [11] L.A. Mohammed,
K.A. Jasim,
Improvement
the
Superconducting
properties of TlBa2 Ca2 Cu3xNix O 9-
δ
superconducting
compound
by
partial substitution of copper with 29 Ekhlas Hassan, Haider MJ. Haider JOURNAL OF KUFA–PHYSICS | Vol. 15, No. 1 (2023) [19] V. Mote, Y. Purushotham, B. Dole,
Williamson-Hall
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Temperature Tc and the Lattice
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L. 5. REFERENCES Mohammed,
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δ
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Digest Journal of Nanomaterials &
Biostructures (DJNB), 17 (2022). [15] M.K. Kamil, K.A. Jasim, Calculating
of crystalline size, strain and Degree
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Estado de exceção: origem e estrutura topológica
|
INTERthesis
| 2,015
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cc-by
| 7,478
|
Resumo Este artigo objetiva fornecer, panoramicamente, uma reflexão sobre a origem
histórica e os contornos teóricos do estado de exceção para que se possa fortalecer
a luta contra os espaços de exceção existentes dentro das democracias. A partir da
obra Estado de exceção de Giorgio Agamben, buscou-se o ponto de apoio crítico
para a leitura do texto histórico de Théodore Reinach, De l’état de siège, e, ao
mesmo tempo, o aporte teórico para explicar a estrutura topológica do estado de
exceção. Dentro desta metodologia, em um primeiro momento, abordou-se a história
do estado de exceção, vislumbrando, enfim, que este está mais próximo da tradição
democrático-revolucionária do que da tradição absolutista. Em um segundo
momento, analisou-se o funcionamento e a topologia ímpar do estado de exceção,
bem como a importância fundamental da decisão soberana para a instauração de
situações excepcionais. Ao final, afirmou-se a precisão certeira da tese de Walter
Benjamin: a exceção tornou-se a regra. Seria tão ou mais proveitoso, portanto,
pesquisar, não se um governo é ou não democrático, mas sim os espaços de
exceção existentes dentro das democracias. Palavras-chave: Estado de exceção. Debate histórico. Democracia. Política. Giorgio
Agamben. ESTADO DE EXCEÇÃO: ORIGEM E ESTRUTURA TOPOLÓGICA Lucas Moraes Martins1 http://dx.doi.org/10.5007/1807-1384.2015v12n1p157 http://dx.doi.org/10.5007/1807-1384.2015v12n1p157 1 Doutor em Direito pela Universidade Federal de Minas Gerais. Professor na Faculdade de Direito do
Centro Universitário UNA, Belo Horizonte e no curso de Direito da Faculdade Santo Agostinho,
Montes Claros, MG, Brasil. Email: lucasmoraesmartins@hotmail.com 1 INTRODUÇÃO Comumente, medidas excepcionais de governo são associadas à tradição
antidemocrática. Se este ponto for verdadeiro, nada mais estranho ao entendimento
o fato de medidas excepcionais se fazerem presentes com tanta frequência,
justamente no surgimento das democracias. Por exemplo, o ex-presidente do Egito,
Mohamed Morsi, jurando sob os princípios da liberdade, justiça e democracia,
publicou, em 22 de novembro de 2012, uma declaração constitucional na qual
afirma, no artigo 2o, serem os decretos presidenciais insuscetíveis de análise judicial
(EGITO, 2012). Afirma ainda, no artigo 6o, ser factível ao presidente tomar as ações
necessárias para proteger o país e os objetivos da Revolução Egípcia (EGITO, Esta obra foi licenciada com uma Licença Creative Commons - Atribuição 3.0 Não
Adaptada. 1 Doutor em Direito pela Universidade Federal de Minas Gerais. Professor na Faculdade de Direito do
Centro Universitário UNA, Belo Horizonte e no curso de Direito da Faculdade Santo Agostinho,
Montes Claros, MG, Brasil. Email: lucasmoraesmartins@hotmail.com Esta obra foi licenciada com uma Licença Creative Commons - Atribuição 3.0 Não
Adaptada. Esta obra foi licenciada com uma Licença Creative Commons - Atribuição 3.0 Não
Adaptada. 158 2012), iniciada em 25 de janeiro de 2011, que depôs o governo de Hosni Mubarak,
no poder desde a década de oitenta. Não apenas durante o surgimento, mas também na manutenção das
democracias, medidas excepcionais são amplamente utilizadas, frequentemente
justificadas pela urgência e necessidade. Por exemplo, o USA Patriot Act de 2001,
cujo nome oficial é Uniting and Strengthening America by Providing Appropriate
Tools Required to Intercept and Obstruct Terrorism (USA PATRIOT) Act of 2001
(EUA, 2001), permite ao Attorney General manter detido o estrangeiro suspeito de
envolvimento com atividades que ponham em risco a segurança interna dos Estados
Unidos. Na redação original da lei, esta indefinite detention do estrangeiro, ou
melhor, a detenção indefinida do estrangeiro, frise-se, realizada à margem das
regras processuais normais e fundada tão somente na suspeita de envolvimento
com atos danosos à segurança nacional, poderia durar até sete dias (EUA, 2001). O que estes e outros exemplos trazem à reflexão é que o estado de exceção,
na verdade, parece ser mais uma criação da tradição democrático-revolucionária,
iniciada com a Revolução Francesa, do que uma decorrência da tradição absolutista. Se, dentro das democracias, medidas excepcionais encontram-se frequentemente
como práticas usuais de governo, deve-se, portanto, repensar a história e a relação
entre estado de exceção e a tradição democrática. 1 INTRODUÇÃO A ideia do estado de sítio fictício
ou político com a noção de suspensão da Constituição, ou das normas
constitucionais protetoras da liberdade individual, marcaria a origem (Der Ursprung),
o salto original, do estado de exceção. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 2 A TRADIÇÃO DEMOCRÁTICO-REVOLUCIONÁRIA Na história político-jurídica francesa, a transição conceitual do estado de sítio
militar ou efetivo (état de siège effectif) ao estado de sítio político ou fictício (état de
siège fictif) demonstra quão fluidos são os limites da legalidade diante de situações
excepcionais. Originalmente, o estado de sítio sempre foi uma instituição militar,
condicionado a algumas situações de ataque efetivo ou real às fortificações
permanentes (places de guerre, praça-forte) ou posto militar. Todavia, o estado de
sítio (état de siège) não se confundia com o estado de guerra (état de guerre). O R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 159 decreto de 8-10 de julho de 1791 da Assembleia Constituinte francesa diferenciava
três “états” em que a praça-forte ou o posto militar poderiam se encontrar: estado de
paz (état de paix), estado de guerra (état de guerre) e estado de sítio (état de siège),
conforme ensina Reinach (1885, p.92-93). No estado de paz, as autoridades civis e militares atuavam separadamente,
cada uma dentro das esferas de suas atribuições. Já no estado de guerra, a
autoridade civil deveria agir de acordo com a autoridade militar e cumpriria as
requisições castrenses. O état de guerre era determinado por um decreto do corpo
legislativo por proposição do rei. Na ausência da Assembleia, o estado de guerra
poderia ser decretado provisoriamente pelo próprio rei, sob a responsabilidade dos
ministros, devendo haver uma deliberação posterior do corpo legislativo, mantendo-
se a proclamação real. Por outro lado, no état de siège, para manter a ordem
interna, todas as funções – não apenas as faculdades executivas, mas também as
constitucionais – de que a autoridade civil era investida pela constituição, passavam
ao comandante militar, que as exerceria exclusivamente sob responsabilidade
própria (REINACH, 1885, p.93). Diferentemente do estado de guerra, o estado de sítio derroga duplamente os
princípios da Constituição, pois suspende explicitamente a separação de poderes e,
implicitamente, o direito ao juízo natural (REINACH, 1885, p.93). Por envolver a
suspensão da separação dos poderes e do juízo natural, o estado de sítio não
poderia ser decretado por ordem real, nem mesmo por um decreto legislativo, como
ocorria com o estado de guerra, ou seja, não bastava um decreto ou uma lei para se
instituir o estado de sítio. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 2 A TRADIÇÃO DEMOCRÁTICO-REVOLUCIONÁRIA O estado de sítio, ao menos no decreto de 8-10 de julho de 1791, não poderia
ser creatio ex nihilo, isto é, criado do nada por decreto ou lei, pois seu surgimento
estava condicionado ao aparecimento real de uma situação fática de premente
necessidade, com pressupostos delimitados faticamente, quais sejam, o ataque
efetivo ou real a uma fortificação permanente (places de guerre) ou posto militar. Outra situação específica na qual poderia haver o état de siège denota claramente a
relação desse état com o plano fático: caso houvesse investidas de tropas inimigas e
as comunicações das praças-fortes com o exterior fossem interrompidas, existiria o
estado de sítio ipso facto (DUGUIT, 1925, p.75; REINACH, 1885, p.93; SCHMITT,
2009, p.236). R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 160 Por isso, o estado de sítio nada mais era do que uma instituição puramente
militar. Este estado de sítio, cujo surgimento estava condicionado às situações
fáticas militares reais, pode ser, por conseguinte, denominado de état de siège
effectif (réel ou militaire) ou estado de sítio efetivo (real ou militar). Ao que parece, a Assembleia Constituinte francesa, repudiando a ideia de
uma guerra civil, não considerou a possibilidade de desviar a aplicação das regras
do estado de sítio efetivo e do estado de guerra contra os rebeldes ou inimigos
internos. Entretanto, a partir das leis de 10 e 19 de frutidor do ano V, 27 de agosto e
4 de setembro de 1797, respectivamente, e com a Constituição de 22 de frimário do
ano VIII, 13 de dezembro de 1799, os contornos do estado de sítio até então
conhecidos foram se alterando. (REINACH, 1885, p.93). As datas citadas são particularmente interessantes neste contexto, pois as
leis de 10 e 19 de frutidor do ano V foram decretadas pelo Diretório Executivo que,
com o golpe de 9 de termidor, executou os dirigentes jacobinos, marcando um
retorno da alta burguesia girondina ao poder. De qualquer forma, apenas a partir
destas duas leis pode-se dizer que existiu na legislação francesa duas espécies de
estado de sítio: o estado de sítio efetivo ou militar, état de siège effetif ou militaire, e
o estado de sitio fictício ou político, état de siège fictif ou politique (REINACH, 1885,
p.97). 2 A TRADIÇÃO DEMOCRÁTICO-REVOLUCIONÁRIA A lei de 10 de frutidor, nos artigos 1o e 2o, equiparou os municípios do interior
(communes de l’intérieur), sob as figuras de état de siège e état de guerre, às
praças-fortes (places de guerre). Em outros termos, igualou as cidades do interior às
praças-fortes. Isso significava dizer que as investidas dos rebeldes passaram a ser
consideradas como ataques de tropas militares inimigas. Entretanto, o modo de
proclamar os estados de guerra e de sítio permanecia o mesmo: o estado de guerra
apenas poderia resultar de uma lei e o estado de sítio somente apareceria com um
ataque real, efetivo (REINACH, 1885, p.94-95). Somente com a lei de 19 frutidor do ano V, com o artigo 39, foi reforçado o
poder do Diretório Executivo, conferindo-lhe o direito de colocar uma cidade em
estado de sítio. Ao se alterar a forma de declaração do estado de sítio, colocava-se
uma nova arma a serviço do executivo, sem nenhum tipo de regulamentação sobre
seu emprego (REINACH, 1885, p.95-96). R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015
p
g
(
,
, p
) R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 161 A possibilidade de declarar o état de siège em qualquer parte do território,
mesmo fora dos casos de ataques reais, era, na realidade, um poder até então
desconhecido pelo Diretório, já que o Executivo nunca havia detido este direito. Segundo Schmitt (2009, p.238), em oposição ao estado de sítio militar, efetivo ou
fático, surgia, portanto, o état de siège fictif ou político, permitindo ao governo a
implantação do estado de sítio caso o considerasse necessário segundo os próprios
interesses políticos, independente da situação fática real. Nesse contexto, após o fim do Diretório e na época do Consulado, já com
Napoleão, a constituição de 22 de frimário do ano VIII inseriu uma nova variante
bastante interessante. Sob o signo da propriedade, igualdade e liberdade, direitos
enunciados explicitamente neste corpo constitucional, o artigo 92 declarava a
possibilidade de suspensão da constituição, em casos de revolta a mão armada ou
de agitações que ameaçassem a segurança do Estado, desde que a suspensão
fosse determinada por lei. O Executivo poderia, através de decreto, determinar a
suspensão da constituição apenas quando o corpo legislativo não estivesse reunido,
convocando-o, simultaneamente, à análise da medida (REINACH, 1885, p.96). R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 2 A TRADIÇÃO DEMOCRÁTICO-REVOLUCIONÁRIA O artigo 92 da Constituição de 22 de frimário do ano VIII, apesar de indicar a
possibilidade de suspender o “império da constituição”, não fez menção expressa ao
estado de sítio, nas modalidades de efetivo ou fictício. Em outros termos, ainda não
havia sido estabelecido o nexo entre suspensão da Constituição e estado de sítio
(SCHMITT, 2009, p.240). Isso fica comprovado pelo fato de o decreto napoleônico
de 24 de dezembro de 1811, que alargou as dimensões do estado de sítio,
consolidando o état de siège fictif, não haver levado em consideração o artigo 92 da
Constituição do ano VIII, e sim a legislação anterior, isto é, o decreto de 1791
(decreto de 8-10 de julho de 1791) e as leis do ano V (leis de 10 e 19 de frutidor)
(REINACH, 1885, p.98). O decreto imperial de 24 de dezembro de 1811, a exemplo do decreto de
1791, fazia distinção entre os três “estados” (paz, guerra e sítio), editando regras
novas sobre como o estado de guerra e o estado de sítio seriam determinados. Com
inspiração na lei de 19 de frutidor do ano V, o estado de sítio poderia ser
determinado por um decreto do imperador ou por ocasião de um ataque poderoso,
cerco, sedição interior ou por reuniões realizadas sem autorização dentro da
circunscrição de alguma fortificação (REINACH, 1885, p.99). Interessante notar que R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 162 a lei de 19 de frutidor do ano V apenas dizia que o poder de colocar uma comuna
em estado de sítio era do Diretório, enquanto o decreto napoleônico de 24 de
dezembro de 1811, além de consolidar este poder, indicava a forma de realizá-lo,
através de um decreto do imperador. Sob o ponto de vista político, o decreto de 24 de dezembro de 1811, feito às
vésperas da campanha contra a Rússia, tinha como principal objetivo preservar as
cidadelas às margens do Rio Reno contra eventuais tumultos da população. Isso
significa que Napoleão tinha em mente não apenas o inimigo externo, mas também
o inimigo interno, ao editar esse decreto. Entretanto, justamente por isso, esse
decreto não deixa de ter um fundo de intenção militar, ainda que tenha consolidado
o état de siège fictif (REINACH, 1885, p.98-99; SCHMITT, 2009, p.240-241). Não se pode afirmar se Napoleão utilizou ou não o estado de sítio como
forma de luta política. 2 No original: “En cambio, [Napoleão] amplió el contenido del estado de sitio mediante el decreto de
24 diciembre de 1811, poniendo así los cimientos para su utilizabilidad política.”. SCHMITT, Carl. La
dictadura. 3a ed. Trad. José Diaz Garcia. Madrid: Alianza, 2009, p.240. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 2 A TRADIÇÃO DEMOCRÁTICO-REVOLUCIONÁRIA “Entretanto, [Napoleão] ampliou o conteúdo do estado de sítio
mediante o decreto de dezembro de 1811, estabelecendo, assim, os cimentos para
a sua utilização política” (SCHMITT, 2009, p.240)2. Ironicamente, o decreto de 24 de
dezembro de 1811, apesar de não ter sido revogado formalmente, figura entre os
atos invocados em 1814 para justificar a deposição de Napoleão (REINACH, 1885,
p.102). Justamente neste período da Restauração, antes do “Governo dos Cem
Dias”, o rei Luís XVIII outorga, em 4 de junho de 1814, a Charte de 1814 que, no
artigo 14, atribuía ao soberano o poder de fazer regulamentos e decretos
necessários para a execução das leis e para assegurar a segurança do Estado
(REINACH, 1885, p.104). Entretanto, o governo real não via nisso a utilização do
poder para casos de necessidade, e sim uma mera expressão da soberania. Se as
ordens reais, mesmo que tão somente a juízo do rei, violavam a lei ou mesmo a
própria Charte, não importava tanto sob o prisma da realeza, pois eram apenas uma
expressão da soberania real e úteis para a segurança da ordem existente
(SCHMITT, 2009, p.246-247). Somente com o Acte additionnel à Constituição do Império, de 22 de abril de
1815, redigido por Benjamin Constant a pedido de Napoleão ao retornar do exílio, o R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015
2 No original: “En cambio, [Napoleão] amplió el contenido del estado de sitio mediante el decreto de
24 diciembre de 1811, poniendo así los cimientos para su utilizabilidad política.”. SCHMITT, Carl. La
dictadura. 3a ed. Trad. José Diaz Garcia. Madrid: Alianza, 2009, p.240. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 163 estado de sítio ganhou menção constitucional expressa. Não obstante, o artigo 66
do Acte restringia sua declaração a uma lei (REINACH, 1885, p.103). A denominada Monarquia de Julho (Gouvernement de Juillet ou Monarchie
de Juillet), período compreendido entre 1830 e 1848, que tinha como objetivo a
implantação de um governo constitucional segundo o modelo inglês, repetiu o artigo
14 da Charte de 1814 no artigo 13 da Constituição de 14 de agosto de 1830, sem,
contudo, mencionar o estado de sítio (REINACH, 1885, p.249; SCHMITT, 2009,
p.104-105). Não obstante, o decreto real de 1o de junho de 1832 declarou estado de
sítio em três distritos. 3 No original: “Malgré l’avis du Conseil d’État, qui proposait de n’admettre la mise hors la loi des
communes d’intérieur qu’en cas de guerre ou d’insurrection déclarée, l’Assemblée, qui avait déjà fait
un usage préventif d’état de siège dans les journées du 29 Janvier et du 13 Juin 1849, décida que
cette mesure pourrait être adoptée toutes le fois que la sûreté d’État paraîtrait l’exiger.” R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 2 A TRADIÇÃO DEMOCRÁTICO-REVOLUCIONÁRIA Outro decreto real de 3 de junho de 1832 também declarou
estado de sítio em várias cidades, sem dar razões e por breve período. Por fim, o
decreto de 6 de junho de 1832 declarou estado de sítio em Paris contra os
amotinados, isto é, o proletariado revolucionário (SCHMITT, 2009, p.250-252). Após a queda da Monarquia de Julho, a Assembleia Nacional estabeleceria
novamente o estado de sítio em Paris, em 24 de junho de 1848, que tinha como
finalidade a proteção da propriedade privada e da Constituição burguesa (SCHMITT,
2009, p.252), conferindo poderes executivos ao general Cavaignac, sendo que este
estado de sítio somente seria encerrado em 19 de outubro de 1848 (REINACH,
1885, p.110-111). A Constituição Republicana de 4 de novembro de 1848 incorporou de modo
definitivo o estado de sítio político ou fictício. Em seu artigo 106, esta Constituição
ordenava uma lei que regulamentasse e determinasse os casos possíveis do estado
de sítio e, através de um decreto de 11 de dezembro de 1848, a Assembleia
legislativa reservou a si o direito de fazer esta lei, culminando na lei de 9 de agosto
de 1849 (REINACH, 1885, p.111). A lei de 9 de agosto de 1849 regulamentou o estado de sítio político
aplicando-o aos casos de perigo iminente para a ordem externa ou interna, ou seja,
possibilitou o uso preventivo do état de siège (AGAMBEN, 2007, p.95; SCHMITT,
2009, p.253-256), isto é, “toda vez que a segurança do Estado parecesse exigir”
(REINACH, 1885, p.112)3. Todavia, a declaração do estado de sítio caberia ao
Parlamento e, na ausência do Legislativo, supletivamente pelo chefe do Executivo, R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015
3 No original: “Malgré l’avis du Conseil d’État, qui proposait de n’admettre la mise hors la loi des
communes d’intérieur qu’en cas de guerre ou d’insurrection déclarée, l’Assemblée, qui avait déjà fait
un usage préventif d’état de siège dans les journées du 29 Janvier et du 13 Juin 1849, décida que
cette mesure pourrait être adoptée toutes le fois que la sûreté d’État paraîtrait l’exiger.” R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 164 que
deveria
convocar
a
Assembleia
simultaneamente
para
analisar
e,
eventualmente, ratificar o decreto (REINACH, 1885 p.112-113). A partir de então,
predomina na tradição francesa o princípio de que o Parlamento se reserva o poder
de declarar o estado de sítio (AGAMBEN, 2007, p.25). R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 2 A TRADIÇÃO DEMOCRÁTICO-REVOLUCIONÁRIA Com base neste histórico, fica claro o porquê de Agamben (2007, p.16)
afirmar ser “importante não esquecer que o estado de exceção moderno é uma
criação da tradição democrático-revolucionária e não da tradição absolutista.”. Afirma, ainda, que o estado de exceção moderno nasceu na época da Revolução
francesa (AGAMBEN, 2007, p.22). Basicamente, os motivos citados por Agamben
para embasar estas afirmações são dois. O primeiro é o surgimento gradual do
estado de sítio político ou fictício, emancipado em relação às situações reais de
guerra, ao qual o estado de sítio militar ou efetivo estava entrelaçado, bastando
recordar, por exemplo, o decreto napoleônico de 24 de dezembro de 1811, das leis
de 10 e 19 de frutidor do ano V e do decreto de 8 de julho de 1791. O état de siège
fictif marca o paradigma da extensão dos poderes da esfera militar em tempo de
guerra à esfera civil. O segundo, a ideia de suspensão da constituição, ou das
normas constitucionais protetoras da liberdade individual, introduzida no artigo 92 da
Constituição de 22 de frimário do ano VIII, possibilitando a declaração de uma
cidade ou determinada região hors la constitution. Conforme Agamben, estes dois
modelos, estado de sítio fictício e suspensão da constituição, com o tempo,
acabaram se fundindo em um único fenômeno denominado estado de exceção
(AGAMBEN, 2007, p.15-16). Agamben pode afirmar que o modelo do estado de sítio fictício e a suspensão
da constituição, surgidos na Revolução Francesa, se convertem em um único
fenômeno porque o estado de exceção, a exemplo do état de siège fictif, não
necessita da existência de uma situação real para ser declarado. Tanto o estado de
exceção como o état de siège fictif se emancipam de situações fáticas, reais e
extraordinárias, para convergirem ao ponto da decisão. Ao final, ambos dependem
exclusivamente de uma decisão. Por outro lado, se “[...] o que é próprio do estado de exceção é a suspensão
(total ou parcial) do ordenamento jurídico [...]” (AGAMBEN, 2007, p.39), o estado de
exceção funciona similarmente ao modelo de suspensão da constituição, introduzido
pelo artigo 92 da Constituição de 22 de frimário do ano VIII. A tradição democrático- R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 inal: “L’histoire des lois de salut public commence en France avec la Révolution de 1789.”. 2 A TRADIÇÃO DEMOCRÁTICO-REVOLUCIONÁRIA 2015 165 revolucionária deixara marcas indeléveis e, por isso, não é arbitrária a afirmação de
Théodore Reinach: “A história das leis de salvação da nação começou na França
com a Revolução de 1789.” (REINACH, 1885, p.89)4. A partir de então, a utilização
de medidas excepcionais como prática governamental grassa na política ocidental. Entretanto, Agamben não se preocupa apenas em demonstrar o tronco de
descendência democrático-revolucionário do estado de exceção. Ao teorizar sobre o
estado de exceção, Agamben deseja, com razão, emancipar sua teoria dos
eventuais argumentos que tentem relacioná-la exclusivamente às situações de
guerra ou militares: o estado de exceção não é um direito especial, como o direito de
guerra ou militar, mas o conceito-limite do próprio ordenamento jurídico (AGAMBEN,
2007, p.15). Por isso, Agamben, ao desenvolver as ideias no livro Estado de
exceção, escolheu não utilizar a denominação “estado de sítio” e sim “estado de
exceção”. Historicamente, “estado de sítio”, assim como “lei marcial”, exprimiam ambos
uma relação com uma situação militar ou de guerra real e, portanto, se revelam
inadequados para descrever a estrutura do fenômeno do estado de exceção. Tanto
é que o estado de sítio necessita do qualificativo “político” ou “fictício” para se fazer
inteligível em um contexto civil (AGAMBEN, 2007, p.15). A escolha de Agamben não é neutra, como ele mesmo adverte. “À incerteza
do conceito corresponde exatamente a incerteza terminológica” (AGAMBEN, 2007,
p.15). Procurando afastar sua teoria de interpretações que a pudessem relacionar a
circunstâncias exclusivamente militares ou de guerra, Agamben (2007, p.15; 2010,
p.164) opta pelo termo “estado de exceção” e se aproxima de duas expressões
comuns na doutrina alemã: Ausnahmezustand (estado de exceção) e Notstand
(estado de necessidade), que se encontram historicamente atreladas, não só a
situações de guerra externa, mas principalmente às ameaças à segurança interna e
à ordem pública no território, conforme o artigo 48 da Constituição de Weimar, e
mesmo após o fim da República de Weimar, com o acréscimo do termo “estado de
necessidade interna” (innere Notstand) (AGAMBEN, 2007, p.28-30). Além disso,
conforme Agamben (2007, p.23) assinala, o estado de exceção desempenhou um
papel mais determinante na Alemanha, naquelas situações em que a segurança
pública ou ordem interna estivessem ameaçadas, do que na França ou na Itália. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015
4 No original: “L’histoire des lois de salut public commence en France avec la Révolution de 1789.”. 2 A TRADIÇÃO DEMOCRÁTICO-REVOLUCIONÁRIA 4 No original: “L’histoire des lois de salut public commence en France avec la Révolution de 1789.”. 166 3 ESTRUTURA TOPOLÓGICA DO ESTADO DE EXCEÇÃO Segundo a tradição jurídica e política, o estado de exceção fundamenta-se na
máxima latina necessitas legem non habet, a necessidade não tem lei, formulação
que já se encontrava no Decretum romano de Graciano (AGAMBEN, 2007, p.40-41)
e no mundo medievo, notadamente em São Tomás de Aquino (SAINT-BONNET,
2001, p.121-123). Juridicamente, a busca pela justificativa do estado de necessidade e,
consequentemente, do estado de exceção, divide-se, basicamente, em duas
correntes teóricas. Há aquelas que restringem os casos de necessidade às
situações previstas em lei e, portanto, procuram inserir o estado de exceção no
âmbito do ordenamento jurídico; e, por outro lado, existe a vertente teórica que
afirma a impossibilidade de positivar os casos de necessidade e, por conseguinte,
considera o estado de exceção um fenômeno extrajurídico, exterior ao ordenamento,
mas essencialmente político (AGAMBEN, 2007, p.38). Para a primeira doutrina, o
estado de exceção seria um direito, ius, e para a segunda, um fato, factum. Entretanto, abre-se um flanco para uma crítica certeira a estas vertentes, ao se
afirmar que o estado de necessidade é norma ou simples fato. Se a medida de necessidade, excepcional, já é norma jurídica, e não fato,
qual o motivo de se exigir sua ratificação e aprovação por uma lei? “Se já era direito,
por que não se torna caduca se não for aprovada pelos órgãos legislativos?”
(AGAMBEN, 2007, p.46). Basta recordar o état de siège fictif ou politique após a lei
de 9 de agosto de 1849, que regulamentou o estado de sítio político aplicando-o aos
casos de perigo iminente, exigindo que a declaração do état fosse feita por lei da
Assembleia ou por decreto do Executivo, desde que ratificado pelo Parlamento. Por outro lado, ao se defender que a situação de necessidade e, por
extensão, o estado de exceção, seja um simples fato, “como é possível que os
efeitos jurídicos da ratificação decorram não do momento da transformação em lei e,
sim, ex tunc?” (AGAMBEN, 2007, p.46). Como pode a lei retroagir e validar as
medidas excepcionais? R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 167 A questão do estado de necessidade não resolve satisfatoriamente a
estrutura topológica do estado de exceção, porque, geralmente, a necessidade é
intuída como uma situação objetiva (AGAMBEN, 2007, p.46). R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 3 ESTRUTURA TOPOLÓGICA DO ESTADO DE EXCEÇÃO Todavia, longe de
resolver-se em critérios objetivos ou de se constituir em uma situação objetivamente
posta, a necessidade implica um juízo subjetivo relativo ao escopo que se pretende
atingir (PALLIERI, 1950, p.141). As circunstâncias normais e ordinárias tornam-se necessárias e excepcionais
apenas via uma decisão que lhes atribuam estes qualificativos. O problema está em
considerar o estado de necessidade e o estado de exceção como fatos objetivos ou,
por outro lado, como direitos, explícitos ou implicitamente positivados no
ordenamento jurídico. Mesmo o jurista Pallieri, que entreviu a impossibilidade da
necessidade se reduzir a uma situação objetiva – afirmando ser o conceito
inteiramente subjetivo –, parece perder as próprias reflexões de vista, ao dizer que o
estado de exceção não pode surgir da necessidade, mas da interpretação da
Constituição. Ocorre que a Constituição italiana não previa o instituto do estado de
exceção e Pallieri (1950, p.283-284), negando a necessidade, mas louvando a
norma, cai em um intricado raciocínio para ancorar as limitações excepcionais dos
direitos individuais ao ordenamento jurídico italiano, afirmando que uma medida
excepcional encontraria respaldo na interpretação da Constituição. Se o estado de exceção não é nem um direito (ius) e nem tampouco um
simples fato (factum), e se nem o estado de necessidade consegue delimitar
integralmente seus contornos teóricos, caberia, então, perguntar qual seria a pedra
angular deste fenômeno: a noção de que a necessidade é, de fato, um conceito
totalmente subjetivo, relativo ao objetivo que se deseja alcançar, como afirmou
Pallieri (1950, p.140), apesar de não resolver os limites teóricos do estado de
exceção, aponta para um princípio fundamental, isto é, a necessidade, assim como
a exceção, se reduzem, em última instância, a uma decisão (AGAMBEN, 2007,
p.47). Se decidir implica emitir um juízo sobre alguma coisa, pode-se afirmar que na
necessidade, como na exceção, a decisão tem que recair sobre o indeterminado. Isso significa dizer que a decisão recai sobre uma zona de indeterminação em que
fato (factum) e direito (ius) se tornam indiscerníveis, em que vida e norma se
mesclam em uma zona cinzenta. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 3 ESTRUTURA TOPOLÓGICA DO ESTADO DE EXCEÇÃO 2015 168 Se o patamar de indeterminação ou indiscernibilidade entre ius e factum,
entre nómos e anomia, entre direito e vida, é o locus sobre o qual recai
incessantemente a decisão, deve haver, então, um mecanismo que possibilite a
articulação entre o que está dentro do ordenamento (o elemento jurídico, nómos) e o
que está fora dele (o elemento metajurídico, anomia). O mecanismo que articula e
mantém juntos estes dois aspectos, instituindo um patamar de indiscernibilidade
entre nómos e anomia, é o estado de exceção (AGAMBEN, 2007, p.130). Justamente por criar um limiar entre fato (factum) e direito (ius), o estado de
exceção possibilita a incidência da decisão. Obviamente, só se pode decidir sobre
aquilo que ainda é incerto, indeterminado. Se o que é inerente ao estado de exceção
é a suspensão (total ou parcial) do ordenamento jurídico (AGAMBEN, 2007, p.39),
justamente este movimento de suspensão é que perpetra uma zona ambígua e
incerta, na qual os “procedimentos de fato, em si extra ou antijurídicos, transformam-
se em direito e na qual as normas jurídicas se indeterminam em mero fato”
(AGAMBEN, 2007, p.45). Primo Levi relata em Os afogados e os sobreviventes um acontecimento
elucidativo, capaz de exemplificar este ponto. Segundo Levi, no campo de
concentração, diante da escassez de bens muitas vezes essenciais à subsistência,
tais como alimentos, o furto e contrabando destes produtos produziam uma atividade
de tráfico ou troca muito ativa. Existiam, narra Levi, dois tipos de contrabando que
estabeleciam uma ligação entre a economia interna do campo e a economia do
mundo exterior (LEVI, 1988, p.81). Uma destas espécies de contrabando consistia na troca de produtos do
campo pelos Häftlinge, os prisioneiros, com os trabalhadores externos, os operários
civis. O tráfico com os trabalhadores civis era um aspecto “característico do Campo
de Trabalho e determina sua vida econômica. Constitui, por outro lado, um crime
expressamente previsto pelo Regulamento do Campo [...].” (LEVI, 1988, p.83). O
contrabando de materiais pertencentes ao Campo, ao que parece, era
extremamente reprimido pelos SS e pela direção do Campo (LEVI, 1988, p.84). Entretanto, havia uma segunda forma de contrabando que consistia na
receptação de produtos da fábrica com os quais os Häftlinge lidavam diariamente. Como a fábrica estava fora do Campo, sob a responsabilidade das autoridades civis,
o roubo destes produtos era punido por estas autoridades. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 3 ESTRUTURA TOPOLÓGICA DO ESTADO DE EXCEÇÃO 2015 169 Exatamente aqui surge a ambiguidade, ou melhor, a figura da indeterminação
entre fato e direito. Enquanto o roubo e receptação dos produtos da fábrica eram
punidos pelas autoridades civis, eram autorizados e incentivados pelos SS. Por
outro lado, o roubo dos produtos do Campo com fins de tráfico externo, severamente
punido pelos SS, era considerado pelos civis como operação normal de troca. Diante
disso, Levi convida o leitor à seguinte reflexão: “Desejaríamos, agora, convidar o
leitor a meditar sobre o significado que podiam ter para nós, dentro do Campo, as
velhas palavras ‘bem’ e ‘mal’, ‘certo’ e ‘errado’.” (LEVI, 1988, p.87). Vale ressaltar
que a noção de estado de exceção não implica simplesmente a aplicação de
medidas excepcionais em detrimento das normas, isto é, não se pode reduzir a
disposição topológica entre norma e exceção a uma simples exclusão – ou/ou (Ou a
norma se aplica, excluindo a exceção; ou a exceção se aplica, excluindo a norma). A exceção não habitaria simplesmente a exterioridade da norma sem
qualquer relação com esta. Para Agamben, a “norma se aplica à exceção
desaplicando-se” (AGAMBEN, 2010, p.24) e isso implica que a norma mantém uma
relação com a exceção na forma de sua suspensão. Em outros termos, o estado de
exceção surge como uma situação resultante da suspensão da norma. Através do
mecanismo da exceção, pelo qual o direito é suspenso e o fato eliminado, produz-
se, portanto, “um patamar de indiscernibilidade em que factum e ius se atenuam um
ao outro” (AGAMBEN, 2007, p.47). Esta indeterminação absoluta entre fora e dentro,
entre anomia e nómos, entre vida e norma, entre factum e ius indica o paradoxo –
também denominado de paradoxo da soberania – através do qual se pode entrever,
precisamente, a estrutura topológica do próprio estado de exceção: “Estar-fora e, ao
mesmo tempo, pertencer [...].”(AGAMBEN, 2007, p.57; AGAMBEN, 2005, p.105;
AGAMBEN, 2006, p.131-136). Ainda, como conseqüência da suspensão da norma,
no espaço de exceção há também a inobservância da norma, ou melhor, a
impossibilidade de distinção entre observância e transgressão do direito
(AGAMBEN, 2005, p.105). Se o direito, no estado de exceção, se encontra em sua
forma de suspensão, implicando a coincidência com a realidade, torna-se impossível
observar a lei. Um comportamento que aparentemente segue a lei, pode, ao
contrário, significar uma transgressão e vice-versa. O exemplo trazido por Levi
ganha um brilho ilustrativo peculiar exatamente neste ponto. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 5 No original: “Souverän ist, wer über den Ausnahmezustand entscheidet.”. 3 ESTRUTURA TOPOLÓGICA DO ESTADO DE EXCEÇÃO Justamente essa
impossibilidade de distinção entre observância e transgressão do direito que faz Levi R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 170 convidar o leitor à reflexão sobre os significados que as palavras “bem” e “mal”,
“certo” e “errado” poderiam ter para aqueles que habitavam o Campo. A simples
suspensão do ordenamento jurídico, a criação de um espaço de exceção no qual
surge um limiar de indistinção entre fato (factum) e direito (ius), faz com que se torne
impossível articular entre o lícito e o ilícito (AGAMBEN, 2005, p.106). Entretanto, no estado de exceção, se ordenamento jurídico (dentro) e vida
(fora) se encontram em uma zona na qual se indeterminam, mas são
constantemente delimitados, deve haver algo capaz de realizar esta separação e
articulação entre estes dois elementos. Isto é, deve haver alguém capaz de garantir
a ancoragem do estado de exceção ao ordenamento jurídico que se encontra
suspenso. Este alguém é o soberano. Aqui, as atenções devem se voltar para a
afirmativa de Schmitt, segundo a qual “Soberano é quem decide sobre o estado de
exceção” (SCHMITT, 2004, p.13)5. Cabe à decisão soberana, portanto, decidir quais
situações são excepcionais e quais medidas devem ser adotadas diante da
excepcionalidade, podendo, para tanto, suspender provisoriamente a eficácia do
ordenamento jurídico. Se o estado de exceção faz parte da tradição democrático-revolucionária, não
se deve estranhar, portanto, que medidas excepcionais se façam cada vez mais
presentes nos governos democráticos, como uma prática gerencial que “governa o
curso das coisas, adaptando-se a cada vez, em seu intento salvífico, à natureza da
situação concreta com que deve medir forças.” (AGAMBEN, 2011, p.64). Entender
este ponto é crucial para não se perder de vista os alcances teóricos de uma das
teses desenvolvidas no livro Estado de exceção: o estado de exceção que,
antigamente, era temporal, converteu-se, nos dias atuais, em uma técnica de
governo (AGAMBEN, 2014, p.3). A teoria do estado de exceção, como ressalta o
próprio Agamben (2014, p.3), “não pode ser um fim ou um objetivo em si mesmo,
senão que tem que estar inscrita em um conjunto mais amplo, no contexto das
tecnologias de governo”. Assim como na expressão estado de exceção, a palavra “estado” seria mais
bem traduzida como “situação”, o vocábulo alemão Ausnahmezustand, algumas
vezes traduzido como estado de exceção, indica, na verdade, uma situação
(Zustand) emergencial, excepcional. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 3 ESTRUTURA TOPOLÓGICA DO ESTADO DE EXCEÇÃO Em Ausnahmezustand, Zustand (estado) se 5 No original: “Souverän ist, wer über den Ausnahmezustand entscheidet.”. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 171 diferencia de Staat (estado), como presente, por exemplo, na expressão estado de
direito (Rechtsstaat). Seria tão ou mais interessante, portanto, pesquisar, não se um
governo é ou não democrático, mas sim os espaços de exceção existentes dentro
das democracias. Diante desta coincidência entre os paradigmas do governo e do
estado de exceção, afinal, tudo leva à crer que Walter Benjamin (2007, p.133)
estava certo ao afirmar, nas teses sobre o conceito de história (Über den Begriff der
Geschichte), que o estado de exceção tornara-se a regra. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 4 CONCLUSÃO O artigo buscou apresentar os contornos históricos do estado de exceção,
bem como pontuar a topologia deste fenômeno, apontando, ao final, para a
importância moderna deste estudo. O estado de exceção se alinha mais à tradição
democrático-revolucionária do que à tradição absolutista, porquanto sua origem
pode ser vislumbrada na confluência da ideia do estado de sítio fictício ou político
com a noção de suspensão da Constituição, ambas fruto da Revolução Francesa. Originalmente, o estado de sítio não poderia ser criado do nada por decreto
ou lei, pois seu surgimento estava condicionado ao aparecimento real de situações
fáticas. A utilização política do estado de sítio, emancipado em relação às situações
fáticas reais, é fruto da tradição revolucionária, bastando recordar o decreto
napoleônico de 24 de dezembro de 1811, das leis de 10 e 19 de frutidor do ano V e
do decreto de 8 de julho de 1791. Juntamente com este estado de sítio político, a
ideia de suspensão da constituição foi também introduzida pela tradição
revolucionária, por meio do artigo 92 da Constituição de 22 de frimário do ano VIII,
possibilitando a declaração de uma cidade ou determinada região hors la
constitution: a confluência desses destes dois institutos, estado de sítio político e a
possibilidade de suspensão da constituição, na modernidade, fez surgir o fenômeno
único do estado de exceção. Apesar da tentativa moderna de justificar a exceção pela necessidade
(necessitas legem non habet), o princípio da necessidade não esclarece os
contornos teóricos do estado de exceção, porque este, longe de se figurar como
uma situação objetiva, apresenta-se como fruto de uma decisão. A esta caberia,
portanto, a instauração do estado de exceção e a aplicação das medidas que devem R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 172 ser adotadas diante da excepcionalidade, podendo, inclusive, suspender
provisoriamente a eficácia do ordenamento jurídico. Com a suspensão do
ordenamento jurídico o que se pretende capturar é a própria anomia, e isso leva à
conclusão – afinal, uma monstruosa ironia – de que o direito necessita intimamente
e incessantemente de um momento de violência, do anomos. Se as ideias revolucionárias de suspensão da constituição e do estado de
sítio político se erigiram em mecanismos da tradição democrático-revolucionária
para permitir a adaptação de governos às situações concretas, estes dois
fenômenos foram herdados pela modernidade na figura do estado de exceção. 4 CONCLUSÃO Se,
modernamente, a prática governamental ocidental se vale, amplamente, de medidas
excepcionais para governar o curso dos fatos, esta coincidência entre os
paradigmas do governo e do estado de exceção confirmam a tese de Walter
Benjamin: a exceção tornou-se a regra. Proveitoso seria, portanto, pesquisar,
também, os espaços de exceção existentes dentro das democracias. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 173 Abstract This article aims to provide, panoramically, a reflection on the historical origins and
theoretical contours of the state of exception. Thus, we can strengthen the fight
against the existing spaces of exception within democracies. From the book State of
Exception by Giorgio Agamben, we sought the critical support for reading the
historical text of Théodore Reinach, De l'état de siège. Agamben's book also
provides the theoretical contribution to explain the topological structure of the state of
exception. Within this methodology, at first, it was explained the history of the state of
exception and, finally, it was asserted that this is closer to the democratic-
revolutionary tradition than the absolutist tradition. In a second step, we analyzed the
functioning and the unique topological structure of the state of exception as well as
the fundamental importance of sovereign decision for the establishment of
exceptional situations. In conclusion, it was stated the unerring accuracy of Walter
Benjamin’s thesis: the exception has become the rule. It would be equally or more
useful, therefore, to look not whether a government is democratic or not, but the
spaces of exception within democracies. p
p
Keywords: State of exception. Historical debate. Democracy. Politics. Giorgio
Agamben. REFERÊNCIAS AGAMBEN, Giorgio. Entrevista com Giorgio Agamben. Entrevistadora: Flávia Costa. Revista do Departamento de Psicologia, UFF, Niterói, v.18, n.1, jan./jun. 2006. Disponível
em:
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0104-
80232006000100011 Acesso em: 24 nov. 2012. AGAMBEN, Giorgio. Estado de exceção. Trad. Iraci D. Poleti. 2a ed. São Paulo:
Boitempo, 2007. Resumen Este artículo tiene como objetivo proporcionar, panorámicamente, una reflexión
sobre los orígenes históricos y los contornos teóricos del estado de excepción para
que se pueda fortalecer la lucha contra los espacios de excepción existentes dentro
de las democracias. Con lo libro de Giorgio Agamben, Estado de excepción,
buscamos el punto de apoyo fundamental para la lectura del texto histórico de
Théodore Reinach, De l'état de siège, y al mismo tiempo, la contribución teórica para
explicar la estructura topológica del estado de excepción. Dentro de esta
metodología, en un primer momento, se explica la historia del estado de excepción,
vislumbrando, finalmente que esto está más cerca de la tradición democrático-
revolucionaria que de la tradición absolutista. En un segundo momento, se analizó el
funcionamiento y la estructura topológica única del estado de excepción, así como la
importancia fundamental de la decisión soberana como fuente de la creación de
situaciones excepcionales. Finalmente, se declaró la exactitud infalible de la tesis de
Walter Benjamin: la excepción se ha convertido en la regla. Sería tanto o más útil,
por lo tanto, no descubrir si un gobierno es democrático o no, pero descubrir los
espacios de excepción existentes en las democracias. p
p
Palabras clave: Estado de excepción. Debate histórico. Democracia. Política. Giorgio Agamben. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 174 REFERÊNCIAS LEVI, Primo. É isto um homem?. Trad. Luigi Del Re. Rio de Janeiro: Rocco, 1988. LEVI, Primo. É isto um homem?. Trad. Luigi Del Re. Rio de Janeiro: Rocco, 1988. PALLIERI, G. Balladore. Diritto Costituzionale. 2° ed. Milano: Dott. A. Giuffré, 1950. AGAMBEN, Giorgio. Estado de exceção. Trad. Iraci D. Poleti. 2a ed. São Paulo:
Boitempo, 2007. AGAMBEN, Giorgio. Estado de exceção e genealogia do poder. Trad. Daniel Arruda
Nascimento. Revista brasileira de estudos políticos. UFMG, Belo Horizonte,
v.108,
jan./jun. 2014. Disponível
em:
http://www.pos.direito.ufmg.br/rbep/index.php/rbep/article/view/P.0034-
7191.2014v108p21 Acesso em: 20 jul. 2014. AGAMBEN, Giorgio. Homo sacer: o poder soberano e a vida nua I. 2a ed. Trad.:
Henrique Burigo. Belo Horizonte: UFMG, 2010. AGAMBEN, Giorgio. O Reino e a glória: uma genealogia teológica do governo e da
economia. Trad.: Selvino J. Assmann. São Paulo: Boitempo, 2011. AGAMBEN, Giorgio. The time that remains: a commentary on the Letter to the
Romans. Trad. Patrícia Dailey. Stanford: Meridian, 2005. BENJAMIN, Walter. Über den Begriff der Geschichte. In BENJAMIN, Walter. Erzählen: Schriften zur Theorie der Narration und zur literarischen Prosa. Frankfurt
am Main: Suhrkamp, 2007. DUGUIT, Léon. Traité de droit constitutionnel: les libertés publiques. 2a ed. Tomo
5. Paris: Ancienne librairie fontemoing, 1925. R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015
EGITO. Constitutional declaration, November 22, 2012. Disponível em:
http://english.ahram.org.eg/NewsContent/1/64/58947/Egypt/Politics-/English-text-of-
Morsis-Constitutional-Declaration-.aspx Acesso em: 12 dez. 2012. EGITO. Constitutional declaration, November 22, 2012. Disponível em:
http://english.ahram.org.eg/NewsContent/1/64/58947/Egypt/Politics-/English-text-of-
Morsis-Constitutional-Declaration-.aspx Acesso em: 12 dez. 2012. 175 EUA. Uniting and Strengthening America by Providing Appropriate Tools
Required to Intercept and Obstruct Terrorism (USA PATRIOT) Act of 2001. Disponível
em:
http://www.gpo.gov/fdsys/pkg/PLAW-107publ56/pdf/PLAW-
107publ56.pdf Acesso em 28 jan. 2014. PALLIERI, G. Balladore. Diritto Costituzionale. 2° ed. Milano: Dott. A. Giuffré, 1950. REINACH, Théodore. De l’état de siège: étude historique et juridique. Paris : F. Pichon, 1885. SAINT-BONNET, François. L’état d’exception. Paris: Universitaires de France,
2001. SCHMITT, Carl. La dictadura. 3a ed. Trad. José Diaz Garcia. Madrid: Alianza, 2009. SCHMITT, Carl. Politische Theologie: Vier Kapitel zur Lehre von der Souveränität. 8a ed. Berlim: Duncker & Humblot, 2004. SCHMITT, Carl. Politische Theologie: Vier Kapitel zur Lehre von der Souveränität. 8a ed. Berlim: Duncker & Humblot, 2004. SCHMITT, Carl. Politische Theologie: Vier Kapitel zur Lehre von der Souveränität. 8a ed. Berlim: Duncker & Humblot, 2004. Artigo:
Submetido em 28.07.2014
Aceito em 30.01.2015 R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015 R. Inter. Interdisc. INTERthesis, Florianópolis, v.12, n.1, p.157-175, Jan-Jun. 2015
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Textile-based pressure sensors for step detection: a preliminary assessment
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Textile-based pressure sensors for step detection: a preliminary
assessment To cite this article: A G Ferreira et al 2018 IOP Conf. Ser.: Mater. Sci. Eng. 459 012041 View the article online for updates and enhancements. This content was downloaded from IP address 193.137.92.77 on 17/12/2018 at 16:26 IOP Conference Series: Materials Science and Engineering IOP Conference Series: Materials Science and Engineering This content was downloaded from IP address 193.137.92.77 on 17/12/2018 at 16:26 Aegean International Textile and Advanced Engineering Conference (AITAE 2018)
IOP Conf. Series: Materials Science and Engineering459 (2019) 012041
IOP Publishing
doi:10.1088/1757-899X/459/1/012041 Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd
1 Textile-based pressure sensors for step detection: a
preliminary assessment A G Ferreira1,2, A P Catarino2, J L Monteiro1 and A M Rocha2
1University of Minho, School of Engineering, Centro Algoritmi, Guimarães, Portugal
2University of Minho, School of Engineering, Centre of Textile Science and
Technology, Guimarães, Portugal A G Ferreira1,2, A P Catarino2, J L Monteiro1 and A M Rocha2
1University of Minho, School of Engineering, Centro Algoritmi, Guimarães, Portugal
2University of Minho, School of Engineering, Centre of Textile Science and
Technology, Guimarães, Portugal Email: id4541@alunos.uminho.pt Email: id4541@alunos.uminho.pt Abstract. This paper presents the development and performance assessment of textile-based
sensor based on a three layer architecture for the step detection. Two different transducing
elements (EeonTex™ LG-SLPA and velostat) and electrodes (Satatex Techniktex P-130 and
Elitex yarns) were selected for the construction of the sensors. The performance of the
resulting sensors was assessed based on a dynamometer cyclic compression/decompression test
with different compressions loads and at different speeds. Additionally, a real-life experiment
was conducted to evaluate the sensor response during walking. The results show that all
sensors configurations have a non-linear resistance-force relation. The best sensor
configuration for the step detection was the combination of EeonTex™ LG-SLPA as a
transducing element and the Elitex yarns for the electrodes. In this configuration, the resistance
magnitude varies in an order of hundreds of kohms between the stance and the swing phases. 1. Introduction Position estimation based on the Pedestrian Dead Reckoning (PDR) technique suffer from cumulative
error due to the drift on the inertial sensors measurements. Typically, to prevent the exponential
growth of the error on the position estimation, the PDR-based systems apply the zero-velocity update
(ZUPT) technique. This technique takes advantage of the human gait to minimize the drift impact and
correct the position estimate. I.e., every time the foot is in touch with the ground (stance phase), the
inertial sensors readings are considered drift and the pedestrian position is corrected based on the
magnitude of those readings. However, the ZUPT technique relies on the definition of thresholds on
sensors’ readings (e.g., stance duration, acceleration and angular velocity magnitudes) [1]. These
thresholds make the performance user- and motion-dependent, characteristics that are undesired in
Indoor Positioning Systems (IPSs) for emergency responders [2]. In this paper, we study the viability of embedding textile-based pressure sensors on a sock to detect
the stance phase. To do that, different materials for both the electrodes and the sensing element were
used and the performance of the resulting sensors was assessed based on two different experiments. Besides improving the stance detection of the conventional ZUPT methods, the proposed solution
is wearable, comfortable, easy-to-use, and cheaper than solutions with pressure sensors embedded on
the insole [3]. 1 Aegean International Textile and Advanced Engineering Conference (AITAE 2018)
IOP Conf. Series: Materials Science and Engineering459 (2019) 012041
IOP Publishing
doi:10.1088/1757-899X/459/1/012041 Aegean International Textile and Advanced Engineering Conference (AITAE 2018)
IOP Conf. Series: Materials Science and Engineering459 (2019) 012041
IOP Publishing
doi:10.1088/1757-899X/459/1/012041 2. Materials and Methods In this section, the assembly details and materials used for the construction of the pressure sensor
prototypes as well as the equipment and experimental setup used/designed to assess their performance
are described. 2.2. Configuration of the Sensors’ Prototypes To assess the best combination of materials (electrodes and sensing element), three different sensor
configurations were tested. They differ on the piezoresistive element (EeonTex™ LG-SLPA and
velostat) and the conductive materials (Satatex Techniktex P-130 and a silver coated yarn from Elitex)
used. Table 1 shows the materials used for the construction of each pressure sensor prototype, which
are labeled as S1, S2 and S3. Table 1. Description of the materials used for the construction of
each pressure sensor prototype. Sensor
Label
Conductive Materials
Piezoresistive Sensing
Element
S1
Elitex yarns
EeonTex™ LG-SLPA
S2
Satatex Techniktex P-130
EeonTex™ LG-SLPA
S3
Satatex Techniktex P-130
Velostat 2.1. Pressure Sensor Prototype Figure 1 shows the sketch and the fabrication details of the pressure sensors developed. The sensors
are produced in three layers: the top and bottom layer are made with conductive materials (fabric and
yarn knitted on the sock), and the middle layer is a piezoresistive substrate. The sensing element is
placed on the sock’s heel and conductive leads connect the sensor to the acquisition device (placed on
the user’s ankle). The dimensions of the sensing element and electrodes are 10x10 mm and 7.5x7.5 mm, respectively. The dimension of the electrodes is smaller than the sensing element to prevent any short-circuit
between the two conductive layers. Figure 1. A sketch of the three layer textile-based pressure sensor with the conductive leads embedded
on a sock. Socks fabric
Piezoelectric
layer
Dry electrodes with Figure 1. A sketch of the three layer textile-based pressure sensor with the conductive leads embedded
on a sock. 2.2. Configuration of the Sensors’ Prototypes 2.3. Experimental Setups p
p
Two types of experiments were conducted to assess the viability of using the pressure sensors for step
detection. In the first experiment, a uniaxial dynamometer (Houndsfield, H100KS) and a digital
multimeter (Agilent, 34410A) were used to characterize the electrical behavior of the pressure sensors
during a 10 cycle compression/decompression test. This setting aims to study the sensor’s response, in
terms of electrical resistance, when it is submitted to different pressures. Therefore, for each sensor
configuration, three different compression cycles (2-136N, 2-173N, and 2-210N) were applied. These
compression forces corresponds, based on the ratio between the heel and dynamometer probe areas, to
a body weight of 55, 70, and 85 kg, respectively. The application of different compression forces aims Two types of experiments were conducted to assess the viability of using the pressure sensors for step
detection. In the first experiment, a uniaxial dynamometer (Houndsfield, H100KS) and a digital
multimeter (Agilent, 34410A) were used to characterize the electrical behavior of the pressure sensors
during a 10 cycle compression/decompression test. This setting aims to study the sensor’s response, in
terms of electrical resistance, when it is submitted to different pressures. Therefore, for each sensor
configuration, three different compression cycles (2-136N, 2-173N, and 2-210N) were applied. These
compression forces corresponds, based on the ratio between the heel and dynamometer probe areas, to
a body weight of 55, 70, and 85 kg, respectively. The application of different compression forces aims 2 Aegean International Textile and Advanced Engineering Conference (AITAE 2018)
IOP Conf. Series: Materials Science and Engineering459 (2019) 012041
IOP Publishing
doi:10.1088/1757-899X/459/1/012041 Aegean International Textile and Advanced Engineering Conference (AITAE 2018)
IOP Conf. Series: Materials Science and Engineering459 (2019) 012041
IOP Publishing
doi:10.1088/1757-899X/459/1/012041 to study how the sensor respond to different body weights and if it possible to detect steps for all body
weights and determine the wearer’s weight based on the sensor output. Additionally, three different
speeds (5, 25, and 50 mm/min) were set to evaluate the dynamic sensor response and to assess if the
sensor is able to detect steps at different walking speeds. For the dynamometer experiment, a total of
27 individual tests were performed, 9 per each sensor configuration. to study how the sensor respond to different body weights and if it possible to detect steps for all body
weights and determine the wearer’s weight based on the sensor output. 2.3. Experimental Setups Additionally, three different
speeds (5, 25, and 50 mm/min) were set to evaluate the dynamic sensor response and to assess if the
sensor is able to detect steps at different walking speeds. For the dynamometer experiment, a total of
27 individual tests were performed, 9 per each sensor configuration. On the other hand, the second experiment consisted on asking a volunteer (male, 30 years old,
70kg) to wear a sock with the pressure sensor embedded, perform five consecutive steps and then
stopped with the heel on the ground. This experiment was repeated five times for each sensor
configuration and the sensor output was recorded using the same digital mutimeter (Agilent, 34410A). This experiment aims to evaluate the sensor response on a real scenario, since the dynamometer
cannot replicate the velocities experienced during walking (≈1m/s). 3. Results and Discussion During the stance phase, the heel presses the piezoresistive material and the sensor’s resistance
decrease. In the swing phase, the pressure between the heel and the footwear decreases and the
resistance of the sensor increase. This resistance variation allows the step detection and higher its
variation better the stance phase can be detected. 3.1. Dynamometer Experiment Figure 2 shows the typical response of the developed pressure sensors during a dynamometer
compression cycle. As can be seen in the figure, the resistance-force relation is non-linear (resembles
to an exponential function), which is in line with the findings of similar research [4,5]. Since the goal
of this work is to detect steps, this behavior of the sensor is beneficial as it allows to easily detect the
step, the difference on the sensors resistance between the stance and swing phases can be in the order
of hundreds of kilo ohms (figure 2). As expected, when the sensor is compressed (stance phase) its
resistance decrease and increases during the decompression phase (swing phase). Additionally, the
sensor response during the 10 compression/decompression cycles is almost the same, meaning that the
sensor output is repeatable. Figure 2. Example of a sensor output for the 10 cycles of compression/decompression for the
dynamometer experiment. This graph illustrates the response of the S1 sensor configuration, for a
simulated user weight of 70 kg (173 N) and a test velocity of 25 mm/min. Figure 2. Example of a sensor output for the 10 cycles of compression/decompression for the
dynamometer experiment. This graph illustrates the response of the S1 sensor configuration, for a
simulated user weight of 70 kg (173 N) and a test velocity of 25 mm/min. Figure 3 shows the resistance versus time percentage plots for all sensors configurations, weights
and speeds. The time of the plots was converted to a percentage for an easier comparison between the
experiments performed under different speeds. Based on the results obtained, the S1 sensor configuration stand out as the best option to detect the
steps as the resistance magnitude changes in the order of thousands of kilo ohms between the
compression and decompression cycle. Whereas the sensor configuration S2 has a variation magnitude
in the order of hundreds of kilo ohms and the S3 configuration in the order of hundreds of ohms. Additionally, unlike S2 and S3 configurations, the response of the S1 configuration is almost the same 3 Aegean International Textile and Advanced Engineering Conference (AITAE 2018)
IOP Conf. Series: Materials Science and Engineering459 (2019) 012041
IOP Publishing
doi:10.1088/1757-899X/459/1/012041 Aegean International Textile and Advanced Engineering Conference (AITAE 2018)
IOP Conf. Series: Materials Science and Engineering459 (2019) 012041
IOP Publishing
doi:10.1088/1757-899X/459/1/012041 for different speeds. 3.1. Dynamometer Experiment For the S2 and S3 configurations the magnitude of the sensor response at lower
speed is lower than for higher speeds. This phenomenon can be related with the limitations of the
dynamometer. I.e., for higher speeds the applied force is higher than the threshold defined (e.g., see
figure 2). for different speeds. For the S2 and S3 configurations the magnitude of the sensor response at lower
speed is lower than for higher speeds. This phenomenon can be related with the limitations of the
dynamometer. I.e., for higher speeds the applied force is higher than the threshold defined (e.g., see
figure 2). Although the resistance magnitude seems to decrease during the 10 compression/decompression
cycles, it is not possible to state that the sensor is not repeatable since the dynamometer is not capable
of applying the same force for each compression/decompression cycle. For lower velocities (5
mm/min) the sensors’ magnitude is almost the same. Regarding the different forces applied, by
analysing the graphs of figure 3 does not seem that they have a significant impact of the sensor output. However, more tests with better equipment should be conducted to assess if it is possible to determine
the wearer’s body weight based on the sensor output. Figure 3. Resistance versus time percentage plots for the different sensors configurations, simulated
users’ weight, and test velocities. Figure 3. Resistance versus time percentage plots for the different sensors configurations, simulated
users’ weight, and test velocities. Acknowledgments g
This work was partially financed by FEDER funds through the Competitively Factors Operational
Programme—COMPETE and by national founds through FCT -Foundation for Science and
Technology within the scope of Project POCI-01-0145-FEDER-007136 and Project PEst-
OE/EEI/UI0319/2014. Authors would like to thanks André Paiva and Sérgio Branco for their
collaboration in sensors production and data acquisition, respectively. 4. Conclusions In this paper, three types of textile-based pressure sensors were proposed and evaluated for the stance
detection. These sensors were evaluated on a dynamometer compression/decompression cycle test and
by a real-life experiment. In this paper, three types of textile-based pressure sensors were proposed and evaluated for the stance
detection. These sensors were evaluated on a dynamometer compression/decompression cycle test and
by a real-life experiment. As demonstrated in this paper, the steps can be easily detected by means of a textile-based pressure
sensor integrated on a sock. From the three configurations evaluated, the combination of Elitex yarns
as conductive materials and EeonTexTM LG-SLPA as the piezoresistive element provided the best
results. In this first assessment, many relevant aspects of sensor construction have been investigated
and a detailed knowledge about the sensor behavior has been acquired. As future work, the sensors will be fully characterized in a more advanced dynamometer and more
sensor configurations will be tested. Namely, the creation of 3D structures with conductive yarn to
replace the three layer architecture and simulate the piezoresistive behavior of the sensing elements
used in this work. Additionally, the textile-based pressure sensor will be integrated into PDR system
[6] to assess its viability in the drift control of the inertial sensors. 3.2. Walking Experiment g
p
Figure 4 shows the results of the tests for the walking experiment. As can be seen in the figure, the
best results were obtained for the S1 configuration, followed by the S2 and S3 configurations. Compared with the results obtained from the dynamometer tests, it is possible to see that the resistance
magnitude for all sensor configurations decreased significantly. This phenomenon can be explained by
the existence of a continuous force applied to the sensor due to its placement between the heel and
shoe. Nevertheless, the stance and the swing phases can be clearly distinguished, validating the use of
these pressure sensors for step detection. Additionally, we can see that a peak appears in the beginning of the swing phase. This peak refers
to the heel off moment and can be explained by the diminution of the pressure applied by the heel on
the sensor, which is later restored during the swing phase. Moreover, for all the sensor configurations,
the resistance when the heel is pressing the sensor is very stable and its magnitude after the five steps
is very close to the initial value. This means that the sensor is capable of providing reliable results for
stance detection. 4 4 Aegean International Textile and Advanced Engineering Conference (AITAE 2018)
IOP Conf. Series: Materials Science and Engineering459 (2019) 012041
IOP Publishing
doi:10.1088/1757-899X/459/1/012041 IOP Publishing doi:10.1088/1757-899X/459/1/012041 Figure 4. Resistance versus time plots for each sensor configuration while a volunteer performs five
consecutive steps. Figure 4. Resistance versus time plots for each sensor configuration while a volunteer performs five
consecutive steps. [1]
Jimenez AR, Seco F, Prieto JC, Guevara J. Indoor pedestrian navigation using an INS/EKF
framework for yaw drift reduction and a foot-mounted IMU. 2010 In: 2010 7th Workshop on
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Jimenez AR, Seco F, Prieto JC, Guevara J. Indoor pedestrian navigation using an INS/EKF
framework for yaw drift reduction and a foot-mounted IMU. 2010 In: 2010 7th Workshop on
Positioning, Navigation and Communication p. 135
[2]
Ferreira AFG, Fernandes DMA, Catarino AP, Monteiro JL. Localization and Positioning
Systems for Emergency Responders: A Survey. 2017 IEEE Communications Surveys &
Tutorials 19 2836
[3]
Truong P, Lee J, Kwon A-R, Jeong G-M. Stride Counting in Human Walking and Walking
Distance Estimation Using Insole Sensors. 2016 Sensors 16 823 framework for yaw drift reduction and a foot-mounted IMU. 2010 In: 2010 7th Workshop on
Positioning, Navigation and Communication p. 135
[2]
Ferreira AFG, Fernandes DMA, Catarino AP, Monteiro JL. Localization and Positioning
Systems for Emergency Responders: A Survey. 2017 IEEE Communications Surveys &
Tutorials 19 2836
[3]
Truong P, Lee J, Kwon A-R, Jeong G-M. Stride Counting in Human Walking and Walking References [1]
Jimenez AR, Seco F, Prieto JC, Guevara J. Indoor pedestrian navigation using an INS/EKF
framework for yaw drift reduction and a foot-mounted IMU. 2010 In: 2010 7th Workshop on
Positioning, Navigation and Communication p. 135 [1]
Jimenez AR, Seco F, Prieto JC, Guevara J. Indoor pedestrian navigation using an INS/EKF
framework for yaw drift reduction and a foot-mounted IMU. 2010 In: 2010 7th Workshop on
Positioning, Navigation and Communication p. 135 g
g
p
[2]
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Systems for Emergency Responders: A Survey. 2017 IEEE Communications Surveys &
Tutorials 19 2836 g
g
p
[2]
Ferreira AFG, Fernandes DMA, Catarino AP, Monteiro JL. Localization and Positioning
Systems for Emergency Responders: A Survey. 2017 IEEE Communications Surveys &
Tutorials 19 2836 [3]
Truong P, Lee J, Kwon A-R, Jeong G-M. Stride Counting in Human Walking and Walking
Distance Estimation Using Insole Sensors. 2016 Sensors 16 823 [3]
Truong P, Lee J, Kwon A-R, Jeong G-M. Stride Counting in Human Walking and Walking
Distance Estimation Using Insole Sensors. 2016 Sensors 16 823 5 Aegean International Textile and Advanced Engineering Conference (AITAE 2018)
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doi:10.1088/1757-899X/459/1/012041
[4]
Dinparast Tohidi S, Zille A, Catarino AP, Rocha AM. Effects of Base Fabric Parameters on the
Electro-Mechanical Behavior of Piezoresistive Knitted Sensors. 2018 IEEE Sensors Journal
18 4529
[5]
Carvalho H, Yao Y, Gonçalves LM. Flexible force sensors for e-textiles. 2017 In: IOP
Conference Series: Materials Science and Engineering 254 p. 072007
[6]
Ferreira AG, Fernandes D, Monteiro JL, Catarino AP, Rocha AM. A Pedestrian Positioning
System integrated into a Cyber-Physical System for Emergency Responders’ Monitoring. 2018 In: 2018 International Conference on Indoor Positioning and Indoor Navigation (IPIN) Aegean International Textile and Advanced Engineering Conference (AITAE 2018)
IOP Conf. Series: Materials Science and Engineering459 (2019) 012041
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doi:10.1088/1757-899X/459/1/012041 [4]
Dinparast Tohidi S, Zille A, Catarino AP, Rocha AM. Effects of Base Fabric Parameters on the
Electro-Mechanical Behavior of Piezoresistive Knitted Sensors. 2018 IEEE Sensors Journal
18 4529 [4]
Dinparast Tohidi S, Zille A, Catarino AP, Rocha AM. Effects of Base Fabric Parameters on the
Electro-Mechanical Behavior of Piezoresistive Knitted Sensors. 2018 IEEE Sensors Journal
18 4529 [5]
Carvalho H, Yao Y, Gonçalves LM. Flexible force sensors for e-textiles. 2017 In: IOP
Conference Series: Materials Science and Engineering 254 p. [4]
Dinparast Tohidi S, Zille A, Catarino AP, Rocha AM. Effects of Base Fabric Parameters on the
Electro-Mechanical Behavior of Piezoresistive Knitted Sensors. 2018 IEEE Sensors Journal
18 4529 Aegean International Textile and Advanced Engineering Conference (AITAE 2018)
IOP Conf. Series: Materials Science and Engineering459 (2019) 012041
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doi:10.1088/1757-899X/459/1/012041 f
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[6]
Ferreira AG, Fernandes D, Monteiro JL, Catarino AP, Rocha AM. A Pedestrian Positioning
System integrated into a Cyber-Physical System for Emergency Responders’ Monitoring.
2018 In: 2018 International Conference on Indoor Positioning and Indoor Navigation (IPIN) [5]
Carvalho H, Yao Y, Gonçalves LM. Flexible force sensors for e-textiles. 2017 In: IOP
Conference Series: Materials Science and Engineering 254 p. 072007 References 072007 [6]
Ferreira AG, Fernandes D, Monteiro JL, Catarino AP, Rocha AM. A Pedestrian Positioning
System integrated into a Cyber-Physical System for Emergency Responders’ Monitoring. 2018 In: 2018 International Conference on Indoor Positioning and Indoor Navigation (IPIN) 6 6 6
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https://www.emerald.com/insight/content/doi/10.1108/SASBE-01-2021-0016/full/pdf?title=digital-twin-based-progress-monitoring-management-model-through-reality-capture-to-extended-reality-technologies-drx
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English
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Digital twin-based progress monitoring management model through reality capture to extended reality technologies (DRX)
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Smart and sustainable built environment
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cc-by
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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/2046-6099.htm The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/2046-6099.htm Abstract Purpose – The purpose of this research is to develop a generic framework of a digital twin (DT)-based
automated construction progress monitoring through reality capture to extended reality (RC-to-XR). Design/methodology/approach – IDEF0 data modeling method has been designed to establish an
integration of reality capturing technologies by using BIM, DTs and XR for automated construction progress
monitoring. Structural equation modeling (SEM) method has been used to test the proposed hypotheses and
develop the skill model to examine the reliability, validity and contribution of the framework to understand the
DRX model’s effectiveness if implemented in real practice. p
p
Findings – The research findings validate the positive impact and importance of utilizing technology
integration in a logical framework such as DRX, which provides trustable, real-time, transparent and digital
construction progress monitoring. Practical implications – DRX system captures accurate, real-time and comprehensive data at construction
stage, analyses data and information precisely and quickly, visualizes information and reports in a real scale
environment, facilitates information flows and communication, learns from itself, historical data and accessible
online data to predict future actions, provides semantic and digitalize construction information with analytical
capabilities and optimizes decision-making process. p
p
g p
Originality/value – The research presents a framework of an automated construction progress monitoring
system that integrates BIM, various reality capturing technologies, DT and XR technologies (VR, AR and MR),
arraying the steps on how these technologies work collaboratively to create, capture, generate, analyze, manage
and visualize construction progress data, information and reports. Keywords Automated construction progress monitoring, Building information modeling, Reality capture,
Digital twins, Extended reality Paper type Research paper Smart and Sustainable Built
Environment
Vol. 12 No. 1, 2023
pp. 200-236
Emerald Publishing Limited
2046-6099
DOI 10.1108/SASBE-01-2021-0016 200 Received 31 January 2021
Revised 22 March 2021
6 April 2021
Accepted 6 April 2021 Ibrahim Yitmen
Construction Engineering and Lighting Science, J€onk€oping University,
J€onk€oping, Sweden © Sepehr Alizadehsalehi and Ibrahim Yitmen. 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 Digital twin-based progress
monitoring management model
through reality capture to extended
reality technologies (DRX)
Sepehr Alizadehsalehi
Project Management Program, Department of Civil and Environmental Engineering,
Northwestern University, Evanston, Illinois, USA, and SASBE
12,1 200 1. Introduction Based on Navonand Sacks (Navonand Sacks, 2007),the mostcost-effective andefficientway to
monitor construction projects’ progress and measure their performance is to automate the
process. So far, researchershave studied several emerging field data acquisition technologies to
automate project inspections either by adopting a single technology or putting a combination
of different technologies together (Alizadehsalehi and Yitmen, 2016; Asadi et al., 2021;
Rasoolinejad et al., 2020). Some of these technologies/tools are laser scanners, image-based © Sepehr Alizadehsalehi and Ibrahim Yitmen. 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 systems, radio frequency identification (RFID), wireless sensors (WSs), Global Positioning
Systems (GPS), unmanned aerial vehicles (UAVs) and ultra-wideband (UWB). Among these
technologies, numerous scholars adopted a 3D laser scan and nominated it as the most
reliable available technology to capture 3D data for construction projects due to its speed and
high precision (Perez-Perez et al., 2021). 3D laser scanning, n-D BIM and object recognition
framework (Scan-to-BIM) together offer a visually and detailed comprehensive assessment of
the as-built state of construction projects within the project performance improvement
context (Bosche et al., 2015). To develop a
generic
framework of a
DT 201 Notwithstanding the significant impact of laser scanning data in the automation of
construction progress monitoring, their applications are not rich, as they are limited to
providing data solely by surface recognition. 3D point cloud data can only capture and
measure the elements’ surface, and they have lacks of reflecting the project’s quality of built
elements (Golparvar-Fard et al., 2019). Various researchers were adopting embedded sensing
to assess conditional data of parts and elements of construction projects (Alizadehsalehi and
Yitmen, 2016). Advancements in the generation of 3D geometric as-built data with laser
scanner technologies and collecting various types of quality data regarding the project
environment by using embedded advanced wireless sensors is an opportunity to explore
projects comprehensively. Combining project as-planned models and as-built data from
reality data capturing technologies to produce rich project data models can improve
performance control and project management. 1. Introduction Through this integration, decision-makers can
find the chance to get access to data from laser scanners and installed sensors (as-built),
besides the planned design, schedule and other required data in BIM space (as-planned). On the other hand, gathering, saving, analyzing and managing these huge and complex data
to get the simple, accurate, fast and real-time progress monitoring report needs a smart/
intelligent system that can continuously learn and updates itself from multiple sources such as
historical archive data (Boje et al., 2020). The IoT technologies and related systems act to collect
real-timethroughouttheedgecomputingdevicesandthesmartgateway.IoTenablesaccessand
connection tointelligence dataand isinterlinked with digital twin(DT)anddigital models, which
virtually represent their physical counterparts. As an umbrella term, cognitive computing
consists of technologies, including big data, artificial intelligence (AI), machine learning (ML),
deep learning (DL) and cognitive algorithms that tryto simulate human thought processes in the
computer model throughDT technology. DT consists of physical anddigital products, including
connections between the two products (data that streams between them) (Dawood et al., 2020). Meanwhile, all data that created and captured digital data need to visualize on a real scale for all
stakeholders in different stages of projects. Extended reality (XR) technologies can pull
stakeholders in multidimensional directions with endless opportunities to create innovative and
unique experiences of project status at any time (Alizadehsalehi et al., 2020). q
p
p j
y
(
,
)
In order to overcome current progress monitoring limitations, there is a need for a holistic
framework for digital automated n-dimensional surface and quality progress monitoring
based on available technologies. The main contribution to the knowledge domain is the
insight into a DRX solution’s functionality and its ability to visualize the comprehensive data
included in a BIM system and generates real-time interactive project visualization that helps
create a common understanding among significant stakeholders. Furthermore, the
synthesization of industry professionals’ questionnaire insights and comments further
enables analysis of integrability enablers and professionals’ perception of the technological
ecosystem’s usability. Knowledge of the interoperability potential between reality capturing
data, BIM, DT, IoT and XR technologies could also contribute to the development of progress
monitoring management practice. The result is improved communication regarding design,
construction and operation and maintenance processes. Figure 1.
Research framework 2.1 BIM-based construction progress monitoring 2.1 BIM-based construction progress monitoring
According to the Project Management Body of Knowledge (PMBOK), controlling and
monitoring a construction project consists of those processes required to track, review and
orchestrate the progress and performance of a project, identify any areas in which changes to
the plan are required and initiate the corresponding changes (Guide, 2001). These processes
involve the measurement of the progress through inspections (as-built) and the comparison
with the project plan (as-planned) in order to validate the predicted performance. The overall
purpose of monitoring is to ensure effectively managed results and outputs by measuring
and assessing project performance (Lin and Golparvar-Fard, 2020). Measurement of work in
progress on construction sites is vital for construction project management, and as this
process directly affected different performances of a project such as time, cost, quality and
safety of the project. It is considered one of the most challenging tasks due to the complexity
and interdependency of activities (Arif and Khan, 2021). Traditional progress tracking practice depends on visual inspections and daily or weekly
reports created based on those inspections to ensure that work meets contract specifications
and schedule (Golparvar-Fard et al., 2009). This traditional practice relies heavily on the
inspectors’ personal judgment, observational skills and expert weekly follow-up with a high
probability of incomplete and inaccurate reports. However, if a project is poorly designed or
based on faulty assumptions, even the best monitoring is unlikely to ensure its success. So,
the AEC industry-accepted BIM, as a fast and accurate project progress tracking system,
provides a suitable basis for automated construction progress monitoring. SASBE
12,1 1. Introduction The proposed study’s main objective
is to develop a framework of a DT-based reality capture to the extended reality (DRX) project
progress management model through process modeling and IDEF0 language to provide a project central system, understand/validate the importance, strengths and challenges of the
proposed system. This article also provides a roadmap for future efforts toward
implementing a DRX-based management system in all construction industry stages in the
automation of construction progress monitoring. p
g
g
Figure 1 shows the structure of the article. The theoretical background about BIM-based
construction progress monitoring, reality capturing technologies, DT and XR is provided. Then, the proposed DRX model for construction progress management using process
modeling and IDEF0 modeling approach is presented. In the third stage, the research
hypotheses and a quantitative method involving data collection and measures are explained
using structural equation modeling (SEM) method, hypotheses are tested and the reliability of
the factors is validated. Finally, the theoretical contributions and managerial implications are
discussed, the proposed model’s strengths and challenges and limitations presented and
future research opportunities are suggested. 202 2. Theoretical background
This section outlines the key related works in four main areas relevant to this paper:
(1) BIM-based construction progress monitoring; (2) reality capturing technologies (RC) in
construction; (3) digital twins (DTs) and (4) extended reality (XR) in construction industry. 2.1 BIM-based construction progress monitoring 2.1 BIM-based construction progress monitoring Background
Strengths and weaknesses of DRX based on:
- Literature on BIM-based progress monitoring
- Previous academic publications
- Authors’ experiences
- Conclusion and discussion
- Future roadmap/work
- Construction management specialist
- Literature on Reality Capturing technologies
- Literature on Digital Twin (DT)
- Literature on Extended Reality technologies
- Virtual Reality (VR)
- Create hypotheses
- Create DRX process modeling framework
- Create DRX IDEF0 model
- Structural Equation Modeling (SEM)
Stage 1:
Problem
identification
Stage 2:
Methodology
Stage 3:
Evaluation of
model
Stage 4:
Data analysis
Stage 5:
Disseminate
- Survey of construction professionals
Evaluation of the proposed DRX model
- Mixed Reality (MR)
- Augmented Reality (AR) Background
- Literature on BIM-based progress monitoring
- Literature on Reality Capturing technologies
- Literature on Digital Twin (DT)
- Literature on Extended Reality technologies
- Virtual Reality (VR)
- Mixed Reality (MR)
- Augmented Reality (AR) Stage 5:
Disseminate BIM is a comprehensive digital representation of a building comprising the 3D geometry of
all its components and a semantic description of the component types and their relationships
(Kim et al., 2020b). BIM is critically essential for progress monitoring in three different aspects:
producing as-planned data, providing as-built data and comparing these two together
(Machado and Vilela, 2020). As-planned data are construction progress-monitoring baseline. Nowadays, BIM can bind information associated with AEC contracts of a project as as-planned
data repositories facilitate accessing geometrical data, visualizing designed schedule and
information management about the progress. This is why BIM is accepted as a platform
providing a rich and valuable data source for executing automated project progress monitoring
(Kim et al., 2020a). A well-designed BIM model in the construction phase can analyze
construction operations by enabling project managers to determine site management
strategies, help to better communication and collaboration between project team members,
facilitate contractor coordination and plan access routing and site logistics (Hamledari et al.,
2021; Kopsida and Brilakis, 2020). BIM is considered a perfect launching platform for
construction projects due to the capability for modeling existing building conditions. To develop a
generic
framework of a
DT 203 2.2 Reality capturing technologies for construction progress monitoring
To generate as-built data, the construction field data capturing process requires various
types of data (as required) from a construction project site (Braun et al., 2020). Data
acquisition technologies are currently being widely used to capture the as-built information of
exiting and ongoing construction projects. 2.1 BIM-based construction progress monitoring These data can then be transmitted to the central SASBE
12,1 receiver through a wireless medium. As shown in Table 1, so far, considerable research and
works have carried out different automated data capturing technologies, single or in
integration with other data acquisition technologies, with or without BIM, toward data
gathering. receiver through a wireless medium. As shown in Table 1, so far, considerable research and
works have carried out different automated data capturing technologies, single or in
integration with other data acquisition technologies, with or without BIM, toward data
gathering. 2.3 Digital twin in the built environment
A DT is a digital replica of a living or nonliving physical entity, and it refers to a digital replica
of potential and actual physical assets (physical twin), devices, systems, processes, places and
people that can be used for different aims (Boje et al., 2020). This virtual twin exists only as
software rendered by computing power (El Saddik, 2018). The DT concept started monitoring
high-value, safety-critical pieces of machinery such as aircraft engines in flight and industrial
processes. Generally, this system has three primary levels of the digital master, the digital
shadow and the DT. The digital master is created parallel to product development and is
based on the results of development. This level includes the virtual prototypes with their
simulation and behavior models and the product’s variants and configurations. With the
beginning of an individual’s life real instance of the product, the digital shadow arises. These
real objects cast a digital shadow into the virtual world. Simply data such as identifiers and
more complex data such as condition data, production data, maintenance data or usage data
can be viewed. Depending on the DT’s purpose, it is described which data must be taken into
account to enable the intended functions. With the combination of the digital master
information and digital shadow data, the result is the DT. Simulations and calculations can
now be carried out based on the DT. Besides, automated service offerings based on the DT
can also be generated to link products and services. 2.1 BIM-based construction progress monitoring In the context of comparing as-planned data and
as-built data, clash detection and visualizing discrepancies, BIM provides a very suitable,
powerful and accurate baseline. In addition, BIM helps the communication and coordination
of design and construction processes, real-time cost estimation and the automated creation of
bills of quantity (Almukhtar et al., 2021; Elghaish et al., 2020). So far, researchers have introduced numerous approaches and methods to create as-built
BIM models for progress monitoring of construction projects (Nahangi and Haas, 2015). Most
of them support a specific purpose of project control, such as physical status or the intended
part’s environmental condition. A laser scanner, UAV and digital camera are the most
common reality capture technologies used to generate images, videos or 3D point cloud data
(Rausch and Haas, 2021). Many research types considered laser-scanning systems a
promising technology for as-built data acquisition due to their high precision (Czerniawski
et al., 2021; Pica and Abanda, 2021). The laser scanning surveying technique’s significant
benefit is that it facilitates accurate, rapid and detailed 3D data of objects for use in numerous
applications (Aryan et al., 2021). These models make it possible to access much of the
necessary geometric and visual data. Airborne 3D scanners (UAV-Scanners) are unique
because of their ability of the scanner to detach from the drone. Flexibility and various UAV-
based scanners enable project stakeholders to utilize one scanner in the air and another on
land. These laser technologies users can scan different scenarios, from large areas such as
cities and railways, areas with no light such as tunnels and hard-to-document regions. y
g
g
Wireless sensors (WSs) are low-cost, reliable and small devices able to perform a sensing
task to monitor physical or environmental conditions. Typically, each WS node has the
following different principal parts of (1) a radio transceiver, (2) microcontroller, (3) an
electronic circuit for interfacing with the sensors and (4) an energy source such as a battery or
any type of embedded form of energy harvesting. Sensor nodes differ in size and cost, based
on the required data complexity. These specifications can affect the necessary resources, such
as memory, computational speed, energy and communications bandwidth. WSs are equipped
with one or more sensors, and they can collect data like temperature, humidity, sound,
pressure, tension, lighting, gas level, vehicular movement monitoring, critical events
detection, etc. (Rashid and Rehmani, 2016). 2.1 BIM-based construction progress monitoring DTs have various features such as real-
time (gather and present real-time data of physical assets), analytics (store data, run
continuous analytics from historical data and provide useful insight), simulations (utilize to
run various data-driven simulations), visualization (overlay real-life and live 3D BIM models,
images and videos of the physical asset and also foundation for immersive visualizations),
automation (a bidirectional system that can manage the behavior of physical assets) and
predictions (provide predictions of assets’ future behaviors using historical data and
analytics of various scenarios assets). 204 y
)
The concept of DT is gaining currency in the construction industry (Al-Sehrawy and
Kumar, 2020). DT is an up-to-date and dynamic model of a physical asset or facility. It includes all the structured and unstructured information about projects that can be shared
among team members. DTs help the AEC industry model, simulate, understand, predict and
optimize all aspects of a physical asset or facility. Without DTs, most optimization is reactive
based on after the fact alarms rather than predictive alerts. The design phase can create a
solution virtually and accurately render it operational before a single physical action is taken
(Deng et al., 2021). In this stage, a DT is used to create the optimum solution. It results from
detailed simulations and rich data that specify the best architecture, configuration, materials
and cost (Kan and Anumba, 2019). Then we can simulate that solution under different types
of real scenarios. Based on the data provided, the design can then be modified. A DT can be
used in the build phase to provide the construction specifications or parametric estimates to
different providers (Shirowzhan et al., 2020). In this way, a DT can be an asset in streamlining
the procurement process. Besides, and importantly, sensors are applied to the physical object
to collect and transmit data back to its virtual replica during the build. At this point, with
enough sensors, the virtual twin is providing all relevant data about the state of the physical
twin. For example, an MEP section or structural element of a building can accurately render
its temperature, vibration, strength and so much more in its DT. All of this becomes possible
because of increasingly better digital technologies that include faster computers, better
telemetry and communication of measurements from a collection point to receiving
equipment, smaller, more accurate sensors, data management, and AI (Sacks et al., 2020). Table 1.
Advanced reality
capturing technologies
for construction
progress monitoring 2.1 BIM-based construction progress monitoring BIM-based construction progress monitoring
No
References
Integrated
with BIM
Field data capturing technologies
Image-
based
Laser
scanning
(LS)
Radio frequency
identification (RFID)
Ultra-
wideband
(UWB)
Global
Positioning
System (GPS)
Wireless
sensor (WS)
Unmanned aerial
vehicle (UAV)
1
Guven and Ergen
(2021)
X
*
*
2
Hamledari et al. (2021)
X
*
*
3
Puri and Turkan
(2020)
X
*
4
Lin et al. (2019a)
X
*
5
Han and
Golparvar-Fard
(2017)
X
*
*
*
6
Tuttas et al. (2016)
X
*
*
7
Behnam et al. (2016)
X
*
*
8
Irizarry and Costa
(2016)
X
*
*
9
Bosche et al. (2015)
X
*
10
Teizer (2015)
X
*
*
*
11
Lin et al. (2015)
X
*
*
12
(Son et al., 2015)
X
*
*
13
Shahi et al. (2014)
X
*
*
14
Tuttas et al. (2014)
X
*
15
Dimitrov and
Golparvar-Fard
(2014)
X
*
16
Han and
Golparvar-Fard
(2014b)
X
*
(continued)
Table 1. Advanced reality
capturing technologies
for construction
progress monitoring
To develop a
generic
framework of a
DT
205 BIM-based construction progress monitoring
No
References
Integrated
with BIM
Field data capturing technologies
Image-
based
Laser
scanning
(LS)
Radio frequency
identification (RFID)
Ultra-
wideband
(UWB)
Global
Positioning
System (GPS)
Wireless
sensor (WS)
Unmanned aerial
vehicle (UAV) 205 BIM-based construction progress monitoring
No
References
Integrated
with BIM
Field data capturing technologies
Image-
based
Laser
scanning
(LS)
Radio frequency
identification (RFID)
Ultra-
wideband
(UWB)
Global
Positioning
System (GPS)
Wireless
sensor (WS)
Unmanned aerial
vehicle (UAV)
17
Han and
Golparvar-Fard
(2014a)
X
*
18
Bosche et al. (2013)
X
*
19
Zhang and Arditi
(2013)
X
*
20
Turkan et al. (2013)
X
*
21
Turkan et al. (2012)
X
*
22
Shahi et al. (2012)
X
*
Table 1. SASBE
12,1
206 BIM-based construction progress monitoring
No
References
Integrated
with BIM
Field data capturing technologies
Image-
based
Laser
scanning
(LS)
Radio frequency
identification (RFID)
Ultra-
wideband
(UWB)
Global
Positioning
System (GPS)
Wireless
sensor (WS)
Unmanned aerial
vehicle (UAV)
17
Han and
Golparvar-Fard
(2014a)
X
*
18
Bosche et al. (2013)
X
*
19
Zhang and Arditi
(2013)
X
*
20
Turkan et al. (2013)
X
*
21
Turkan et al. (2012)
X
*
22
Shahi et al. (2012)
X
*
Table 1. 2.1 BIM-based construction progress monitoring SASBE
12,1
206 206 An abundance of data is being collected and fed back to its DT over a digital thread during
operations. Backed by AI, the DT can identify and even predict maintenance issues before
they happen (Boje et al., 2020). It has become a data-informed model of a physical system. This
compelling feature reduces cost since it is typically cheaper to conduct maintenance than
repair it after facing issues proactively. Finally, this continuous real-time feed of data can help
with optimization (Angjeliu et al., 2020). That is, improve its performance by enabling the
system to automatically modify its behavior or prompt a human’s manual intervention. DTs
have become particularly ubiquitous in the IoT world. These Internet-connected electronics
collect and produce data and services and interact and communicate with each other and
central systems (Greif et al., 2020). The data collected from these devices create detailed
knowledge, enabling capabilities. Diverse applications of DT, based on the latest research,
have been shown in Table 2. To develop a
generic
framework of a
DT 207 As stated, the DT concept offers numerous magnificent opportunities. However, various
challenges need to be adequately addressed to achieve their full advantages. Providing real-
time connectivity to assets that might constantly be moving or in remote areas is challenging. Besides, it required constant connectivity with significant bandwidth and computation
power to gain value that is not a trivial issue. A DT requires accurate and small different types of sensors. Managing and maintaining
these sensors in different conditions is a challenging and complex process. All used
technologies and platforms of DTs, in most cases, need to have long life cycles, and they need
proprietary design, simulation or analytics software. This means that a cautious
determination of the technologies to be used becomes a real fundamental success factor in
the long run. In addition, DT’s implementation needs a set of skills for which a set of personnel
must be trained appropriately. DT gathers data and intellectual capital, which becomes more
valuable over time; it is vital to ensure data management systems’ security to avoid data loss
or other possible damage. Finally, like other technologies, it needs firms and their
stakeholders’ culture changes to accept this massive digital transformation. No
References
Applications
1
Deng et al. 2.1 BIM-based construction progress monitoring (2021)
Research on evolution of BIM to DTs in built environment applications
2
Pan and Zhang (2021)
build a data-driven DT framework based on BIM, IoT and data mining for
advanced project management
3
Bosch-Sijtsema et al. (2021)
Applications of digital technologies in AEC industry
4
Hasan et al. (2021)
Investigating construction machinery operation and work tracking through
AR and DT
5
Camposano et al. (2021)
Exploration on how AEC/FM practitioners describe DTs of built assets
6
Hou et al. (2021)
Reviews the applications and challenges of DTs in construction safety
7
Boje et al. (2020)
Reviews the multi-faceted applications and limitations of BIM and need for
construction DT
8
Liu et al. (2020)
Building indoor safety management
9
Rausch et al. (2020)
Use of computational algorithm to support DTs in construction
10
Dawood et al. (2020)
Review, develop and implement DT, VR, AR and BIM in AECO
11
G€otz et al. (2020)
Asset life cycle management
12
Alonso et al. (2019)
SPHERE platform for improving the buildings energy performance
13
Mathot et al. (2019)
Developed and discussed the next generation parametric system Packhunt.io
with BIM, DT and mixed reality (XR) technologies
14
Khajavi et al. (2019)
Discussed DT for building life cycle management
15
Lu et al. (2019)
Proposed the DT-based smart asset management framework
16
Kaewunruen and Lian
(2019)
DT for sustainability-based lifecycle management for railway turnout
systems
Table 2. Diverse applications of
DT-based on the latest
research in AEC
industry 2.4 Visualization by extended reality (XR) 2.4 Visualization by extended reality (XR)
XR extends reality, and it means that it is still up for interpretation (Khairadeen Ali and Park,
2020). XR includes VR, AR, MR and similar reality-altering technologies that have been using
immersive realities. VR is the classic immersive experience where we put on glasses and
earphones and other devices to replace the real world with a virtual one in this completely
simulated or virtual environment. The VR environments shut out the real world and
everything, including the user (avatar), is virtual content. These models can be 360-degree
photos or 360-degree video or n-D models. This is captured environments or situations that
someone is standing in there and can experience that scenario or situation. VR tends to be
interactive experiences, so this is the idea of interacting with objects or props or the
environment itself. 2.1 BIM-based construction progress monitoring It is typically in an enclosed head-mounted display with surround audio
(Khalili, 2021). AR is literally reality augmented with computer-generated content typically
viewed through smartphones, tablets or glasses (Machado and Vilela, 2020). In AR, digital
content is on top of the user’s real world. The real world and whatever experience the AR
device adds besides can be seen (Garbett et al., 2021). This data can be 3D stereoscopic or 2D,
or a mix of the two. The idea of being in a real environment, but being able to receive
information or real data as to what is happening in the real situation as it is happening. MR is
very similar to AR, with higher interaction between virtual contents and the real world,
making the user experience more realistic (Alizadehsalehi et al., 2019a). Not only do we see the
computer-generated content mixed with reality but we can interact with that content, and
that content can interact with the real world. This is where the interactive heads-up displays
of science fiction fit. Users can feel virtual content with hands to scale what it is, and it feels
like a user touching that content as seen in reality. Table 3 summarizes the latest research that
used VR, AR and MR technologies, with or without BIM, for different AEC industry
applications. SBE
1
8 208 3. Methodology
Thi
i gy
This section presents a framework developed to improve automation in construction
progress monitoring management referred to as the DRX model. The framework’s processes
and sub-processes are explained in detail and presented in process modeling and IDEF0
models. A comprehensive evaluation of the framework’s DRX model is performed by
conducting a questionnaire survey to 326 respondents and assessing their responses in light
of similar experiences. All respondents were project managers, construction managers, VDC
coordinators, 4D planners and BIM managers. Finally, the strengths and challenges of DRX
drawn from the study are presented. 3.1 Overview of proposed DRX model for construction progress management
As demonstrated in Figure 2, this study aims to use reality capturing technologies with BIM
as a powerful approach, real-time connectivity to DTs to analyze and optimize data, and
visualize models on the XR environment on a real scale. Such a combination can be extended
to a new generation of construction management performance monitoring. This combination
includes using reality capturing technologies data, BIM, DTs and XR. Integration and
utilization of these technologies in each under-construction project enable the stakeholders to
collect real-time and precise actual data automatically, manage and optimize generated and
captured data, visualize them and get the most comprehensive progress control. The input
mechanisms or data sources in the DRX model are divided into two levels: creating an as-
planned model and acquiring an as-built model. In creating the as-planned stage, the project
design team, including the architect team, structural engineer team, the MEP team and the
administration team, must identify information that should be included in BIM models, which No
References
VR
AR
MR
Integated
with BIM
Applications
1
Dai et al. (2021)
*
X
Increasing safety communication on
construction sites
2
Hasan et al. (2021)
*
X
Investigating construction machinery
operation and work tracking through AR
and DT
3
Bosch-Sijtsema et al. (2021)
*
*
*
X
Applications of digital technologies in AEC
industry
4
Piroozfar et al. (2021)
*
X
Study on the application of AR technologies
for urban utility infrastructure
5
Alizadehsalehi et al. (2020)
*
*
*
X
Review of XR technologies in AEC industry
and presented an implementation workflow
within a NASA-Mars habitat case study
6
Wu et al. (2020)
*
*
*
X
Review and proposed various applications
of BIM-XR
7
Cheng et al. Table 3.
XR technologies in
AEC industry 3. Methodology
Thi
i These
created and captured data can be input in different exchange formats such as RVT, TXT,
DWG XYZ E57 etc
3D
4D
5D
6D
Digital Twin
Extended Reality (XR)
Mobile phone-Tablets-
Computers
ML
Historical data
AI
IoT
Big
Data
nD
Design
Scheduling
Specification/
required quality
Actual cost
Actual timeline
Actual design
Actual quality
Actual cost
Facility
Energy
... n-D
Facility
Energy
... n-D
As-Planned Model
As-Built Model
Virtual Space
Real Space
Cognitive
Computing
Cloud
Computing
Revision required
Revision required
Recapturing data required
Progress monitoring plan
LAV
Laser Scanner
Wireless Sensor
Network
INPUT
CREATE
ACQUIRE
Data Sources
Building Information Model
(BIM)
Building Information Model
(BIM)
Managing, Analytics, Simulation,
and Prediction Engine
Data access
PROCESS
OUTPUT
Figure 2. Overall approach
of DRX
SASBE
12,1
210 Digital Twin
ML
Historical data
AI
IoT
Big
Data
Cognitive
Computing
Cloud
Computing
Revision required
Progress monitoring plan
Managing, Analytics, Simulation,
and Prediction Engine 210 multiple layers can represent. During the pre-construction phase, BIM has the potential to
retain the connections between designs, models, scheduling, cost and other items activities so
that any design or resource change can be reflected in the estimate, schedule, etc. The
acquiring as-built model is capturing/collecting data, but various types of reality capturing
technologies such as laser scanner, digital cameras, UAVs and wireless sensors. These
created and captured data can be input in different exchange formats such as RVT, TXT,
DWG, XYZ, E57, etc. multiple layers can represent. During the pre-construction phase, BIM has the potential to
retain the connections between designs, models, scheduling, cost and other items activities so
that any design or resource change can be reflected in the estimate, schedule, etc. The
acquiring as-built model is capturing/collecting data, but various types of reality capturing
technologies such as laser scanner, digital cameras, UAVs and wireless sensors. These
created and captured data can be input in different exchange formats such as RVT, TXT,
DWG, XYZ, E57, etc. These various created and captured data (big data) integrate into a common data
environment (CDE) in a BIM-based DT engine to be managed, analyzed and simulate in the
process stage. In the process stage of DRX, DT, with the help of AI, IoT, machine learning and
cognitive computing based on the project execution plan, defined level of development (LOD),
created information and historical archive data, a progress monitoring plan was created to
show all critical places to monitor, exact timeline and required report. 3. Methodology
Thi
i (2020)
*
X
Applications in the AECO Industry
8
Wen and Gheisari
(2020)
*
X
Facilitate communication in the AEC
domain
9
Rahimian et al. (2020)
*
X
Monitoring of construction projects
10
Cheng et al. (2020)
*
X
Applications of MR in the AECO Industry
12
Alizadehsalehi et al. (2019a)
*
X
Integration of BIM, lean construction and
MR
13
Davidson et al. (2019)
*
X
To facilitate real-time creation of bill of
quantities during the design phase
14
Alizadehsalehi et al. (2019b)
*
X
Design and construction education
15
Potseluyko and
Rahimian (2019)
*
*
X
Improving business and technical
operations within timber frame self-build
housing sector
16
Du et al. (2018)
*
X
Collaborative decision-making
17
Shi et al. (2018)
*
Construction safety
18
Mo et al. (2018)
*
X
Construction safety training
19
Chalhoub and Ayer
(2018)
*
X
Prefabrication
20
Olorunfemi et al. (2018)
*
Construction safety communication
21
Chalhoub et al. (2018)
*
X
Site survey
22
Chu et al. (2018)
*
X
Evaluate the effectiveness of BIM and AR
23
Hou et al. (2017)
*
*
Operation, maintenance, productivity and
safety
24
Du et al. (2017)
*
X
Collaborative decision-making
25
Azhar (2017)
*
X
Construction safety
26
Klempous et al. (2017)
*
X
Construction safety
27
Paes et al. (2017)
*
Improving the understanding of
architectural 3D models
28
Haggard (2017)
*
X
Benefits and challenges for VR in
construction industry
Note(s): VR5Virtual Reality; AR 5 Augmented Reality; MR 5 Mixed Reality; R 5 Review; F 5 Framework;
S 5 Survey; CS 5 Case Study; I5 Interview
Table 3. XR technologies in
AEC industry
To develop a
generic
framework of a
DT
209 multiple layers can represent. During the pre-construction phase, BIM has the potential to
retain the connections between designs, models, scheduling, cost and other items activities so
that any design or resource change can be reflected in the estimate, schedule, etc. The
acquiring as-built model is capturing/collecting data, but various types of reality capturing
technologies such as laser scanner, digital cameras, UAVs and wireless sensors. 3. Methodology
Thi
i Various available APIs
can be used in this stage to customize the DT. The final stage of the DRX model is the output or data access. In this stage, XR
technologies help responsible teams visualize the model in a real scale environment before
construction started to see all possible progress monitoring scenarios to choose the best one
in terms of safety, quality, speed, price and ease. The results of a progress comparison study
between as-planned data and as-built data performances are ultimately visualized in the BIM
environment and create different real-time progress reports. These comparison data can be
seen on a real scale environment by XR devices or regular smartphones, computers or tablets. Figure 2.
Overall approach
of DRX 3.2 IDEF0 for DRX construction progress management (A0) The development of a management system for any practical purpose should be based on
understanding the process details. IDEF0 is one of the methodologies that can describe
manufacturing functions and the one that can provide a mechanism for the communication of
complex concepts through the simple use of boxes and arrows (Waissi et al., 2015). IDEF0 is
an acronym for ICAM definition for function modeling, where ICAM stands for integrated computer-aided manufacturing. In this study, an IDEF0 data modeling method has been
designed to establish an integration of reality capturing technologies using BIM, DTs and XR
for automated construction progress monitoring. This model implemented in this research, as
shown in Figure 3, is composed of five major processes: generate a digital as-planned model
(node A01), generate a digital as-built model (node A02), data management process (node
A03), visualization (node A04) and analysis progress monitoring (node A05). computer-aided manufacturing. In this study, an IDEF0 data modeling method has been
designed to establish an integration of reality capturing technologies using BIM, DTs and XR
for automated construction progress monitoring. This model implemented in this research, as
shown in Figure 3, is composed of five major processes: generate a digital as-planned model
(node A01), generate a digital as-built model (node A02), data management process (node
A03), visualization (node A04) and analysis progress monitoring (node A05). To develop a
generic
framework of a
DT (1)
Creating digital as-planned model based on goals, budget, specifications, LOD and
standards, BIM knowledge and DT mechanisms (A01). 211 (2)
Generating digital as-built model with sensors, laser scanners and cameras (A02). There are as-planned and as-built data and data that need to be registered, combined
and managed. (3)
Data management which use DT, CC and software analytics (A03). (4)
Visualization stage that use XR technologies (A04). (5)
Analyzing progress monitoring to see the deviations between as-built and as-planned
models and optimizing our decision-making processes (A05). (5)
Analyzing progress monitoring to see the deviations between as-built and as-planned
models and optimizing our decision-making processes (A05). This model focuses on the design and built stages of a project called pre-construction and
construction stages. The top level of the IDEF0 model consists of one single box (A0), which
illustrates the whole DRX construction progress management process. 3.2 IDEF0 for DRX construction progress management (A0) The primary outputs
are a more comprehensive, precise and real-time progress visualization and reports ready for
use by the client, inspectors and other related stakeholders of projects to see the status of a
project and find easily understand the deviation between as-built and as-planned remotely
from out of job site or at the job site to make the best decisions. j
j
The DRX process is based on massive, cumulative, real-time, real-world BIM and field
measurements data. As highlighted in the DRX IDEF0 model, one of the primary stages is
defining the level of detail and complexity of a results representation that refers to LOD. This
LOD is defining all goals, requirements and objectives of the project. The LOD can be
changed as the model moves away from the viewer or other metrics such as object
importance, viewpoint-relative speed or situation. These processes are presented in Table 4 as a node index, a listing showing all nodes in an
IDEF0 model in the outline order. Each of the five processes and their sub-processes is
explained in the following sections. Figure 3.
IDEF-0 representation
of DRX Model 3.3 Digital as-planned BIM model (A01) On the second level of the DRX model (A01), a more comprehensive view of the progress
monitoring management process is described using four different activities as shown in
Figure 4. In the activity, digital as-planned model (A01), the process of generating a digital
as-planned BIM model is defined. This activity is composed of seven (7) major processes:
create a design model (A011); real-time visualization (A012); creating a schedule (A013);
creating cost estimation (A014); optimizing the created BIM model by DTs mechanisms
(A015); identifying monitoring goals (A016) and creating a plan for progress monitoring in
the construction stage (A017). (1)
Creating a design model and visualization is a kind of loop that helps designers draw,
visualize, check the design through XR technologies and get a high-quality design
(A011 and A012). (2)
After drawing a high-quality plan, add a timeline and cost of the project (A013 and
A014). 3.3 Digital as-planned BIM model (A01) SASBE
12,1 SASBE
12,1 212 Digital Twins-based
construction
management
Design
A0
Digital as-planned
model
A01
Digital as-built
model
A02
Data
management
A03
Visualization
A04
Analysis progress
monitoring
A05
Digital Twin-based Reality Capture-to-
Extended Reality (DRX) progress
monitoring management
Simulation
Construct
Start
Concept
BIM Knowledge
Digital Twin mechanism
2D (concept-detail) design
Pre-construction schedule
Standards/Regulations
Specifications
LOD
Budget
Goals
WBS
Optimization process
Design change required
Recapturing data required
Registered, federated
combined, and
coordinated models
Immersed models of
integration
Progress monitoring reports
Deviation between as-built
and as-planned data
Decision making
Construction managers/
Superintendents
Inspector
BIM coordinator/modeler
Safety managers
Specialist contractor
Project managers/
Designer/Architectural team
Client
IFC as-planned BIM model
progress monitoring plan
Physical/conditional
capturedas-built
BIM model
Operate
Maintenance
Renovation
Disposal
End
Note(s): Red Lines and arrows = Stakeholders; Blue lines and arrows = Information exchanges; Black lines, arrows, and boxes = Processes holders; Blue lines and arrows = Information exchanges; Black lines, arrows, and boxes = Processes y
g
Specialist contractor
Note(s): Red Lines and arrows = Stakeholders; Blue lines and arrows = Information exchanges; Black lines, arrows, and boxes = Processes ines and arrows = Information exchanges; Black lines, arrows, an p
Note(s): Red Lines and arrows = Stakeholders; Blue lines and arrows = Information exchanges Note(s): Red Lines and arrows = Stakeholder Diagram
reference
Description and activities included
A0 Digital Twin-based Reality Capture to Extended Reality progress monitoring management (DRX)
A01 Digital As-planned Model
A011 Create a design model
A012 Visualization
A013 Create Schedule
A014 Create cost estimation
A015 Optimize the model (DT)
A016 Identify monitoring goals
A017 Plan for progress monitoring in the construction stage
A0171 Define monitoring scopes
A0172 Define monitoring timelines
A0173 Define monitoring locations
A02 Digital As-built Model
A021 Create/update the reality capture to BIM execution plan (RCBEP)
A022 Pre-capturing process
A023 Data capturing
A0231 Photo tour capturing
A0232 3D point cloud capturing
A0233 Capturing sensors data
A024 Registration and modeling
A0241 Mini map creation
A0242 Scans registration
A0243 Modeling
A025 Quality assurance
A0251 QA photo tour
A0252 QA captured 3D point cloud vs 3D as-built model
A0253 QA model vs client’s requirements
A0254 QA through XR
A03 Data Management
A031 Create common data environment (CDE)
A032 Alignment of as-planned and as-built models
A033 Data analysis
A04 Visualization
A041 Create XR model
A042 Import XR model to XR device
A043 Immerse in XR environment
A05 Analysis Progress Monitoring
Table 4. 3.3 Digital as-planned BIM model (A01) Node index for DRX
framework
To develop a
generic
framework of a
DT
213 (4)
At this stage, an optimized plan is ready to identify the monitoring goals and then
create the progress monitoring plan. It is known what is critical for the project, so the
responsible teams define a progress monitoring plan based on scopes, timeline, and
locations. Then, the required sensors are put, drones are sent, scanners or cameras are
used to capture progress monitoring data (A016 and A017). (4)
At this stage, an optimized plan is ready to identify the monitoring goals and then
create the progress monitoring plan. It is known what is critical for the project, so the
responsible teams define a progress monitoring plan based on scopes, timeline, and
locations. Then, the required sensors are put, drones are sent, scanners or cameras are
used to capture progress monitoring data (A016 and A017). The main outputs are generated final and optimized digital as-planned model and a detailed
progress monitoring plant to use at the construction stage by various stakeholders such as
inspectors, project managers and superintendents. In this framework, stakeholders can
visualize the model on a real scale and identify their future progress monitoring goals based
on the project’s critical points. Defining the pre-construction stage’s monitoring goals helps
architects, the BIM team and other responsible stakeholders create a comprehensive and
accurate progress-monitoring plan from the pre-construction stage. The DRX model needs to
identify and know the progress monitoring objectives. 3.4 Create progress as-built model (A02) 3.4 Create progress as-built model (A02)
The second phase (A02), as seen in Figure 5, is the generating of the as-built model, and the
activities in this section are: create/update the RCBEP document (A021); pre-capturing
process (A022); data capturing (A022); registration and modeling (A024); and quality
assurance (A025). 3.4 Create progress as built model (A02)
The second phase (A02), as seen in Figure 5, is the generating of the as-built model, and the
activities in this section are: create/update the RCBEP document (A021); pre-capturing
process (A022); data capturing (A022); registration and modeling (A024); and quality
assurance (A025). 215 (1)
A Reality Capture to BIM Execution plan (RCBEP) is created based on as-planned
model and progress monitoring plan from the previous section and information from
job site (A021). (1)
A Reality Capture to BIM Execution plan (RCBEP) is created based on as-planned
model and progress monitoring plan from the previous section and information from
job site (A021). (2)
The pre-capturing process is needed to go through RCBEP to conduct review project
objectives and create a capturing plan, including all logistics. (A022). (2)
The pre-capturing process is needed to go through RCBEP to conduct review project
objectives and create a capturing plan, including all logistics. (A022). (3)
Data capturing from the job site contains capturing photo 360s, point clouds and
gathering sensors data (A023). (3)
Data capturing from the job site contains capturing photo 360s, point clouds and
gathering sensors data (A023). (4)
Registration, combining and generating a single file included all data together and, if
required, convert to a 3D BIM model (A024). (4)
Registration, combining and generating a single file included all data together and, if
required, convert to a 3D BIM model (A024). (5)
The generated file is needed to go through quality assurance to ensure that the
captured and registered file is correct (A025). (5)
The generated file is needed to go through quality assurance to ensure that the
captured and registered file is correct (A025). 3.5 Data management (A03) As shown in Figure 6, the activities included in the data management stage (A03) are Create
C
D t E
i
t (CDE) (A031) Ali
t f A
l
d
d A b ilt (A032)
d As shown in Figure 6, the activities included in the data management stage (A03) are Create
Common Data Environment (CDE) (A031); Alignment of As-planned and As-built (A032) and
Data analysis (A033). In the next step, by different software and application and also
available programming interfaces (APIs), the as-built and as-planned data can be aligned and
coordinated in a single scale, format, and a shared CDE (A031) and (A032). The system then
uses AI, ML and cognitive computing to choose the correct information to manage and
analyze this massive amount of information and data (A033). An intelligent construction
progress monitoring system can provide any amount of data or information in real-time to the
project stakeholders. With DT, all stakeholders are able to harness, analyze, interpret and
give them unique, actionable insights for those that manage, operate and experience
buildings and infrastructure projects. These sections optimize created and generated data to
enhance the project performance by having all these data and knowledge connected. As the broad majority of target systems have multiple variables and multiple data
streams, the DT requires cognitive computing, AI and ML. Therefore, the system at this stage
is able to do various cloud analyses, such as clash detection. The system is also able to
analyze the available historical data from previous projects to help stakeholders make quick,
accurate decisions. In the end, data can export in various formats required to use by the
stakeholders, which have been mentioned in the RCBEP document mentioned in section
(A021). DT technology is one of the essential Industry 4.0 technologies currently started and
improving every day. 3.3 Digital as-planned BIM model (A01) For instance, what parts, elements and
attribution of components need to be monitored, what accuracy and level of details are Digital as-planned
model
A01
Create a design
model
A011
Visualization
A012
Create schedule
A013
Create cost
estimation
A014
Optimize the
model (DT)
A015
Identify
monitoring goals
A016
Plan for progress monitoring
in the construction stage
A017
Define monitoring
scopes
A0171
Define
monitoring
timelines
A0172
Define
monitoring
locations
A0173
IFC as-planned BIM model
Progress monitoring plan
Real-time Corrections
by designer
Required design
corrections by designer
Required design
corrections by designer
Required design
corrections by designer
2D (concept-detail) design
Pre-construction schedule
WBS
5D BIM model
4D BIM model
3D BIM model
Estimation software
or original authoring
software
Scheduling software
or original authoring
software
Related real-time XR
software or plugin
Historical data (Big data)
Artificial intelligence (AI)
Cognitive learning
Machine learning (ML)
Inspector
BIM coordinator/modeler
Safety managers
Specialist contractor
Project managers/Construction
managers/Superintendents
Designer/Architectural team
Client
Note(s): Red Lines and arrows = Stakeholders; Blue lines and arrows = Information exchanges; Black lines, arrows, and boxes = Processes
Figure 4. IDEF-0 representation
of DRX Model
(Generating As-
planned model)
SASBE
12,1
214 SASBE
12,1 Note(s): Red Lines and arrows = Stakeholders; Blue lines and arrows = Information exchanges; Black lines, arrows, and boxes = Processes 214 Note(s): Red Lines and arrows = Stakeholders; Blue lines and arrows = Information exchanges; Black lines, arrows, and boxes = Processes ines and arrows = Information exchanges; Black lines, arrows, and boxes = Processes required for each element and which view must be inspected. The determination of
monitoring objectives is the primary act toward the determination of proper monitoring
techniques and technologies. To develop a
generic
framework of a
DT required for each element and which view must be inspected. The determination of
monitoring objectives is the primary act toward the determination of proper monitoring
techniques and technologies. Figure 5.
IDEF-0 representation
of DRX Model
(Generating As-
built model) Figure 6.
Data management
process at the as-built
generation stage Figure 7.
Visualization of as-
planned and as-built
data to monitor data at
XR environments 3.6 Visualization (A04) Visualization involves as-planned and as-built data to monitor data at the real scale in various
VR, AR and MR environments. Various available hardware, software and plugin (APIs) help
stakeholders to visualize models. The BIM-to-XR development framework encompasses SASBE
12,1 SASBE
12,1 A02
Create/update the reality
capture-to-BIM execustion
plan (RCBEP)
A021
Pre-Capturing
Process
A022
Data Capturing
A023
Photo tour
capturing
A0231
3D point cloud
capturing
A0232
Capturing sensors
data
A0233
Mini Map creation
A0241
Scans
registration
A0242
Combining LS and
WSs data
A0242
Modeling
A0243
QA Photo tour
A0251
QA captured 3D
Point cloud vs 3D as-
build Model
A0252
QA Model vs client’s
requirements
A0253
QA XR
A0254
Registration and
Modeling
A024
Quality assurance
A025
Digital as-built model
RCBEP: As-planned model
with inspection goals
IFC as-planned BIM model
Information from site
Progress monitoring plan
Different type of
digital cameras
Different
type of LSs
Recapturing required
Embedded
sensors
Inspector
BIM coordinator/modeler
Safety managers
Specialist contractor
Project managers/Construction
managers/Superintendents
Raw actual as-built
physical and
conditional data
Registered, federated,
combined, and coordinated
physical and conditional data
Photo tour
Available documents
Available plans
Related cloud-based
platform
Note(s): Red Lines and arrows = Stakeholders; Blue lines and arrows = Information exchanges; Black lines, arrows, and boxes = Processes
Figure 5. IDEF-0 representation
of DRX Model
(Generating As-
built model)
SASBE
12,1
216 Note(s): Red Lines and arrows = Stakeholders; Blue lines and arrows = Information exchanges; Black lines, arrows, and boxes = Processes 216 To develop a
generic
framework of a
DT three main activities: create an XR model (A041); import an XR model to an XR device (A042)
and immerse users in an XR environment (A043). As shown in Figure 7, an XR model is
determined according to specifications, BIM knowledge and ideas from designers and
stakeholders (node A03). At this stage, the model is ready to use by different types of XR
devices and applications (node A04). The created XR model can then be used to analyze the 217 Create Common Data
Environment (CDE)
A031
Data management
A03
Alignment of As-
planned and As-built
A032
Data analysis
A033
Project managers, Construction
managers, superintendents
BIM coordinator/modeler
Specialist contractor
Navisworks
Historic archive data
Revit
Related software
Artificial intelligence
Machine learning
Cognitive computing
Note(s): Red Lines and arrows = Stakeholders; Blue lines and arrows = Information
exchanges; Black lines, arrows, and boxes = Processes
Figure 6. 3.6 Visualization (A04) Data management
process at the as-built
generation stage
217 Data management Data management
A03 Create Common Data
Environment (CDE)
A031 Alignment of As-
planned and As-built
A032 A031 A032 Data analysis Note(s): Red Lines and arrows = Stakeholders; Blue lines and arrows = Information
exchanges; Black lines, arrows, and boxes = Processes XR model
XR model
Related software
or plugin
Related devices,
sensors, and
software
XR applications
(layer)
Create XR model
A041
A042
A04
A043
Specialist contractor
Designer/
Architectural team
Client
BIM coordinator/modeler
Project managers/Construction
managers/superintendents
Inspector
Visualization
Import XR model
to XR device
Immerse in XR
environment
Note(s): Red Lines and arrows = Stakeholders; Blue lines and arrows = Information
exchanges; Black lines, arrows, and boxes = Processes
F
Visualizat
planned and
data to monito
XR envir Visualization Create XR model Create XR model
A041 A041 XR model Import XR model
to XR device A043
Immerse in XR
environment A043 Note(s): Red Lines and arrows = Stakeholders; Blue lines and arrows = Information
exchanges; Black lines, arrows, and boxes = Processes overall design and evaluate alternative design decisions to take corrective actions on design
and make better design decisions (node A05), as seen in Figure 7. overall design and evaluate alternative design decisions to take corrective actions on design
and make better design decisions (node A05), as seen in Figure 7. Figure 8.
Hypothesized
SEM model 4. Evaluation of the proposed model The developed system DRX integrates all the proposed technologies currently existing, but is
on their path of evolution. The real applications for each of these emerging technologies have
been described one by one. However, as technologies such as DT is not fully adapted to be
used specifically for the construction process in the AEC industry, as the last part of the
research, the reliability, validity and contribution of the proposed framework to understand
the effectiveness of the DRX model when implemented in real practice. 218 4.1 Research hypotheses
Figure 8 displays the correlations among the four major aspects in the DRX model, based on
the extensive literature review and the detailed IDEF0 diagram: AEC digital technologies, 4D
building information modeling (BIM), digital twins and automated project progress
monitoring. The subsequent four hypotheses aim to explain the relationship among the construct
measures and the other variables and the theoretical background to these relationships. To develop a
generic H1. Leveraging of AEC digital technologies facilitates generation and simulation of 4D
BIM model. generic
framework of a
DT H1. Leveraging of AEC digital technologies facilitates generation and simulation of 4D
BIM model. g
framework of a
DT H2. Utilizing 4D BIM simulations enhance automated project progress monitoring. H3. Facilitating 4D BIM models constitute the generation of digital twins. H4. The digital twins enrich the simulated data and empower automated project
progress monitoring performance. 219 219 H3. Facilitating 4D BIM models constitute the generation of digital twins. H3. Facilitating 4D BIM models constitute the generation of digital twins. H4. The digital twins enrich the simulated data and empower automated project
progress monitoring performance. 219 H4. The digital twins enrich the simulated data and empower automated project
progress monitoring performance. H4. The digital twins enrich the simulated data and empower automated project
progress monitoring performance. 4.2 Sampling and data collection Using a computerized self-administered questionnaire (CSAQ), questionnaires with the
designed IDEF0 model were sent to the project management professionals such as BIM
managers, 4D planners, VDC coordinators, construction managers and project managers in
the consulting and contracting firms. The sample covers only large firms, constructing a
typical project with a value greater than 5m USD, carrying out 10–25 projects per year or
having more than 100–200 employees, whereas an engineering consulting company is
designing and controlling a typical project with a value greater than 5m USD, carrying out
25–50 projects per year or having more than 25–50 employees are defined as large. A total of
500 individuals were contacted to participate in the research, and a total of 326 participated
and completed the survey (65% response rate from the contacted firms). This indicates that
the sampling procedure was effective and that the respondents perceived the research as
relevant and worthwhile. The respondents were asked to rate the extent of agreement with
each statement based on a “five-point Likert scale” of 1 (strongly disagree) to 5 (strongly
agree). Figure 9 shows the respondents’ percentages to the survey from in the USA, UAE,
Sweden, Denmark and Canada with different professions. Out of 326, 50 respondents were
project managers with the highest representation of 20.25% and contributing to the USA
survey, 26 respondents. Fifty respondents were construction managers having the lowest
representation of 15.34% and contributing to the survey from Denmark, which is six Figure 9. Distribution of
respondents according
to regions,
background, and
professions Figure 9. Distribution of
respondents according
to regions,
background, and
professions respondents. The hypotheses regarding the contingent and direct effects were tested utilizing
the collected data by the survey. respondents. The hypotheses regarding the contingent and direct effects were tested utilizing
the collected data by the survey. SASBE
12,1 4.3 Analysis of findings
LISREL 8.8 statistical software package has been used to test and examine the hypothesized
models, as shown in Figure 8. First, “Descriptive statistics and Pearson correlation
coefficients” were calculated. Then the SEM was analyzed, a statistical methodology that
takes a hypothesis-testing approach to analyze a structural theory’s bearing on some
phenomenon. SEM’s basic steps include “specifying a model based on theory, determining
how to measure constructs, collecting data, and analyzing data that include overall model fit
statistics and parameter estimates”. 4.2 Sampling and data collection The resulting goodness-of-fit
statistics revealed that comparative fit index (CFI) 5 0.933, GFI 5 0.918, AGFI 5 0.913,
NFI 5 0.926, NNFI 5 0.922 and root-mean-square error of approximation (RMSEA) 5 0.066. The reason behind using different packages is that if the vast majority of the indexes indicate
a good fit, then there is probably a good fit. The results of the GFI, AGFI, CFI, NFI and NNFI
Variables
Mean
SD
1
2
3
4
AEC digital technologies
4.83
0.84
1.000
4D building information modeling (BIM)
4.84
0.86
0.627**
1.000
Digital twins
4.85
0.88
0.683**
0.705***
1.000
Automated project progress monitoring
4.86
0.89
0.724***
0.748***
0.769***
1.000
Note(s): *ρ < 0.1, **ρ < 0.05, ***ρ < 0.01, n 5 326
Table 5. Descriptive statistics
and Pearson
Correlation analysis 4.3 1 Descriptive statistics
Table 5 demonstrates the means and standard deviations, as well as the inter-factor
correlation matrix, for the study variables with the aim of valuating the significance level of
the relationships. Pearson correlation analysis was used to validate the interrelationships
between the dependent and independent variables tested in this study. All of the constructs
are interrelated and focus on “AEC Digital Technologies”, “4D Building Information
Modeling (BIM)”, “Digital Twins (DT)” and “Automated project progress monitoring”. A ρ value can express the significance of a relationship. When the ρ value is < 0.05, the
relationship between the two sets of ratings is considered to be significant. The correlation
matrix examination points which there were notable and positive linear associations between
factors representing the variables “AEC Digital Technologies”, “4D Building Information
Modeling (BIM)”, “Digital Twins” and “Automated project progress monitoring”. g (
)
g
p
j
p
g
g
4.3.2 SEM analysis and goodness-of-fit test. SEM techniques were utilized to perform the
path analyses that helped test the relationships between the model constructs (hypotheses)
and the model’s goodness of fit (Kumar et al., 2020). The results showed that all standardized
loadings on the relative constructs were greater than 0.5 (ρ < 0.001). The overall fit of the
model was significant (χ2 5 169.23 with df 5 89; ρ < 0.01). The resulting goodness-of-fit
statistics revealed that comparative fit index (CFI) 5 0.933, GFI 5 0.918, AGFI 5 0.913,
NFI 5 0.926, NNFI 5 0.922 and root-mean-square error of approximation (RMSEA) 5 0.066. 4.2 Sampling and data collection The model analysis specifies how latent variables or
hypothetical constructs depend upon or are indicated by the observed variables. To measure
the validity of the constructed model and the fit and suitability of the assumed causal
relationships to the actual data, “Goodnessof- Fit Index (GFI)”, “Adjusted Goodness-of-Fit
Index (AGFI)”, “Comparative Fit Index (CFI)”, “Normed Fit index (NFI)”, “Non-Normed Fit
Index (NNFI)” and “Root-Mean-Square Error of Approximation (RMSEA)”, and the ratio of
“Chi-square” to the “Degree of Freedom” ( χ2/df ) were used. Goodness-of-fit indices evaluate
whether the assumed model fits the empirical data, so they are significant indicators of the
validity and reliability of the model. 220 Index (AGFI) , Comparative Fit Index (CFI) , Normed Fit index (NFI) , Non-Normed Fit
Index (NNFI)” and “Root-Mean-Square Error of Approximation (RMSEA)”, and the ratio of
“Chi-square” to the “Degree of Freedom” ( χ2/df ) were used. Goodness-of-fit indices evaluate
whether the assumed model fits the empirical data, so they are significant indicators of the
validity and reliability of the model. 4.3 1 Descriptive statistics
Table 5 demonstrates the means and standard deviations, as well as the inter-factor
correlation matrix, for the study variables with the aim of valuating the significance level of
the relationships. Pearson correlation analysis was used to validate the interrelationships
between the dependent and independent variables tested in this study. All of the constructs
are interrelated and focus on “AEC Digital Technologies”, “4D Building Information
Modeling (BIM)”, “Digital Twins (DT)” and “Automated project progress monitoring”. A ρ value can express the significance of a relationship. When the ρ value is < 0.05, the
relationship between the two sets of ratings is considered to be significant. The correlation
matrix examination points which there were notable and positive linear associations between
factors representing the variables “AEC Digital Technologies”, “4D Building Information
Modeling (BIM)”, “Digital Twins” and “Automated project progress monitoring”. 4.3.2 SEM analysis and goodness-of-fit test. SEM techniques were utilized to perform the
path analyses that helped test the relationships between the model constructs (hypotheses)
and the model’s goodness of fit (Kumar et al., 2020). The results showed that all standardized
loadings on the relative constructs were greater than 0.5 (ρ < 0.001). The overall fit of the
model was significant (χ2 5 169.23 with df 5 89; ρ < 0.01). 4.2 Sampling and data collection The reason behind using different packages is that if the vast majority of the indexes indicate
a good fit, then there is probably a good fit. The results of the GFI, AGFI, CFI, NFI and NNFI Variables
Mean
SD
1
2
3
4
AEC digital technologies
4.83
0.84
1.000
4D building information modeling (BIM)
4.84
0.86
0.627**
1.000
Digital twins
4.85
0.88
0.683**
0.705***
1.000
Automated project progress monitoring
4.86
0.89
0.724***
0.748***
0.769***
1.000
Note(s): *ρ < 0.1, **ρ < 0.05, ***ρ < 0.01, n 5 326
Table 5. Descriptive statistics
and Pearson
Correlation analysis exceeded the threshold value of 0.90, and the hypothesized model revealed a good fit. A ratio
of model fit statistics based on degrees of freedom below 3 indicates an adequate model fit
(χ2/df 5 1.893). The hypothesized model in Figure 8, thus, can be classified as closely fitting
the data. Table 6 lists the results of the goodness-of-fit measures for the hypothesized model. Overall, goodness-of-fit measures all indicate very favorable fitness judgments for the
hypothesized model. To develop a
generic
framework of a
DT 4.3.3 Hypothesis test. To test Hypotheses 1 through 4, the hypothesized model was tested
using “LISREL 8.8”, where the paths between the AEC digital technologies and 4D building
information modeling (BIM) (H1), 4D building information modeling (BIM) and automated
project progress monitoring (H2), 4D building information modeling (BIM) and digital twins
(H3), digital twins and automated project progress monitoring (H4) were estimated. The
hypotheses regarding the relationships were tested based on the “associated t statistics”. “T values exceeding” 1.65, 1.98 or 2.576 were considered significant at the 0.10, 0.05 and 0.01
levels, respectively. AEC digital technologies, 4D building information modeling (BIM) and
digital twin all significantly and positively influenced (ρ < 0.05) automated project progress
monitoring (with values of H1 5 0.82, t value 5 4.72, H2 5 0.83, t value 5 4.75, H3 5 0.84,
t value 5 4.78, and H4 5 0.85, t value 5 4.80, respectively). Thus, hypotheses 1 through 4 were
supported. Table 7 lists the results of the parameter estimates of the hypothesized model. Considering the standardized parameter estimates, the results show that four hypothesized
relationships were classified as significant and accepted. 221 p
g
p
“Digital Twins —Automated project progress monitoring” had the highest significance
with a path coefficient of 0.85, revealing that 4D BIM data administration modeling
procedures contribute to digital twin attainment. 4.2 Sampling and data collection “4D Building Information Modeling (BIM)
— Digital Twin” had the second highest significance with a path coefficient of 0.84, revealing
that 4D BIM data administration methods provide an incentive to enhance digital progress
monitoring. Table 8 lists the “standardized structural coefficients” of the variables, AEC Statistics
Fitness criteria
Values
Fitness judgment
χ2
ρ < 0.01
169.23 (ρ 5 0.000 < 0.01)
Yes
RMSEA
<0.08 (<0.05 is excellent, and <0.08 is good)
0.066
Yes (good)
GFI
>0.90
0.918
Yes
AGFI
>0.90
0.913
Yes
NFI
>0.90
0.926
Yes
NNFI
>0.90
0.922
Yes
CFI
>0.90
0.933
Yes
χ2/df
<2.00
1.893
Yes
Hypothesized model
Parameter
coefficient
t-value
Construct relationship
H1: AEC digital technologies →4D building information modeling (BIM)
0.82**
4.72
H2: 4D building information modeling (BIM) →automated project progress
monitoring
0.83**
4.75
H3: 4D building information modeling (BIM) →digital twins
0.84***
4.78
H4: Digital twins →automated project progress monitoring
0.85***
4.80
Note(s): Fit Indices: χ2 5 169.23, df 5 92, χ2/df 5 1.893 GFI 5 0.918, CFI 5 0.933, AGFI 5 0.913, NFI 5 0.926,
NNFI 5 0.922, RMSEA 5 0.066
**ρ < 0.05, and ***ρ < 0.01
Table 6. Overall goodness-of fit
measures for the
hypothesized model
Table 7. 4.2 Sampling and data collection The variables “Automation”,
“Self-learning”, “Diagnosis”, “Intelligent decision-making”, “Remote decision-making”,
“Clash detection”, “Data integration”, “Feedback driving” and “Collaboration performance”
with values of 0.87, 0.87, 0.87, 0.87, 0.86, 0.86, 0.86 and 0.86 have the highest factor loading,
respectively. Most significantly, the high path coefficient values in the structural model
validate the empirical modeling work. 223 Hypothesis 1: It posits that leveraging of AEC digital technologies facilitates the
generation and simulation of the 4D BIM model. As presented in Figure 8, the path
coefficients of 0.82, which describe the hypothesized model relationships, were statistically
positive and significant. H1 implies which various reality capture technologies such as laser
scans and sensors can improve the accuracy and timeliness of the data collected from sites to
generate and simulate the 4D BIM model. The process can be significantly improved by
utilizing and combining the captured data with BIM as an effective visualization, modeling
and management source. This combination yields a collection of accurate, complete and
reliable field data models from the architectural, structural and MEP part of any construction
project. Hypothesis 2: It posits that utilizing 4D BIM simulations enhance automated project
progress monitoring. As presented in Figure 8, the path coefficients of 0.83, which describe
the hypothesized model relationships, were statistically positive and significant. This
proposes that, when a high-level n-D as-planned model of the work to be tracked is available,
the previous data included in the model can be leveraged for automated progress monitoring. The level at which the developed 4D model gives access to accurate information at the correct
time and in the precise place is pivotal for decision-making and ensuring that a project is
delivered according to pre-defined parameters established at its onset. Provide a consistent
platform for representing as-built, as-planned and progress discrepancy data that facilitates
communication and reporting methods. It is used to visualize the architectural, structural and
MEP parts at any stage of project monitoring. This progress monitoring level leads to a
unified system for a more robust automated comparison of as-planned and as-built data. Hypothesis 3: It posits that the facilitating 4D BIM models constitute the generation of
digital twins. As displayed in Figure 8, the path coefficients of 0.84, which describe the
hypothesized model relationships, were statistically positive and significant. 4.2 Sampling and data collection Parameter estimates
for SEM Variables and references
Observed variables/
indicators
Parameter
Standardized
structural
coefficient
AEC digital technologies (Bosch-Sijtsema
et al., 2021; Dinis et al., 2020; Han and
Golparvar-Fard, 2017; Rahimian et al., 2020;
Salehi and Yitmen, 2018)
3D laser scanning (LS)
λ1
0.84***
Image-based modeling
λ2
0.81***
Wireless sensor (WS)
λ3
0.78**
Simulation and virtual
reality (VR)
λ4
0.83***
Mobile interfaces and
augmented reality (AR)
λ5
0.82***
Mixed reality (MR)
λ6
0.79**
4D building information modeling (BIM)
(Alizadehsalehi and Yitmen, 2019; Braun
et al., 2018, 2020; Golparvar-Fard et al., 2009,
2015; Kropp et al., 2018; Puri and Turkan,
2020; Tuttas et al., 2017)
Creating 3D geometric
object-based model data
γ1
0.81***
Creating 3D as-built data
γ2
0.82***
Identify parametric
features in 3D model
γ3
0.80***
Data sorting and
categorizing
γ4
0.79**
Creating sequencing and
schedule data
γ5
0.83***
Data connection
γ6
0.79**
4D visualization and
simulation
γ7
0.84***
Clash detection
γ8
0.85***
Digital twins (Aheleroff et al., 2020; Al-
Sehrawy & Kumar, 2020; Boje et al., 2020;
Bosch-Sijtsema et al., 2021; Dawood et al.,
2020; G€otz et al., 2020; Lin and Golparvar-
Fard, 2020; Lu et al., 2020; Meza et al., 2021;
Pan and Zhang, 2021; Sommer et al., 2019)
Data integration
β1
0.85***
Data management
β2
0.84***
Intelligent decision-
making
β3
0.86***
Optimization operation
β4
0.82***
Data analysis
β5
0.81***
Feedback driving
β6
0.85***
Digital mapping
β7
0.84***
Data fusion
β8
0.83***
Diagnosis
β9
0.86***
Computing
β10
0.83***
Self-learning
β11
0.86***
Prognosis
β12
0.82***
Redesign
β13
0.81***
Perception
β14
0.84***
Automated project progress monitoring
(Braun et al., 2020; Golparvar-Fard et al.,
2009; Lin and Golparvar-Fard, 2020; Puri
and Turkan, 2020; Rahimian et al., 2020)
Environmental condition
data
ω1
0.83***
Physical data collection
ω2
0.84***
Remote inspection
ω3
0.81***
Remote decision-making
ω4
0.85***
Data management
ω5
0.82***
Monitoring time
ω6
0.83***
Monitoring quality
ω7
0.82***
Monitoring cost
ω8
0.83***
Automation
ω9
0.86***
Practical
ω10
0.80**
Future potential
ω11
0.82***
Stakeholder satisfaction
ω12
0.84***
Communication quality
ω13
0.80**
Collaboration
performance
ω14
0.85***
Note(s): ***ρ < 0.01 222 Note(s): ***ρ < 0.01 To develop a
generic
framework of a
DT digital technologies, 4D building information modeling (BIM), digital twins and automated
project progress monitoring representing the magnitudes that reflect the relative importance
of the relationships with their identified important references. 5. Discussion and implications
5.1 Theoretical contributions 224 This research attempted to introduce a framework for enhancing the automation of progress
monitoring and control of construction projects’ types and sizes. Due to the capability of the
emerging technologies and methodologies for capturing and integrating data from the
construction site, this research developed and introduced a DRX management model. The developed system DRX integrates all the proposed technologies currently existing, but is
on their path of evolution. The proposed DRX model gathered these technologies from
different domains, which each can address and solve some part of identified construction
progress monitoring gaps of AEC industry. This research contributed to how these emerging
technologies work collaboratively together as a DRX system to create, capture, analyze,
manage and visualize construction progress data, information and reports. g
p
g
,
p
This research has made a contribution toward developing a DRX project progress
management model based on process modeling and IDEF0 language; developing a guideline
of using various available technologies within the framework of DRX and examining the
importance, strengths and challenges of DRX model. DRX is able to optimize data and
information for the decision-making process at the pre-construction stage; capture precise,
real-time, and comprehensive data at the construction stage; create a CDE for all created data
at the pre-construction stage and captured data at construction stage; analyze data and
information based on the requirements of any type and size of a construction project;
visualize information and reports in a real scale environment at any stages of a project;
facilitate data and information flows and communication; constant reality feedback to plan;
provide visual verification; learn from itself, historical data and accessible online data to
predict the reliability of plans and optimize decision-making process. Another significance of the current study would be the detailed outcome of all the
developed DRX model’s executive processes. This comprehensive model is entirely ready to
adapt in terms of integrability and operability to a BIM platform to be used by AEC
professionals. The findings at this research are consistent with the previous studies and author’s
assumptions regarding DRX model of framework. Supporting the automated assessment of
construction projects in progress utilizing extensive acquisitions of site information has the
potential to improve the productivity of construction project monitoring significantly (Dinis
et al., 2020; Puri and Turkan, 2020; Sanhudo et al., 2020). 4.2 Sampling and data collection This proposes
that advancing in 4D BIM integrated with digital twins, that is the underline and federate for
digital construction help to synchronize their changes and keep them up-to-date with the
changing reality of the assets as the project progress. DTs offer a key, analytic edge to BIM
systems. By amassing data from various sources and combining them with an n-D model,
construction team members get vital access insight into each component. With the aid of DT
abilities, BIM models are evolving to become “living,” automatically updated representations
of physical assets they represent. BIM creates a n-D virtual model of project that it contains
much of the information that a digital twin would require. Hypothesis 4: It posits that the digital twins enrich the simulated data and empowers the
performance of automated project progress monitoring. As shown in Figure 8, the path
coefficients of 0.85, which describe the hypothesized model relationships, were statistically
positive and significant. This proposes that the 4D BIM creating the perfect basis for a digital
twin platform. Real-time automated progress monitoring analysis based on actual operating
conditions can be undertaken by incorporating real-time data like laser scanners, digital
cameras and/or sensors. While 4D BIM provides procedures, technologies and data schemas
facilitating a standardized semantic representation of construction projects components/
systems, the DT conveys a more holistic process-oriented characterization of the complex
management and optimization process by leveraging data flows for automated construction SASBE
12,1 project progress monitoring. This system provides superior performance, enabling
automated and robust progress control and delivering accurate as-built BIM models to
project stakeholders. 5. Discussion and implications
5.1 Theoretical contributions Many of the engineering managers,
in discussions regarding this research, noted that field data capturing technologies like laser
scanner, digital camera and wireless sensors have high positive influences in the progress
monitoring of construction projects. They believe that advancement in automatic field data
acquisition systems will enable more accurate collection of data and knowledge about
processes and operations on site. Nevertheless, these procedures for taking site information
do not support the desired results or completeness for automated progress monitoring
(Alizadehsalehi and Yitmen, 2019). With the advancement in processing, sensing, computer
technologies and communication capabilities of reality capturing technologies, they have
become more affordable and modern remote sensing systems for construction projects
progress monitoring (Han et al., 2018; Omar and Nehdi, 2016; Pucko et al., 2018). This indicates
that progress monitoring has the ability to fully and automatically capture data, except the
technologies need an approach to be completed. These cutting-edge technologies offer diverse
advantages in supporting construction site monitoring. This endorses the scope of the use of field data capturing technologies for automated project progress monitoring, as
described above. To develop a
generic
framework of a
DT The findings are congruent with the literature. To accomplish the information-based
integrated construction project management system, automated progress measurement is
necessary (Alizadehsalehi and Yitmen, 2019). On-time, accurate and efficient progress
monitoring is required for a construction management system to move forward and be
operative and productive. A systematic, well-organized and comprehensive approach/
framework for progress monitoring needs to be developed to manage project progress
effectively (Braun et al., 2020; Lin and Golparvar-Fard, 2020; Puri and Turkan, 2020). Current
different BIM-based field data capturing technologies are perhaps the most notable change in
progress monitoring. The BIM-based automated progress monitoring systems utilize the
collected 4D/BIM-based as-built data and compare it with the 4D/BIM-based as-designed data
to recognize the discrepancies between them well-interpreted approach. This timely and
efficient data analysis and visualization of differences between 4D/BIM-based as-built data
and 4D/BIM-based as-designed data in an early step provide possibilities to prevent potential
expected delays and enable appropriate corrective actions. This indicates that project
managers need a robust monitoring method/system that assures the most updated design,
up-to-date schedule, cost and progress performance data delivered and presented in a timely
and comprehensive way. That supports the scope of the BIM-based field data capturing
technologies for the automated project progress monitoring described previously. 5. Discussion and implications
5.1 Theoretical contributions They visually recognize issues and SASBE
12,1 make informed decisions to resolve the problems in real-time, on-site. XR technologies are
emerging to digitalize the AEC industry, making it significantly more productive and
effective. Construction management teams will have better construction schedule control and
decision-making through the DRX system, saving money and time for a modular
construction project. This process needs a system to share data and collaboration
information and enable real-time communication between various users. The findings reinforce the literature that precise and effective monitoring, analysis and
visualization of a project’s structural parts of as-built state ongoing projects are key
components of effective project evaluation (Alizadehsalehi et al., 2020; Kan and Anumba,
2019; Lin and Golparvar-Fard, 2020). DTs give project team members access to as-built and
as-planned BIM models that are continuously synced in real-time. DRX is advancing rapidly
beyond n-D BIM and DT, due to the confluence of 4D visualization, reality capturing
technologies, XRs, providing an immersive and integrated view of all infrastructure assets
above ground, on the surface and below ground to observe and monitor. With cognitive
computing applications like AI and ML and computing analytics, projects provide insights
and analytics visibility to improve progress monitoring processes more effectively with
anticipating issues before they arise and react more swiftly with confidence. In the form of the
DRX model, these technology integrations envisage the automation of monitoring tasks
through a real-time DT, enabling experts to conduct inspections remotely, vastly increasing
productivity and leveraging scarce knowledge resources. This provides all project
stakeholders to continuously monitor progress against the schedule laid out in a 4D/BIM-
based environment precisely and comprehensively. 226 5.2 Managerial implications
The DRX system presented in this research automates and enhances the time-consuming
management task’s accuracy by capturing fast and precisely, computing construction
progress process, updating project information automatically, presenting in a real scale and
transferring reports and data quickly by cloud. The quantitative analysis of the data collected
from 326 project managers, construction managers, VDC coordinators, 4D planners and BIM
managers indicates that there is a willingness to use the DRX model of management. Based
on the result of this analysis, clash detection, data integration, intelligent decision-making,
feedback driving, diagnosis, self-learning, remote decision-making, automation and
collaboration performance are the essential features of the DRX model. 5. Discussion and implications
5.1 Theoretical contributions The summary of
the strengths and challenges of the proposed system based on literature review, author’s
experiences and construction management specialist are shown in Table 9. 5. Discussion and implications
5.1 Theoretical contributions 225 The findings are congruent with literature in that for construction progress monitoring,
using DT proposes always having access to as-planned and as-built data and models that are
synced continuously in real-time (Boje et al., 2020; Braun et al., 2020; Kan and Anumba, 2019). BIM provides technologies, procedures and data schemas to facilitate a standardized
semantic representation of building systems. A DT is a connection between a real-world
object and its digital representation, which consistently utilizes data from data-capturing
technologies and various sensors located on a job site to establish a virtual object’s
representation (Boje et al., 2020). It conveys a more holistic socio-technical and process-
oriented characterization of the complex artifacts involved by leveraging the cyber-physical
bi-directional data flows’ synchronicity. Furthermore, BIM lacks semantic completeness in
areas such as control systems, including sensor networks, social systems and urban artifacts
beyond buildings’ scope, thus requiring a holistic, scalable semantic approach that factors in
dynamic data at different levels (Braun et al., 2020). This allows project stakeholders to
continuously monitor the project’s progress versus the schedule laid out in a 4D-BIM model. This also provides the model predictive control/monitoring method to make the best
decisions with forwarding simulation, beginning with the project’s current state (Rahimian
et al., 2020). Therefore, project stakeholders can continuously analyze various ways of
operations and estimate/determine their corresponding cost functions and probabilities to
pick the most optimal judgment/decision (or adjustment) for what they must do next. p
p
j
g
(
j
)
y
The findings are consistent with the literature in that transforming complex data into
simple and straightforward XRs visualizations environment gives context to critical
information on the job site (Alizadehsalehi et al., 2020; Kopsida and Brilakis, 2020; Lin et al.,
2019b). With BIM-to-XR workflow provides interactive visualization of the proposed design
with an immersive experience and human scale. Users can interact with the 3D information
model on top of the physical space by either superimposing the different design options
within the existing job site condition or reading additional information that does not exist in
the real-world (Alizadehsalehi et al., 2020). The visualization of complex data/model with XR
technologies makes it easy to observe spatial relationships more precisely to recognize and
mitigate structural and MEP clashes earlier. XR technologies enhance construction projects’
profitability and productivity by facilitating collaborative communication among project
team members and other stakeholders (Boje et al., 2020). SASBE
12,1 6. Conclusions
Thi
d This study presented an automated construction progress monitoring system called DRX. The integration of BIM, various reality capturing technologies (laser scanner and wireless
sensors), DT and XR technologies (VR, AR, and MR) have been shown by IDEF0 modeling. It
arrays steps on how these technologies work collaboratively to create, capture, generate,
analyze, manage and visualize construction progress data, information and reports. The SEM
method has been used to test the proposed hypotheses and develop the skill model to examine
the reliability, validity and contribution of the framework to understand the DRX model’s
effectiveness if implemented in real practice. Finally, this research highlighted the strengths
and challenges of DRX. As a core part of the DRX system, DT helps manage, simulate,
analyze as-planned and as-built data and optimize and predict a project’s functionality in
various scenarios. With the aid of data and feedback, both physical and simulated models can
develop autonomy and learn from their environments. 6. Conclusions
Thi
d However, this implementation will Strength and challenges
Brief explanation
Cost
Strengths
Reduced manpower required for progress status capture on site
Reduced manpower required for recording captured data in the office
Reduced cost of errors and recaptured data
Gives client more necessary info and confidence when selecting contractors
Reduced cost of management plan
Reduced cost of change control
Challenges
Cost of purchasing RC-to-BIM, BIM-to-XR and DT software
Cost of purchasing UAV, camera, sensors and XR technologies
Cost of software and hardware upgrading
Cost of additional time to design and implement DRX progress model
Cost of hiring additional employees to implement BIM by designers
Cost of hiring engineering team (computer science)
Cost of training of existing employees in the office and onsite
Cost of hiring additional employees to implement the DRX system
Cost of additional time to design the DRX design model
Cost of hiring reality capturing team to capture data
Quality
Strengths
Capturing all blind points of the project
Automation of progress control process
Construction progress rule and code checking
Early progress planning on pre-construction stage
Monitoring worker motion and worker location
Automatic layout of as-planned and as-built
Better understanding of project
Identification of potential quality collapse
High quality progress meeting with all visualization information
Reduced information loss in data exchange
Automatic identification of quality issues
Real-time checking of progress conditions
Reduced personnel safety hazards
Better monitoring of physical environments (equipment)
Improved construction management
Easily changed work practices
Allows a human-scale clearance check
Challenges
Lack of integration of progress monitoring rule checking into BIM
Decision-making process still needs human input and can be error prone
Lack of trust to comparing as-built and as-planned data by current algorithms
Lack of fully automated system
Different levels of visual training
UAV is easy to fall due to incompetent operator
UAV is easy to fall due to strong wind
The remote-control signal is interfered/blocked by other radio signals or obstacle
UAV may distract the attention of workers and may cause more accidents
Time
Strengths
Real-time and quick monitoring of progress (paperwork eliminated)
Real-time and quick reporting of physical conditions
Real-time and quick reporting of quality conditions
Real-time reporting of progress status
Quick progress issues reporting and investigation
Progress control regulated in contract documents
Real-time reporting of sources of progress issues
Tests the feasibility of an architectural design
Permits real-time virtual collaboration for stakeholders from different locations
Challenges
Long hours to develop a BIM model
Lack of authority and procedures for UAV operations
Lengthy training of employees in BIM, LS, WS, XR and DT skills
Table 9. 6. Conclusions
Thi
d Strengths and
challenges of DRX
To develop a
generic
framework of a
DT
227 Strength and challenges
Brief explanation
Cost
Strengths
Reduced manpower required for pro
Reduced manpower required for rec
Reduced cost of errors and recaptur
Gives client more necessary info an
Reduced cost of management plan
Reduced cost of change control
Challenges
Cost of purchasing RC-to-BIM, BIM
Cost of purchasing UAV, camera, se
Cost of software and hardware upg
Cost of additional time to design an
Cost of hiring additional employees
Cost of hiring engineering team (com
Cost of training of existing employe
Cost of hiring additional employees
Cost of additional time to design the
Cost of hiring reality capturing team
Quality
Strengths
Capturing all blind points of the pro
Automation of progress control pro
Construction progress rule and code
Early progress planning on pre-con
Monitoring worker motion and wor
Automatic layout of as-planned and
Better understanding of project
Identification of potential quality co
High quality progress meeting with
Reduced information loss in data ex
Automatic identification of quality i
Real-time checking of progress cond
Reduced personnel safety hazards
Better monitoring of physical enviro
Improved construction managemen
Easily changed work practices
Allows a human-scale clearance che
Challenges
Lack of integration of progress mon
Decision-making process still needs
Lack of trust to comparing as-built
Lack of fully automated system
Different levels of visual training
UAV is easy to fall due to incompet
UAV is easy to fall due to strong w
The remote-control signal is interfer
UAV may distract the attention of w
Time
Strengths
Real-time and quick monitoring of p
Real-time and quick reporting of ph
Real-time and quick reporting of qu
Real-time reporting of progress stat
Quick progress issues reporting and
Progress control regulated in contra
Real-time reporting of sources of pr
Tests the feasibility of an architectu
Permits real-time virtual collaborati
Challenges
Long hours to develop a BIM mode
Lack of authority and procedures fo
Lengthy training of employees in B
Long time needed to create rules, pr
Cumbersome public policy for gettin Strength and challenges
Brief explanation
Cost
Strengths
Reduced manpower required for progress status capture on site
Reduced manpower required for recording captured data in the office
Reduced cost of errors and recaptured data
Gives client more necessary info and confidence when selecting contractors
Reduced cost of management plan
Reduced cost of change control
Challenges
Cost of purchasing RC-to-BIM, BIM-to-XR and DT software
Cost of purchasing UAV, camera, sensors and XR technologies
Cost of software and hardware upgrading
Cost of additional time to design and implement DRX progress model
Cost of hiring additional employees to implement BIM by designers
Cost of hiring engineering team (computer science)
Cost of training of existing employees in the office and onsite
Cost of hiring additional employees to implement the DRX system
Cost of additional time to design the DRX design model
Cost of hiring reality capturing team to capture data
To develop a
generic
framework of a
DT
227 Quality
Capturing all blind points of the project
Automation of progress control process
Construction progress rule and code checking
Early progress planning on pre-construction stage
Monitoring worker motion and worker location
Automatic layout of as-planned and as-built
Better understanding of project
Identification of potential quality collapse
High quality progress meeting with all visualization information
Reduced information loss in data exchange
Automatic identification of quality issues
Real-time checking of progress conditions
Reduced personnel safety hazards
Better monitoring of physical environments (equipment)
Improved construction management
Easily changed work practices
Allows a human-scale clearance check
Lack of integration of progress monitoring rule checking into BIM
Decision-making process still needs human input and can be error prone
Lack of trust to comparing as-built and as-planned data by current algorithms
Lack of fully automated system
Different levels of visual training
UAV is easy to fall due to incompetent operator
UAV is easy to fall due to strong wind
The remote-control signal is interfered/blocked by other radio signals or obstacle
UAV may distract the attention of workers and may cause more accidents Challenges Time
Real-time and quick monitoring of progress (paperwork eliminated)
Real-time and quick reporting of physical conditions
Real-time and quick reporting of quality conditions
Real-time reporting of progress status
Quick progress issues reporting and investigation
Progress control regulated in contract documents
Real-time reporting of sources of progress issues
Tests the feasibility of an architectural design
Permits real-time virtual collaboration for stakeholders from different locations
Long hours to develop a BIM model
Lack of authority and procedures for UAV operations
Lengthy training of employees in BIM, LS, WS, XR and DT skills
Long time needed to create rules, procedures, and policies
Cumbersome public policy for getting license to fly UAVs in project area Challenges require overcoming numerous obstacles of technological, organizational, commercial and
sociological. 6. Conclusions
Thi
d DT has promising applications within the DRX system, which can help design
precise as-planned models, collect more accurate data, compare as-planned and as-built
models and verify that the completed work is consistent with plans and specifications. DRX
provides trustable, real-time, transparent and digital data/information/reports that will be
helpful for: 228 (1)
Creating high-quality designs (as-planned models): create a design model, and
visualization is a kind of loop that help designer to draw, visualize and check the
design through XR technologies and get a high-quality design. This part of the DRX
management system helps all stakeholders of the projects such as clients, project
managers and designers to check the model step by step, find out its issues and
compare different design scenarios to get the highest quality design and the first
step of the project. (2)
Planning and optimizing various scenarios (as-planned models): as a complete high
fidelity digital representation of a physical asset, DRX with DT provides a safe,
reliable environment to study, inspect and test various scenarios and optimize
strategies before implementation at the job site. (3)
Capturing accurate, real-time and comprehensive data at construction stage
(as-built models): DRX, with the help of various types of reality capturing
technologies such as laser scanner, cameras and sensors, in construction stage, can
capture precisely, real-time and comprehensive as-built data from the timely status
of construction projects based on the pre-defined progress monitoring plan. (4)
Analyzing data and information precisely and quickly (as-planned and as-planned
models): DRX included DT as a powerful and intelligent engine to manage, analyze
and simulate these created and captured data for analyzing data and information
precisely and quickly, which is connected to historical data and work with cognitive
computing and computing analytics. (5)
Visualizing information and reporting in a real scale environment (as-planned and
as-planned models): DRX, with the help of XR technologies (VR, AR and MR), which
is in the data access part of it, can visualize created and captured data and
information in a real scale environment. Immersing in this environment allowed
involved stakeholders to virtually walk on the project, evaluate the model and give
the designer comments to improve the system. (6)
Facilitating information flows and communication (as-planned and as-planned
models): in the context of the DRX model of management, the system facilitates
information flows and communication between as-planned and as-planned models. 6.1 Future research The body of results shows the applicability of the DRX integration for a variety of AEC
analysis scenarios, and it proves the high eventual added value that a DRX would convey. DRX automation system provides the answer to the DT concept requirements in Industry 4.0
deployments. The benefits of DRX to the AEC industry will be long-term, from a lean and
BIM construction process toward smart, dynamic and semantic AEC management systems. Reality capturing technologies such as laser scanners, UAV-based cameras, sensors, besides
XR technologies have proved to be reliable and safe, yet effective options in an industry that
is consistently evolving and integrating novel emerging technologies. In addition, with the
help of cognitive computing, AI, ML and 5G network associated with future hardware and
software advancements, BIM and DT-based systems will be more widely applied in the AEC
industry. y
Further research is required to establish methodologies and best practices for current and
future DRX integration in AEC manners. Using this system’s legal and financial aspects will
also lead to future research of DRX and its integration in the AEC domain. Future research
will also implement this framework on multiple construction projects to analyze the existing
and emerging examples of DRX applications by utilizing objective data to evaluate the long-
term impact of DRX in construction operations. 6. Conclusions
Thi
d (7)
Learning from itself, historical data and accessible online data to predict future
actions: a DRX system with DT continuously learns and updates itself from multiple
sources such as itself, historical data belongs to the company, municipality or
regulations, or online data and to represent its near real-time status, working
condition or position. DRX is able to predict future actions. (8)
Providing semantic and digitalize construction information with analytical
capabilities: DRX converts the captured and created data into semantic concepts,
meaningful translations of the data and thereby bridge the semantic gap between
the computer and humans with the advantage of novel technologies that are
meaningful for construction projects. (9)
Increasing the vision: empower complex construction systems to be intelligently
operated with transparency and while also gaining valuable insights that we need to
operate these systems to their highest efficiency level. (9)
Increasing the vision: empower complex construction systems to be intelligently
operated with transparency and while also gaining valuable insights that we need to
operate these systems to their highest efficiency level. To develop a
generic
framework of a
DT (10)
Optimizing decision-making process: DRX helps in creating and collecting as much
data from pre-construction, construction and historical data as possible and trying
to find the optimal choice. Novel technologies at DRX let the decision-makers have
the opportunities to select the best scenarios from various accepted and high-quality
models and scenarios. (10)
Optimizing decision-making process: DRX helps in creating and collecting as much
data from pre-construction, construction and historical data as possible and trying
to find the optimal choice. Novel technologies at DRX let the decision-makers have
the opportunities to select the best scenarios from various accepted and high-quality
models and scenarios. 229 (9)
Increasing the vision: empower complex construction systems to be intelligently
operated with transparency and while also gaining valuable insights that we need to
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Ib
hi
Yi
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Honokiol Inhibits Proliferation, Invasion and Induces Apoptosis Through Targeting Lyn Kinase in Human Lung Adenocarcinoma Cells
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Frontiers in pharmacology
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ORIGINAL RESEARCH
published: 28 May 2018
doi: 10.3389/fphar.2018.00558 Edited by: Edited by:
Marcello Locatelli,
Università degli Studi “G. d’Annunzio”
Chieti – Pescara, Italy
Reviewed by:
Pei Luo,
Macau University of Science
and Technology, China
Songxiao Xu,
Artron BioResearch Inc., Canada Edited by:
Marcello Locatelli,
Università degli Studi “G. d’Annunzio”
Chieti – Pescara, Italy Honokiol is a natural compound with small molecular structure and extracted from
bark of magnolia trees. The biological activities of honokiol include anti-oxidation, anti-
inflammation as well as anti-tumor. However, their mechanism remains unknown. In this
study, A549 cell line and EGFR-mutant cell line PC-9 with higher expression level of Lyn
than A549 cells were used to assess the anti-tumor effects of honokiol. As shown in
this study, honokiol is an effective drug on inhibiting proliferation and inducing apoptosis
depended on Lyn and EGFR signal pathway regulated by Lyn, and its efficacy is stronger
in PC-9 cells than A549 cells. In addition, this anti-tumor effect in PC-9 cells was
weakened by Lyn-knockdown. Taken together, this study indicated the mechanism of
honokiol on lung adenocarcinoma and provides a possibility of honokiol as an effective
anti-tumor medicine. Honokiol is a natural compound with small molecular structure and extracted from
bark of magnolia trees. The biological activities of honokiol include anti-oxidation, anti-
inflammation as well as anti-tumor. However, their mechanism remains unknown. In this
study, A549 cell line and EGFR-mutant cell line PC-9 with higher expression level of Lyn
than A549 cells were used to assess the anti-tumor effects of honokiol. As shown in
this study, honokiol is an effective drug on inhibiting proliferation and inducing apoptosis
depended on Lyn and EGFR signal pathway regulated by Lyn, and its efficacy is stronger
in PC-9 cells than A549 cells. In addition, this anti-tumor effect in PC-9 cells was
weakened by Lyn-knockdown. Taken together, this study indicated the mechanism of
honokiol on lung adenocarcinoma and provides a possibility of honokiol as an effective
anti-tumor medicine. Reviewed by:
Pei Luo,
Macau University of Science
and Technology, China
Songxiao Xu,
Artron BioResearch Inc., Canada *Correspondence:
Xiao-Jun Yao
xjyao@must.edu.mo
Guo-Ping Li
lzlgp@163.com
Elaine L.-H. Leung
lhleung@must.edu.mo Keywords: honokiol, Lyn kinase, EGFR, apoptosis, Lung cancer Specialty section:
This article was submitted to
Ethnopharmacology,
a section of the journal
Frontiers in Pharmacology Honokiol Inhibits Proliferation,
Invasion and Induces Apoptosis
Through Targeting Lyn Kinase in
Human Lung Adenocarcinoma Cells
Xi Dai1,2, Run-Ze Li1, Ze-Bo Jiang1, Chun-Li Wei1, Lian-Xiang Luo1, Xiao-Jun Yao1*,
Guo-Ping Li3* and Elaine L.-H. Leung1* 1 State Key Laboratory of Quality Research in Chinese Medicines, Macau Institute for Applied Research in Medicine and
Health, Macau University of Science and Technology, Macau, China, 2 Department of Respiratory Medicine, Affiliated
Hospital of Southwest Medical University, Luzhou, China, 3 Department of Respiratory Medicine, The Third People’s Hospital
of Chengdu, The Affiliated Hospital of Southwest Jiaotong University, Chengdu, China INTRODUCTION Lung cancer is the most prevalent type of cancer as well as the most common cause of death from
cancer worldwide (Siegel et al., 2013). According to a global statistic on lung cancer, there are
more than 1.825 million new case and 1.59 million deaths every year, and more than 55% of the
world’s lung cancer cases occur in Asia. In both China and the United States, lung cancer is the
most common incident cancer and the leading cause of cancer death (Siegel et al., 2016), and cause
huge mortality to population. Recently, targeting therapy, such as EGFR tyrosine kinase inhibitors
(TKIs) or VEGF receptor inhibitors, improve response rates, time to progression, and overall
survival. Unfortunately, only about 10% patients with EGFR mutant benefit from TKI and most
of them develop disease progression after a median of 9.2–14.7 months of TKI therapy. Acquired
resistance caused by T790M mutation remains a significant barrier to effective treatment referred to
patients with EGFR-mutant NSCLC (Westover et al., 2018; Wu and Shih, 2018). Additionally, these
therapies lead to side effects which bring pain to patients. Therefore, novel and effective preventive
and therapeutic agents should be developed to combat this major health problem. Received: 15 March 2018
Accepted: 10 May 2018
Published: 28 May 2018 Keywords: honokiol, Lyn kinase, EGFR, apoptosis, Lung cancer Citation: Dai X, Li R-Z, Jiang Z-B, Wei C-L,
Luo L-X, Yao X-J, Li G-P and
Leung EL-H (2018) Honokiol Inhibits
Proliferation, Invasion and Induces
Apoptosis Through Targeting Lyn
Kinase in Human Lung
Adenocarcinoma Cells. Front. Pharmacol. 9:558. doi: 10.3389/fphar.2018.00558 Dai X, Li R-Z, Jiang Z-B, Wei C-L,
Luo L-X, Yao X-J, Li G-P and
Leung EL-H (2018) Honokiol Inhibits
Proliferation, Invasion and Induces
Apoptosis Through Targeting Lyn
Kinase in Human Lung
Adenocarcinoma Cells. Front. Pharmacol. 9:558. doi: 10.3389/fphar.2018.00558 May 2018 | Volume 9 | Article 558 1 Frontiers in Pharmacology | www.frontiersin.org Honokiol Inhibits Lung Cancer Growth Dai et al. Dai et al. Honokiol Inhibits Colony Formation and
Invasion in PC-9 Cells Traditional Chinese medicine (TCM) for cancer treatment
has more than 2000 years history. Concepts of diagnosis
and treatment methods in TCM demonstrate characteristics
and advantages of TCM-based herbal medicines and laid the
foundation or the innovation and development of cancer
treatment with TCM (Liu et al., 2015). Obviously, TCM contains
various natural compounds which are the most prolific source
of leading compounds for drug development. Usually, TCM has
therapeutic efficacy in lung cancer with various biological activity
and minimal side effects. For these reasons, TCM has gained
increasing interest and acceptance worldwide, especially TCM-
based herbal drugs that selectively target molecular pathways
in cancer cells and modulates proliferation and progression of
cancer. On the other hand, current clinical systemic treatments
for NSCLC is low efficacy but high toxicity. TCM-based herbal
medicines may increase efficacy of chemotherapy (McCulloch
et al., 2006). Honokiol was an effective drug to modulate cell proliferation. As
same as the results of MTT assay, honokiol significantly inhibited
the colony formation capacity in PC-9 cells in a concentration
dependent manner (Figure 2A). Moreover, the wound healing
assay and transwell invasion assay were used to evaluate the
impact of honokiol on cell migration and invasion. Consistent
with previous finding from MTT assay and colony formation
assay, we also found that honokiol significantly inhibited the
migration and invasion capacity of PC-9 cells (Figures 2B,C). Honokiol Suppresses Lyn Kinase and
EGFR Signaling Pathway in PC-9 Cells PC-9 cell line is one of the EGFR mutation cell line with
continuous activation of EGFR. In our previous study, we have
found Lyn was an effective regulator on EGFR signaling pathway,
therefore, we detected the effect of honokiol on Lyn and it
mediated EGFR signaling transduction in PC-9 cells. PP2 is a
non-specific Src TKIs, as a positive control in this study. As shown in Figure 3C, honokiol decreased the expression
level of Lyn and its phosphorylation status significantly. In
addition, treatment with honokiol also led to a reduction
of EGFR/PI3K/AKT and STAT3, and their phosphorylation
status. These results indicated that honokiol inhibits PC-9 cell
proliferation, invasion and induces apoptosis through targeting
Lyn kinase and Lyn-mediated EGFR signaling pathway. Honokiol Induces Apoptosis in PC-9
Cells Flow cytometry was used to analyze the percentage of apoptosis
cells after treated with honokiol. Our results showed that
honokiol induced apoptosis in PC-9 cells in a concentration
dependent manner. Images picked from microscope at 40×
magnification also showed deformation, death and floating of
PC-9 cells induced by honokiol (Figures 3A,B). Honokiol is a natural compound isolated from the bark and
root of Magnolia officinalis and used as traditional Chinese
herbal medicine for thousands of years for the treatment of
various diseases. The biological activities of honokiol includes
anti-oxidative, anti-allergic, anti-inflammatory, anti-bacterial,
and many previous studies have indicated that honokiol is
a useful biologically active compound in protecting against
hepatotoxicity, neurotoxicity, thrombosis and angiopathy (Fried
and Arbiser, 2009). Recently, the anti-cancer activities of
honokiol have gained increasing number of attention and various
mechanisms have been reported (Bai et al., 2003; Chen et al.,
2004; Battle et al., 2005; Jiang et al., 2008; Park et al., 2009). However, the mechanisms of antitumor activity of honokiol are
still unclear completely. In this present study, we used A549 cell
line and EGFR-mutant cell line PC-9 with higher expression level
of Lyn than A549 cell line to clarify the in vitro antitumor effects
and treatment mechanism of honokiol. Honokiol Suppresses Lyn Kinase and
EGFR Signaling Pathway in PC-9 Cells
PC-9 cell line is one of the EGFR mutation cell line with
continuous activation of EGFR. In our previous study, we have
found Lyn was an effective regulator on EGFR signaling pathway,
therefore, we detected the effect of honokiol on Lyn and it
mediated EGFR signaling transduction in PC-9 cells. PP2 is a
non-specific Src TKIs, as a positive control in this study. Honokiol Inhibits Cell Proliferation in
Both A549 Cells and PC-9 Cells As shown in Figure 1B, compared with A549 cells which harbors
EGFR wild type, the expression level and activity of Lyn were
markedly increased as well as the expression level and activity of
EGFR in PC-9 cells with EGFR mutation. MTT assay was used
to assess the cytotoxicity of honokiol on A549 cells and PC-9
cells. Honokiol was tested in both cell lines for 24, 48, and 72 h
at an increasing concentration of 0, 20, 40, 60, and 80 µM. The
percentage of cell viability and the IC50 values were analyzed with
GraphPad Prism 5.0 software. As shown in Figure 1A, honokiol
inhibited the proliferation rate of PC-9 cells in a concentration
dependent and time-dependent manner. In addition, honokiol
showed lower cytotoxicity in A549 cells than PC-9 cells. The
IC50 value of PC-9 cells at 24, 48, and 72 h were all lower than
that of A549 cells. This result implied that honokiol has stronger
cytotoxic effect on PC-9 cells with higher expression level of Lyn
kinase (Figure 1B). To further evaluate the role of Lyn kinase for the effects of
honokiol, PC-9 cells were transfected with Lyn small interfering
RNA (Lyn-siRNA) for Lyn knockdown. The expression level of
Lyn kinase was analyzed by Western blots analysis. Figure 4A
showed the expression level of Lyn and p-Lyn were decreased
significantly after administration with Lyn-siRNA. Based on this,
MTT assay was used to assess the cytotoxicity of honokiol on
PC-9 cells with or without Lyn siRNA knockdown. As shown
in Figure 4B, the cytotoxicity of honokiol at a concentration
of 60 µM for 24, 48, and 72 h were all weakened by Lyn
knockdown. More than this, the wound healing assay and
transwell invasion assay also indicated that the inhibition effects
of honokiol at a concentration of 60 µM on migration and
invasion capacity of PC-9 were weakened by Lyn knockdown
(Figures 4C–F). Additionally, Western blots analysis was used May 2018 | Volume 9 | Article 558 Frontiers in Pharmacology | www.frontiersin.org 2 Honokiol Inhibits Lung Cancer Growth Dai et al. Dai et al. FIGURE 1 | Effects of honokiol on cell viability in A549 cells and PC-9 cells. Honokiol Inhibits Cell Proliferation in
Both A549 Cells and PC-9 Cells (A) The percentage of cell viability of PC-9 cells treated with various concentration of
honokiol (0, 20, 40, 60, and 80 µM) for 24, 48, and 72 h, and a comparison of cell viability and IC50 between A549 cells and PC-9 cells at different exposing time in
honokiol. (B) A comparison of expression level of Lyn and EGFR between A549 cells and PC-9 cells, as analyzed by Western blots assay. ∗∗P < 0.05 and
∗∗∗P < 0.01 for comparison between two groups. FIGURE 1 | Effects of honokiol on cell viability in A549 cells and PC-9 cells. (A) The percentage of cell viability of PC-9 cells treated with various concentration of
honokiol (0, 20, 40, 60, and 80 µM) for 24, 48, and 72 h, and a comparison of cell viability and IC50 between A549 cells and PC-9 cells at different exposing time in
honokiol. (B) A comparison of expression level of Lyn and EGFR between A549 cells and PC-9 cells, as analyzed by Western blots assay. ∗∗P < 0.05 and
∗∗∗P < 0.01 for comparison between two groups. apoptosis in chronic lymphocytic leukemia cells through
activation of Caspase-8 and Bax, it also potentiates the
cytotoxicity of anti-leukemia chemotherapeutic drugs (Battle
et al., 2005). Additionally, subsequent reports also found
honokiol induced apoptosis on colon cancer and breast cancer. Honokiol prevents the growth of RKO cells and MDA-MD-231
cells through targeting p53 and KRAS signaling (Wang et al.,
2004; Wolf et al., 2007). Of interest, honokiol was also observed
to induce apoptosis through increasing the expression of Bax,
while decreasing the expression of Bcl-2 in KRAS-mutant lung
cancer cells (Luo et al., 2017). Moreover, one study suggests that
cyclophilin-D is likely a target of the cytotoxic action of honokiol
(Li et al., 2007). In this research, cells from high-grade esophageal
dysplasia were found to highly sensitive to honokiol, but this
sensitivity could be weakened by cyclophilin-D siRNA. On the
other hand, honokiol has been shown to overcome MDR during to detect the protein expression of EGFR/PI3K/AKT and STAT3
in Lyn-knockdown PC-9 cells after treatment with honokiol. As shown in Figure 5A, the expression levels of EGFR, PI3K,
AKT, STAT3 and their phosphorylation status were higher in
honokiol-induced Lyn-siRNA transfected PC-9 cells than in
control cells. This result proved that Lyn played an important
role for honokiol. Honokiol Inhibits Cell Proliferation in
Both A549 Cells and PC-9 Cells Taken together, honokiol is an effective TCM
to inhibit lung cancer growth through targeting Lyn kinase. DISCUSSION Several studies on honokiol have suggested that it has multiple
mechanisms of antitumor properties such as anti-tumor growth,
anti-migration, anti-angiogenesis, or overcoming multiple drug
resistance (MDR) (Kumar et al., 2013). Honokiol induces May 2018 | Volume 9 | Article 558 Frontiers in Pharmacology | www.frontiersin.org 3 Honokiol Inhibits Lung Cancer Growth Dai et al. Dai et al. GURE 2 | Effects of honokiol on colony formation, migration, and invasion in PC-9 cells. (A) Colony formation of PC-9 cells treated with various concentration of
nokiol (0, 20, 40, 60, and 80 µM) for 10 days was assessed. (B) Migration of PC-9 cells treated with various concentration of honokiol into wound was assessed
0, 12, and 24 h post scratch. (C) Invasion of PC-9 cells treated with various concentration of honokiol was assessed for 24 h. ∗P > 0.05, ∗∗P < 0.05 and
P < 0.01 for comparison among groups. FIGURE 2 | Effects of honokiol on colony formation, migration, and invasion in PC-9 cells. (A) Colony formation of PC-9 cells treated with various concentration of
honokiol (0, 20, 40, 60, and 80 µM) for 10 days was assessed. (B) Migration of PC-9 cells treated with various concentration of honokiol into wound was assessed
at 0, 12, and 24 h post scratch. (C) Invasion of PC-9 cells treated with various concentration of honokiol was assessed for 24 h. ∗P > 0.05, ∗∗P < 0.05 and
∗∗∗P < 0.01 for comparison among groups. FIGURE 2 | Effects of honokiol on colony formation, migration, and invasion in PC-9 cells. (A) Colony formation of PC-9 cells treated with various concentration of
honokiol (0, 20, 40, 60, and 80 µM) for 10 days was assessed. (B) Migration of PC-9 cells treated with various concentration of honokiol into wound was assessed
at 0, 12, and 24 h post scratch. (C) Invasion of PC-9 cells treated with various concentration of honokiol was assessed for 24 h. ∗P > 0.05, ∗∗P < 0.05 and
∗∗∗P < 0.01 for comparison among groups. EGFR over-expression and mutation have been demonstrated
as an important factor for growth and progression of lung
adenocarcinoma (Merrick et al., 2006), but treatment efficacies
of EGFR TKIs are weak and limited due to TKIs-related toxicities
or high risk in part of patients (Rosenthal et al., 2014). Therefore,
it’s prudent to develop effective and safe drugs targeting EGFR in
lung adenocarcinoma. DISCUSSION A little part of previous reports indicated
honokiol regulated EGFR and its downstream effectors. Song’s
research indicated that honokiol decrease the expression of EGFR
and its downstream effective proteins (Song et al., 2016). Lee also
reported magnolol inhibits EGFR-mediated signaling pathways
in prostate cancer (Lee et al., 2009). In addition, honokiol was
reported to enhance the efficacy of EGFR TKIs (Leeman-Neill
et al., 2010). cancer progression. In a nude mice xenograft model research,
in combination with honokiol, docetaxel showed a reduction
in tumor volume as well as microvessel count in nude mice
significantly (Shigemura et al., 2007), combination of honokiol
with DDP enhancing the treatment efficacy of lung cancer as well
(Jiang et al., 2008). Subsequent study demonstrated that honokiol
modulated MDR due to down-regulated NF-κB signaling (Ahn
et al., 2006). Honokiol has efficacy to enhance the activation
of TNF-α, in this way, honokiol inhibits activation of NF-κB
and Akt. As a result, honokiol dramatically decreases expression
level of NF-κB target genes, such as VEGF, MMP-9, and COX-2. In summary, honokiol is an effective antitumor agent due to
its multiple function effects and multiple signaling pathways
undergoing its regulation. May 2018 | Volume 9 | Article 558 Frontiers in Pharmacology | www.frontiersin.org 4 Honokiol Inhibits Lung Cancer Growth Dai et al. Dai et al. FIGURE 3 | Honokiol induced apoptosis and down-regulated Lyn and Lyn-mediated EGFR signaling in PC-9 cells. (A) Apoptosis of PC-9 cells treated with various
concentration of honokiol for 24 h was analyzed by flow cytometer. (B) Representative pictures of PC-9 cells treated with various concentration of honokiol for 24 h
were picked with microscope (40×). (C) Lyn, p-Lyn, EGFR, p-EGFR, PI3K, p-PI3K, AKT, p-AKT, STAT3, and p-STAT3 protein expression of PC-9 cells were
detected by Western blots assay after treated with honokiol (0, 20, 40, and 60 µM) for 24 h. The non-specific Src tyrosine kinase inhibitor (TKI) PP2 is the positive
control. ∗P > 0.05 and ∗∗∗P < 0.01 for comparison among groups. FIGURE 3 | Honokiol induced apoptosis and down-regulated Lyn and Lyn-mediated EGFR signaling in PC-9 cells. (A) Apoptosis of PC-9 cells treated with various
concentration of honokiol for 24 h was analyzed by flow cytometer. (B) Representative pictures of PC-9 cells treated with various concentration of honokiol for 24 h
were picked with microscope (40×). DISCUSSION (C) Lyn, p-Lyn, EGFR, p-EGFR, PI3K, p-PI3K, AKT, p-AKT, STAT3, and p-STAT3 protein expression of PC-9 cells were
detected by Western blots assay after treated with honokiol (0, 20, 40, and 60 µM) for 24 h. The non-specific Src tyrosine kinase inhibitor (TKI) PP2 is the positive
control. ∗P > 0.05 and ∗∗∗P < 0.01 for comparison among groups. Lyn is a member of the Src family of non-receptor tyrosine
kinase and an important regulator of immune homeostasis
and pattern recognition receptor-induced responses. Src kinase
family has been reported playing an important role in VEGF
signaling and effecting proliferation and migration of cancer
cell, and Yes and Lyn kinase were highly associated with EGFR
signaling (Munshi et al., 2000; Iida et al., 2013). Previous studies
reported that EGFR promotes DNA replication of cancer cell by
Lyn phosphorylation, and Lyn kinase regulates phosphorylation
of EGFR through PKCβII (Huang et al., 2013; Sutton et al., 2013). In our previous research, Lyn kinase was demonstrated
to be an important factor up-regulating activity of EGFR and
its downstream signaling pathway by phosphorylating tyrosine
845 of EGFR which suggests the therapeutic target for lung
adenocarcinoma. Here, honokiol has stronger cytotoxic effect
on PC-9 cells with higher expression level of Lyn kinase than
A549 cells in this study, and honokiol decreased expression level
of Lyn significantly in a dose-dependent manner. Moreover,
after Lyn silencing, the regulating effect of honokiol on EGFR
and its downstream effectors were weakened. Taken together, May 2018 | Volume 9 | Article 558 Frontiers in Pharmacology | www.frontiersin.org 5 Honokiol Inhibits Lung Cancer Growth Dai et al. FIGURE 4 | Inhibition effects of honokiol on cell proliferation, migration and invasion of PC-9 cells was weakened by Lyn-knockdown. (A) Lyn and p-Lyn protein
expression in wide type PC-9 cells and Lyn-knockdown PC-9 cells were detected by Western blots assay. (B) The percentage of cell viability of PC-9 cells with or
without Lyn-siRNA transfection at 60 µM concentration of honokiol for 24, 48, and 72h. (C,E) Migration of PC-9 cells with or without Lyn-siRNA transfection into
wound was assessed at 0h, 12 and 24 h post scratch at 60 µM concentration of honokiol. (D,F) Invasion of PC-9 cells with or without Lyn-siRNA transfection was
assessed for 24 h at 60 µM concentration of honokiol. ∗P > 0.05, ∗∗P < 0.05 and ∗∗∗P < 0.01 for comparison among groups. DISCUSSION FIGURE 4 | Inhibition effects of honokiol on cell proliferation, migration and invasion of PC-9 cells was weakened by Lyn-knockdown. (A) Lyn and p-Lyn protein
expression in wide type PC-9 cells and Lyn-knockdown PC-9 cells were detected by Western blots assay. (B) The percentage of cell viability of PC-9 cells with or
without Lyn-siRNA transfection at 60 µM concentration of honokiol for 24, 48, and 72h. (C,E) Migration of PC-9 cells with or without Lyn-siRNA transfection into
wound was assessed at 0h, 12 and 24 h post scratch at 60 µM concentration of honokiol. (D,F) Invasion of PC-9 cells with or without Lyn-siRNA transfection was
assessed for 24 h at 60 µM concentration of honokiol. ∗P > 0.05, ∗∗P < 0.05 and ∗∗∗P < 0.01 for comparison among groups. 48, and 72 h, respectively; 10 µl of MTT solution (5 mg/ml)
was added in each well and incubated for another 4 h. Then
100 µl of DMSO was added in each well and incubated for
4 h to dissolve the formazan crystals. Absorbance of plates was
measured at 570 nm with Tecan microplate reader (Morrisville,
NC, United States). we reported for the first time that honokiol inhibits EGFR
and its downstream signaling pathway by targeting Lyn kinase
(Figure 5B). Colony Formation Assay A549 cells and PC-9 cells were obtained from American Type
Culture Collection (ATCC) and cultured at 37◦C in a humidified
atmosphere of 5% CO2 with RPMI-1640 medium, which
supplemented with 10% fetal bovine serum (FBS), 100 units/ml
penicillin and 100 µg/ml streptomycin. Honokiol reagent was
purchased from Selleck Chemicals. The primary antibodies of
Lyn, p-Lyn, EGFR, p-EGFR, PI3K, p-PI3K, AKT, p-AKT, STAT3,
p-STAT3 were purchased from Cell Signaling Technology,
β-Actin was purchased from Santa Cruz Biotechnology. The
secondary antibodies were purchased from Odyssey. PC-9 cells were seeded in dishes with a diameter of 60 mm with
500 cells/dish and cultured overnight of adhesion, then treated
with different concentrations of honokiol (0, 20, 40, 60, and
80 µM) with medium changes per 3 days until visible colonies
formed. The colonies were washed by PBS and fixed with 4%
paraformaldehyde and stained with Wright-Giemsa stain. The
number of colonies formation in each dish was counted. Wound Healing Assay PC-9 cells were seeded in 6-well plates with the concentration of
2 × 105 cells/ml. Scratch wounds were made by scraping the cell
layer with the tip of 10 µl pipette, debris was removed by washing
the cells with 1 × PBS, then three fields (40×) were randomly
picked from scratch wound of each well. After wounding, cells
were exposed to various concentration of honokiol (0, 20, 40, 60, MTT Assay A549 cells and PC-9 cells were seeded in 96-well plates and
cultured overnight of adhesion, then treated with different
concentrations of honokiol (0, 20, 40, 60, and 80 µM) for 24, May 2018 | Volume 9 | Article 558 Frontiers in Pharmacology | www.frontiersin.org 6 Dai et al. Honokiol Inhibits Lung Cancer Growth FIGURE 5 | Effect of honokiol on down regulation of EGFR signaling in PC-9 cells was weakened by Lyn knockdown. (A) EGFR, p-EGFR, PI3K, p-PI3K, AKT,
p-AKT, STAT3, and p-STAT3 protein expression in wide type PC-9 cells and Lyn-knockdown PC-9 cells were detected by Western blots assay after treated with
honokiol (60 µM) for 24 h. (B) Graphical abstract showed the antitumor mechanism of honokiol on lung adenocarcinoma. ∗P > 0.05, ∗∗P < 0.05 and ∗∗∗P < 0.01
for comparison among groups. FIGURE 5 | Effect of honokiol on down regulation of EGFR signaling in PC-9 cells was weakened by Lyn knockdown. (A) EGFR, p-EGFR, PI3K, p-PI3K, AKT,
p-AKT, STAT3, and p-STAT3 protein expression in wide type PC-9 cells and Lyn-knockdown PC-9 cells were detected by Western blots assay after treated with
honokiol (60 µM) for 24 h. (B) Graphical abstract showed the antitumor mechanism of honokiol on lung adenocarcinoma. ∗P > 0.05, ∗∗P < 0.05 and ∗∗∗P < 0.01
for comparison among groups. and 80 µM) for 12 and 24 h, three random fields (40×) were
picked from scratch wound at each exposed time. membrane. Non-specific binding was blocked with 5% no-fat
milk which diluted in 1 × TBST for 1 h and washed three times
with 1 × TBST. The membranes were incubated with primary
antibodies (1:1000) at 4◦C overnight. After removed the extra
primary antibodies and washed three times with 1 × TBST, the
membranes were incubated with secondary antibody (1:1000) for
1 h at room temperature. The bands were visualized by the Pierce
ECL Western Blotting Kit or LI-COR Odyssey Scanner. Apoptosis Assay All of the statistical analyses were conducted by the SPSS 17.0
software and all the data was expressed as Mean ± SEM. Statistical significance differences were evaluated by one-way
ANOVA and using LSD or Tamhane’s method for comparison
tests between two groups. The level of statistical significance was
defined as P < 0.05. PC-9 cells were seeded in 6-well plates and exposed to various
concentration of honokiol (0, 20, 40, 60, and 80 µM) for 24 h. Annexin V-FITC solution and PI solution were used to stain
cells in the dark for 15 min. Each sample was analyzed by flow
cytometry and the percentages of apoptotic cells of every group
were analyzed with Flow J software. Transwell Invasion Assay Transwell membranes (pore size: 8 µm) coated with Matrigel
(BD Biosciences, United States) were re-hydrated for 1h in an
environment of 37◦C in FBS-free RPMI-1640 medium. PC-9
cells were resuspended into FBS-free RPMI-1640 medium with
various concentration of honokiol (0, 20, 40, 60, and 80 µM) and
seeded into the upper chamber at a density of 5 × 105 cells/ml. 600 µl RPMI-1640 medium with 10% FBS was added into the
lower chamber. After 24 h incubation, Transwell membranes
were with 4% paraformaldehyde for 30 min and then stained with
Wright-Giemsa stain. Non-invading cells on the upper surface of
the membrane were removed. Five randomly fields were picked
from every membrane with microscope at 400× magnification. Cell Transfection For Lyn knockdown, PC-9 cells were seeded in 6-well plates. When the cells reached 80% confluence, the medium was
replaced with FBS-free RPMI1640 medium and cells were
transfected with Lyn siRNA and Lipofectamine 2000. The Lyn
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the original author(s) and the copyright owner are credited and that the original
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terms. Copyright © 2018 Dai, Li, Jiang, Wei, Luo, Yao, Li and Leung. 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 are credited and that the original
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marker for separation, then transferred the protein to PVDF X-JY, G-PL, and EL: conceived and designed the study. XD, R-ZL,
Z-BJ, C-LW, and L-XL: performed the experiments. XD and
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The effect of water deficit on inflorescence period at palm oil productivity on peatland
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The effect of water deficit on inflorescence
period at palm oil productivity on peatland
Nanik Ambar Suharyanti1, Kosuke Mizuno1*, and Ahyahudin Sodri1
1 School of Environmental Science, Universitas Indonesia, Central Jakarta, 10430, Indonesia Nanik Ambar Suharyanti1, Kosuke Mizuno1*, and Ahyahudin Sodri1 1 School of Environmental Science, Universitas Indonesia, Central Jakarta, 10430, Indonesia Abstract. Various factors influence palm oil production, including
planting material, environment (climate and soil), and cultivation
management. The environment occupies a crucial position that can affect
crop production potential even with optimal cultivation management. Unfavorable climatic conditions, for instance, drought, can cause water
deficit, which has implications for crop productivity. This paper aims to
get a comparative study for the effect of water deficit on the inflorescence
period, namely the sex determination phase (29 months before harvesting)
and floral abortion (10 months before harvesting). The research was
carried out on oil palm plantations on peatland in Riau, Indonesia, with the
characteristic annual rainfall of 2000 mm/ year. This study employs a
simple regression analysis on panel data of five years and two estates for
data analysis. The result highlights that the increase of a 100 mm water
deficit on oil palm peatland plantation impacts the decrease in productivity
by 6% in the sex determination phase. In contrast, in the floral abortion
phase, the decline in productivity is more significant, namely 7%. The
analysis also indicated that the research deficit had more impact on the
bunch number than the bunch weight. E3S Web of Conferences 211, 05005 (2020)
The 1st JESSD Symposium 2020 E3S Web of Conferences 211, 05005 (2020)
The 1st JESSD Symposium 2020 https://doi.org/10.1051/e3sconf/202021105005 © 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/). * Corresponding author: kosuke.mizuno@ui.ac.id 1 Introduction Oil palm productivity is a synergy between genetic factors (planting material) and the
environment [1-3]. In achieving the sustainability of the palm oil industry, productivity is a
crucial factor, so that it requires a detailed understanding of the factors that influence it
[4-5]. Technologically, planting material and soil is relatively easy to engineer or process,
while climate needs a detailed and in-depth study of natural phenomena. Climate is posited
as a crucial factor in its influence on oil palm productivity [6-9]. Several previous
researchers have made studies on these interactions, including [2, 8]. The study [2] states
four levels of plant productivity, with each factor playing a role at each level (shown in
Figure1). [8] describes a productivity scheme divided into three stages with each
influencing factor (shown in Figure 2). Both researchers stated that plant material and the
environment (climate and soil) are crucial factors that support productivity because they are
positioned at the initial level before cultivation management factors. E3S Web of Conferences 211, 05005 (2020)
The 1st JESSD Symposium 2020
https://doi.org/10.1051/e3sconf/202021105005 E3S Web of Conferences 211, 05005 (2020) https://doi.org/10.1051/e3sconf/202021105005 E3S Web of Conferences 211, 05005 (2020)
The 1st JESSD Symposium 2020 The 1st JESSD Symposium 2020 Fig. 1. Differences in the level of oil palm production
and its factors contribute [2]. Fig. 2. Scheme definitions of factors
that influence the potential for genetic
outcomes and actual results [8]. Fig. 1. Differences in the level of oil palm production
and its factors contribute [2]. Fig. 2. Scheme definitions of factors
that influence the potential for genetic
outcomes and actual results [8]. Fig. 1. Differences in the level of oil palm production
and its factors contribute [2]. Fig. 2. Scheme definitions of factors
that influence the potential for genetic
outcomes and actual results [8]. Among the agro-climatic factors, water supply is the most important for palm oil [10]. Unfavorable climatic conditions such as drought can reduce water supply to cause water
deficits that affect fruit formation and yield [11]. Drought can affect oil palm productivity
by reducing the number formed due to male and female flowering ratios [12]. Oil palm
plants are different from other palm plants that carry male and female flowers at the same
flowering; the sex determination of oil palms is determined during the flowering phase
[13]. 1 Introduction There is a time lag between the effect of environmental stress and its implications for
crop production [6, 7, 8, 14, 15]. Several factors influencing this time lag included soil
conditions and fruit development [15,11]. Previous studies state that an increase in water
deficit of 100 mm in one year can decrease production by 8-10% in the first year after that
and 3-4% in the second year [10, 6]. [8] explained in more detailed that a 100 mm water
deficit could have an impact on FFB production in the flowering phase, namely 1) floral
initiation (1-3% yield loss per 100 mm water deficit), 2) sex determination (3-4% yield
loss), 3) floral abortion (8-10% yield loss). However, the study did not mention in detail the
type of soil. Another study in Ghana showed that the higher the water deficit, the lower the
oil palm production. The study results are described as follows a) yields ranging from 22–
25 tonnes/ha FFB with an annual water deficit of 150 mm, b) 16–18 tonnes/ha FFB with an
annual water deficit of 250 mm, and c) 6–7 tonnes/ha FFB for areas with an annual water
deficit of 400 mm [16]. Based on this, a fundamental study of plant physiology on
environmental factors (water deficit) is required as a basis for drought mitigation
consideration. Thriving in tropical countries, Indonesian oil palm plantations rely heavily on rain as the
primary water source. The application of irrigation techniques is still rare in oil palm
plantations in Indonesia. Oil palm plants need sufficient water conditions, starting from the
nursery stage, maintenance, and before harvest. Understanding the climate and its effects on
oil palm growth is very important to ensure the palm oil industry's sustainability, given the
increasing need for this sector. Oil palm requires climatic conditions suitable for growth, including annual rainfall of
2000 - 3500 mm /year, monthly rainfall > 100 mm, minimum water deficit, and dry spell
[17-18]. Indonesia experienced an unfavorable climate phenomenon in 2015. The climate
anomaly phenomenon, El Nino, has reduced rainfall and increased air temperature, 2 E3S Web of Conferences 211, 05005 (2020)
The 1st JESSD Symposium 2020 https://doi.org/10.1051/e3sconf/202021105005 contributing to drought stress for oil palm crops. Drought in the tropics is closely related to
El Nino, where this climate anomaly can cause reduced or even no rain for a long time [18]. 1 Introduction Water stress will increase male flower production, which underlies the decline in oil palm
production [19, 11]. This condition is a problem for oil palm plantations, especially on
peatlands. Peat soil has irreversible drying properties where it will be difficult to get wet
again if the soil has dried for a certain period [20]. Therefore, the water deficit problem
needs to be studied so that preventive action before the critical period occurs. p
p
The definition of water deficit is the discrepancy between field capacity and the amount
of water retained in the soil at a certain period [8-9]. Water deficit in oil palm plants on
peatlands can occur due to two things, namely climate anomalies and improper water
management (implication of irreversible drying). A climate anomaly is an event that cannot
be avoided, but it can be predicted that further mitigation actions are taken. Water deficit
affects the flowering phase, especially in the sex determination and floral abortion phases
[21, 6]. If preventive efforts are carried out too late, it will impact the fruit formation
process so that the fruit can experience abortion and failure to form bunches. Oil palm
plants experience 4 phases of flowering starting from 1) floral initiation, 2) sex
determination, 3) anthesis, 4) harvest [21, 7]. The effect of stress (including drought) on the
period of plant growth is shown in Figures 3 and 4. Fig. 3. Effect of stress on bunch number in oil
palm to stress [7]. Fig. 4. Effect of eliminating stress on bunch
number in oil palm to stress [7]. This study will discuss the effect of environmental factors, namely water deficit in the
sex determination phase and floral abortion on the production of Oil Palm Fresh Fruit
Bunch. Sex determination is the period of sex formation of fruit that occurs 29 months
before harvesting, while floral abortion is the abortion of bunches, which usually occurs 9-
10 months before harvesting. Floral abortion is the phase between sex determination and
anthesis, which generally occurs 4 - 6 months before anthesis [21, 22, 23, 7, 24]. There are
at least four types of factors that may participate in sex determination, namely 1) abiotic
factors (e.g., water deficit), 2) metabolic factors (e.g., carbon stock), 3) hormone status and
4) genetic factors [25]. 1 Introduction This study aims to get a comparative analysis for the effect of water deficit on the
inflorescence period, namely the sex determination phase (29 months before harvesting) Fig. 3. Effect of stress on bunch number in oil
palm to stress [7]. Fig. 4. Effect of eliminating stress on bunch
number in oil palm to stress [7]. Fig. 3. Effect of stress on bunch number in o Fig. 3. Effect of stress on bunch number in oil
palm to stress [7]. Fig. 4. Effect of eliminating stress on bunch
number in oil palm to stress [7]. Fig. 3. Effect of stress on bunch number in oil
palm to stress [7]. Fig. 4. Effect of eliminating stress on bunch
number in oil palm to stress [7]. This study will discuss the effect of environmental factors, namely water deficit in the
sex determination phase and floral abortion on the production of Oil Palm Fresh Fruit
Bunch. Sex determination is the period of sex formation of fruit that occurs 29 months
before harvesting, while floral abortion is the abortion of bunches, which usually occurs 9-
10 months before harvesting. Floral abortion is the phase between sex determination and
anthesis, which generally occurs 4 - 6 months before anthesis [21, 22, 23, 7, 24]. There are
at least four types of factors that may participate in sex determination, namely 1) abiotic
factors (e.g., water deficit), 2) metabolic factors (e.g., carbon stock), 3) hormone status and
4) genetic factors [25]. This study aims to get a comparative analysis for the effect of water deficit on the
inflorescence period, namely the sex determination phase (29 months before harvesting)
and floral abortion (10 months before harvesting). This paper will make a unique study on
the effect of water deficit on palm oil plantations' productivity, especially peatlands. 3 E3S Web of Conferences 211, 05005 (2020)
The 1st JESSD Symposium 2020 https://doi.org/10.1051/e3sconf/202021105005 Peatland oil palm plantations were chosen because, as the world's biggest palm oil
producer, 25% of Indonesia's oil palm plantations are located on peatlands [26]. Peatland oil palm plantations were chosen because, as the world's biggest palm oil
producer, 25% of Indonesia's oil palm plantations are located on peatlands [26]. The research location was carried out on a peatland oil palm plantation in Riau
Province, Indonesia. Marginal land, such as peat, is relatively more challenging to become
cultivated land than mineral land. 2.4 Data analysis This research employs a quantitative approach with simple regression analysis on five
years' panel data and two estates for data analysis. Simple regression analysis was chosen to
find out how independent variables affect the dependent variable [28]. This study
determined the water deficit in the sex determination period and floral abortion period as
independent variables while the yield of palm oil, bunch number, and bunch weight as
dependent variables. The statistical program used for data processing is Eviews 11. 2.2 Data collection This study utilizes secondary data from private oil palm plantations for production data,
bunch number, and bunch weight for 2015-2019 and rainfall for 2012 - 2018. Rainfall data
will be used to calculate the water deficit value. 2.1 Study area The research was conducted in a private oil palm plantation in Riau Province with a cover
area is 3.109 ha and an average density of 120 stands per hectare. This research used adult
palms aged 8-15 years. This study chose adult palms because it is more susceptible to
drought. The effect of drought is relatively mild on young palms (<8 years) and old palms
(> 15 years) [27]. The type of soil in the observation estate is entirely peat. The average
annual rainfall is 2000 mm / year. 1 Introduction However, if done with proper water management,
peatlands can store water longer than mineral lands. The peatlands will probably gain in the
face of a lousy climate that causes drought. 2.3 Meteorological data Drought stress analysis is carried out by calculating water deficit, using variable climate
components, namely rainfall, rainy days, and evapotranspiration [25]. The annual water
deficits are calculated following [21] based on the method of the Institut de Recherches
pour les Huiles et Oléagineux (IRHO) with Equation 1. B = Res + R – Etp (1) B = Res + R – Etp (1) B is the balance at the end of the period, Res is the soil water reserve at the start of the
period, and R and Etp are the rainfall and potential evapotranspiration during the period. B is the balance at the end of the period, Res is the soil water reserve at the start of the
period, and R and Etp are the rainfall and potential evapotranspiration during the period. 3 Results and discussion 4 4 https://doi.org/10.1051/e3sconf/202021105005 E3S Web of Conferences 211, 05005 (2020)
The 1st JESSD Symposium 2020 This research analyzes the correlation between water deficit and crop production for adult
palm, including the two constituent components, namely the bunch number and average
bunch weight in the efflorescence phase. The primary oil palm production components are
bunch number, bunch weight, and oil content [29]. The environmental conditions strongly
influence bunch numbers during inflorescence, namely sex determination and floral
abortion. Bunch weight is affected by the number of fruits formed from the female flowers
produced during the inflorescence phase [29]. The flowering period in mature palms
generally occurs around 2–3 years [21, 7, 8, 11]. This research highlights two main phases
that affect the success of inflorescence, namely sex determination and floral abortion. Sex
determination is the most critical process in the development of the oil palm. The period is
determined 29 months before harvesting, while the floral abortion is ten months before
harvesting [21, 23]. In this paper, water deficit calculation using the IRHO method is carried out per month
by considering the rainfall, rainy days, and evapotranspiration per month. Following the
IRHO method [21], water balance is the sum of monthly rainfall plus water reserves at the
beginning of the month, minus evapotranspiration per month. At the beginning of the
month, the water reserve comes from the previous month's water reserve or a maximum
200 mm value. Evapotranspiration is assumed to be 150 mm/month if the rainy day is <10
days per month or 120 mm / month if the rainy day is> 10 days/month. The water balance
value shows the availability of water per month. If the water balance is <0 mm, there is a
water deficit; if the water balance is more than 200 mm, the excess water will be stored by
the soil and used for the following month (the maximum value of water reserve and held by
the ground is 200 mm). This study analyzed secondary data on two private oil palm plantations located in Riau
Province and calculates the water deficit value. The calculation results are shown in Figure
5 while simultaneously displaying rainfall and plant productivity to determine their
harmony. Plant productivity in January 2015 is compared with rain and water deficit in the
sex determination phase (29 months before harvesting) or in August 2012 and so on the
following month. E3S Web of Conferences 211, 05005 (2020)
The 1st JESSD Symposium 2020 Figure 5 and Figure 6 show the relationship between rainfall, water deficit, and plant
productivity. Oil palm plants' productivity will increase if accompanied by sufficient water
conditions through adequate rain for the plants, so that a minimum water deficit. Assessment of water adequacy in oil palm plants through water deficit calculations shows
that the distribution of monthly precipitation over a year is essential in the water balance of
oil palm plants [30]. Oil palm, an originate tropical forest plant in West Africa, requires
evenly distributed rainfall per month (there is no dry month, namely precipitation <100 mm
per month) [31]. Fig. 6. The pattern of production distribution of palm oil with precipitation and water deficit in the
floral abortion phase (Source: Author’s Analysis, 2020). Fig. 6. The pattern of production distribution of palm oil with precipitation and water deficit in the
floral abortion phase (Source: Author’s Analysis, 2020). Fig. 6. The pattern of production distribution of palm oil with precipitation and water deficit in the
floral abortion phase (Source: Author’s Analysis, 2020). The productivity of oil palm plants is determined by the number of female flowers in the
flowering phase. To determine the effect in detail, this research conducted a statistical test
by correlating the relationship between water deficit in two critical stages of flowering to
three components: plant productivity, bunch number, and bunch weight. The analysis
results are shown in Table 1. Table 1. Relationship of the effect of water deficit on the inflorescence period. Table 1. Relationship of the effect of water deficit on the inflorescence period. Variable
Water Deficit on The Inflorescence Period
Sex determination
Floral Abortion
c
coefficient
c
coefficient
Yield (ton/ha/annum)
14,27
- 0.008074*
13,93
- 0.009184*
Bunch Number
(bunch/plant)
11,85
- 0.008783*
11,17
- 0.004030ns
Bunch Weight
(kg/bunch)
9,59
+ 0.006067*
10,26
- 0.001753ns
Significance level of effects: n.s., not significant; * significant (p < 10%). Water Deficit on The Inflorescence Per Significance level of effects: n.s., not significant; * significant (p < 10%). 3 Results and discussion Meanwhile, the analysis results in the floral abortion phase (10 months
before harvesting) are shown in Figure 6. In the floral abortion period, productivity in
January 2015 is compared to rainfall and water deficit in March 2014, and continue in the
following months. Fig. 5. The pattern of productivity distribution of palm oil with precipitation and water deficit in the
sex determination phase (Source: Author’s Analysis, 2020). Fig. 5. The pattern of productivity distribution of palm oil with precipitation and water deficit in the
sex determination phase (Source: Author’s Analysis, 2020). 5 5 E3S Web of Conferences 211, 05005 (2020)
The 1st JESSD Symposium 2020 https://doi.org/10.1051/e3sconf/202021105005 3.1 Water deficit and palm oil production Based on the analysis result (Table 1), water deficit in sex determination and floral abortion
phases significantly affected plant yield (p <10%). The floral abortion phase's coefficient
value is greater than that of the sex determination phase, which means that the floral 6 6 E3S Web of Conferences 211, 05005 (2020)
The 1st JESSD Symposium 2020 https://doi.org/10.1051/e3sconf/202021105005 abortion phase's effect will have a more significant impact than that of the sex
determination phase. Water deficit in the floral abortion phase will reduce productivity by
7% for every 100 mm increase in water deficit. In comparison, the water deficit in the sex
determination phase will reduce productivity by 6% for every 100 mm increase in water
deficit. abortion phase's effect will have a more significant impact than that of the sex
determination phase. Water deficit in the floral abortion phase will reduce productivity by
7% for every 100 mm increase in water deficit. In comparison, the water deficit in the sex
determination phase will reduce productivity by 6% for every 100 mm increase in water
deficit. Water deficit can reduce oil palm productivity through low sex ratio and floral abortion
[21]. The higher the sex ratio, the more female flowers. The low sex ratio occurs because
the sex determination period is disturbed so that more male flowers are formed than female
flowers, which will develop into a fruit. However, a high sex ratio does not fully guarantee
high productivity [32]. This case is due to the possibility of abortion of female flowers and
the failure of bunches. Flower abortion occurs due to a lack of carbohydrates for flower
development, water deficit, and over-pruning to stress the plant [21, 32]. Based on the
research results, the decrease in productivity in the floral abortion phase in peatlands is
smaller than the previous research results (without specifying the type of land), which states
that productivity decreases by 10% [6]. This research presume that the decline in oil palm
productivity on peatlands (with proper water management) due to water deficit has a
smaller value than mineral soils. Because, with proper water management, peatlands can
store more water than mineral lands. This assumption is in line with research from [13],
which states that the abortion of oil palm bunches is much smaller (2-13%) on deep
peatlands in Malaysia with proper water management. 3.2 Water deficit and bunch number Bunch number dramatically affects the production of oil palm plants. Previous studies
stated that the bunch number was more correlated with crop production than weight [33]. Therefore, the critical point in the bunch formation period needs to be considered to reduce
yield losses. The number of a bunch per stand is primarily influenced by sex determination
during the development of the inflorescences. That process occurs about 29 months before
the maturity of fruit in mature palms [21, 11]. Previous researchers stated that the variation
in the formation of the bunch number is determined by three mechanisms: changes in the
rate of development of flowers, variations in the sex of flowering, and abortion flowering in
the flowering period [33]. [13] adding another factor that also influences, namely, bunches
failure between anthesis and harvest. The results of the analysis in Table 1 show that the water deficit has significant
implications (p < 10%) for the variable bunch weight in the sex determination phase. In
contrast, in the floral abortion phase, the effect is not significant. The sex determination
phase determines the sex of flowers in which sufficient water conditions will tend to form
more female flowers than male flowers. This female flower will then develop into a fruit to
be harvested. Therefore, the sex determination phase influences the amount of fruit that will
be produced by plants. 3.3 Water deficit and bunch weight Several components contribute to the formation of mature oil palm bunches, including the
number of spikelets, the number of flowers per spikelet, the percentage fruit set, the mean
weight per fruit, and the frame [8]. Among these components, the number of spikelets and
the number of flowers per spikelet are formed in the flowering phase, strongly influenced
by water availability. The statistical analysis in Table 1 shows that the water deficit has only significant
implications in the sex determination phase. Water deficit affects the increase in bunch
weight, but only with a low percentage of 6%. 4 Conclusion Water deficit in the inflorescence period affects decreasing oil palm production on
peatlands. An increase in water deficit by 100 mm in the sex determination phase will
reduce productivity by 6% while in the floral abortion phase, decreasing 7%. Knowledge of
the impact of water deficit on the efflorescence phase is useful for knowing the oil palm
production pattern to predict future production. Besides, planters can also take mitigation
measures to decline output is not too significant. Such mitigation measures are, for
instance, by optimizing fertilization to avoid nutrient imbalances and also by maintaining
soil moisture with cover crop plants. This paper contributes to the study of oil palm
plantation management by providing evidence of the effect of water deficits in the
inflorescence period at oil palm productivity on peatlands. For oil palm plantations on
peatlands, drought mitigation can be pursued with proper water management. This researh
indicate that the water deficit's effect on oil palm plantations on peatlands with appropriate
water management is not as significant as on oil palm plantations on mineral lands. Therefore, it is necessary to carry out further research to be a comparison with this research. Another thing that also needs to be studied in further research is the water deficit on young
palms and old palms to compare adult palms' impact. 7 7 E3S Web of Conferences 211, 05005 (2020)
The 1st JESSD Symposium 2020 https://doi.org/10.1051/e3sconf/202021105005 3.4. Production projection based on precipitation and water deficit Physiologically, oil palm plants need adequate water conditions to support their growth. Knowledge of this critical point of drought stress is required to determine the pattern of oil
palm production so that projections can be made for the next year and mitigation measures
to minimize the percentage decline in production. The production-distribution pattern is essential by planters to determine the production
target in the next production year. Planters must try to take mitigation measures, for
instance, by applying optimal fertilizers and maintaining soil moisture with ground cover
crops to minimize stress to plants. Knowledge of water deficit can also be used as a
consideration for irrigation in plantations. However, the application of this irrigation
technology is not yet famous for oil palm plantations in Indonesia. Empirical data on
Thailand Oil palm Plantations states that daily drip irrigation during the dry season can
increase productivity by up to 50% [34]. Acknowledgements This
study
was
supported
by
Universitas
Indonesia,
with
grant
number
NKB
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https://www doi org/10 1093/aob/mcp214 30. W. Darmosarkoro, I. . Harahap, E. Syamsuddin, Technical culture of oil palm plants in
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(2010) (
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Petdeset let mednarodnega identifikatorja ISBN
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Izvleček Predstavljen je 50-letni razvoj mednarodnega standarda ISO 2108, ki vključuje na-
tančen opis strukture mednarodnega identifikatorja ISBN, njegov namen in rabo ter
poudari njegove prednosti. Pomembno je, da je to enkraten in stalen identifikator za
monografske publikacije v vseh njihovih različicah. Omenjeni so sorodni identifikator-
ji, predstavljen je tudi nastanek in delovanje Slovenske agencije za ISBN. Analizirana
je podatkovna zbirka slovenskih založnikov, ki lahko postane zanimiv vir za razisko-
valce založništva v Sloveniji. Ključne besede: ISO 2108, ISBN, metapodatki, Slovenska agencija za ISBN, podatkovna
zbirka slovenskih založnikov, zgodovinski pregledi Keywords: ISO 2108, ISBN, metadata, Slovenian ISBN Agency, Slovenian Publishers’ Da-
tabase, historical reviews Fifty years of the international identifier ISBN Alenka Kanič Oddano: 27. 7. 2017 – Sprejeto: 11. 9. 2017 Oddano: 27. 7. 2017 – Sprejeto: 11. 9. 2017 1.04 Strokovni članek
1.04 Professional article
UDK 655.535.28(091) 1 Uvod Mednarodna standardna knjižna številka je praznovala petdesetletnico svojega
obstoja. Pravzaprav je težko reči, kdaj se je številka v resnici rodila, saj se razvoj
takega mednarodnega identifikatorja ne zgodi od danes do jutri. Identifikator
sprva ni bil mišljen kot mednarodni identifikator, njegovi začetki segajo daleč
v leto 1965, ko je lastnik največje verige papirnic in knjigarn v Veliki Britaniji
W. H. Smith oznanil, da bo z letom 1967 v svojih poslovalnicah prešel na raču-
nalniško poslovanje. V te namene je predlagal uporabo sistema standardnega
številčenja knjig. K sodelovanju je povabil Gordona Fosterja (1921–2010), irskega
inženirja računalništva, statistika in profesorja na London School of Economics
ter častnega profesorja na Trinitiy College v Dublinu, bivšega vojnega veterana
in strokovnjaka za dešifriranje, da bi s skupino Smithovih strokovnjakov razvil
sistem enotnega označevanja knjig (Gordon Foster, 2017). Vendar je bila zami-
sel o označevanju knjig zanimiva tudi za širšo mednarodno skupnost in o tem
so razpravljali leta 1966 v Berlinu na tretji mednarodni konferenci o raziskavah
knjižnega trga in racionalizaciji v knjigotrštvu. Tam so ugotovili, da je prvi pogoj
za učinkovit avtomatiziran sistem lahko le preprosta in enkratna identifikacij-
ska oznaka za izdano enoto. Tudi v Združenih državah Amerike so razmišljali
podobno, govorili so o narodnem in celo mednarodnem standardu za potrebe
številčenja knjig (ISBN users’ manual, 2001; Kanič, 2017). Abstract The development of the international standard ISO 2108 during the past 50 years is
presented, as well as the detailed structure of the international identifier and its scope. The usage is described and explained, stressing its advantages and benefits. Particular
importance is paid to the fact that it has to be the unique and persistant identifier of
a monographic publication for any of its forms and manifestations, and some related
international identifiers are discussed in this respect. The origin and history of the
Slovenian National ISBN Agency are presented, its tasks and activities, including an
analysis of the Slovenian Publishers’ Directory database which proves to be a good
source for further research in the field of publishing in Slovenia. 199 Alenka Kanič 2 SBN – standardna knjižna številka Med tem časom je v Veliki Britaniji profesor Foster s svojo ekipo razvijal svoj sis-
tem in je, kot je bilo predvideno, v letu 1967 podal svoje končno poročilo. Njegov
sistem je temeljil na nizu devetih števk, ki so označevale založnika in naslov knji-
ge ter so zajemale še kontrolno številko. Celoten niz so poimenovali standardna
knjižna številka (SBN) (Gordon Foster, 2017; ISBN, 2017). Primer standardne knjižne številke: SBN 582-10154-9 Primer standardne knjižne številke: SBN 582-10154-9 V Združenem kraljestvu so bili navdušeni in so sistem takoj sprejeli. To 9-mestno
oznako so uporabljali celo do leta 1974, ko je bila že dalj časa v rabi tudi 10-mest
na oznaka. Prva agencija za SBN je začela delovati pod okriljem založniškega
podjetja J. Whitaker and Sons v Londonu. Navdušenje nad sistemom je bilo nale-
zljivo in vključila se je Mednarodna organizacija za standarde oziroma njen komi-
te TC 46 (Technical Committee 46), ki je najprej razpravljal o britanskem sistemu
in nato ustanovil delovno skupino, katere vodstvo je zaupal Davidu Whitakerju,
ki danes velja za očeta tega identifikatorja. Zaradi večjega števila sodelujočih 200 Knjižnica, 2017, 61(3), 199–211 Petdeset let mednarodnega identifikatorja ISBN institucij tak mednarodni projekt seveda teče malce počasneje, zato sta se bri-
tanski shemi SBN že v letih 1968–1969 pridružili najprej ZDA in Kanada, sledili
sta še Avstralija in Nova Zelandija, torej angleško govoreče države, nato pa so se
jim pridružile še Francija, Danska in Švedska (ISBN Timeline, 2015). To torej ni
bil več samo angloameriški sistem, zato se je ISO/TC 46 odločil, da iz 9-mestnega
niza števk nastane zmogljivejša 10-mestna oznaka. Identifikacijskima oznakama
za založnika in naslov knjige ter kontrolni številki so na začetku dodali še oznako
za državo. Treba je bilo spremeniti tudi ime. Standardna knjižna številka (SBN)
postane mednarodna standardna knjižna številka (ISBN) in od takrat ne spremi-
nja več svojega imena. Posodablja se le še izrazoslovje v zvezi s standardom in
identifikatorjem. Knjižnica, 2017, 61(3), 199–211 3 Razvoj standarda ISBN in drugih sorodnih standardov v
povezavi s knjižničnim gradivom Vsako delo Mednarodne organizacije za standarde in njenih komitejev se zaključi
z objavo mednarodnega standarda. Prva izdaja mednarodnega standarda ISO
2108, ki pokriva področje mednarodne standardne knjižne številke, je bila ob-
javljena leta 1970, od tedaj pa so sledile še štiri izdaje. Najbolj revolucionarna je
bila četrta izdaja iz leta 2005, ko se je 10-mestna oznaka spremenila v 13-mestno
in je obveljala pomembna odločitev, da je s tem identifikatorjem treba označevati
tudi elektronske knjige. V letu 2016 je bila nato pripravljena že peta posodoblje-
na izdaja standarda, ki podrobneje opredeljuje številčenje elektronskih knjig in
pojasnjuje dileme v zvezi z njimi. S prvo izdajo mednarodnega standarda je bila za njegovo nemoteno in pravilno
delovanje ustanovljena Mednarodna agencija za ISBN s sedežem v Berlinu. Po
letih uspešnega delovanja s pomočjo finančnih sredstev Pruske kulturne funda-
cije se je sedež agencije leta 2005 iz Berlina preselil v London, za članice siste-
ma ISBN pa je bila uvedena letna članarina. Delo Mednarodne agencije za ISBN
danes nadzira izvoljeni svet direktorjev, v prvih letih delovanja pa so to funkcijo
opravljali prvopristopniki. To so bili profesorja Hans-Jürgen Ehlers in Robert Mül-
ler iz Nemčije, Emery Koltay in kasneje Michael Cairns iz ZDA ter že omenjeni
David Whitaker. Vse te strokovnjake smo gostili v Ljubljani leta 1996, ko smo
organizirali srečanje vodij nacionalnih agencij. Ti se namreč vsako leto srečujejo,
izmenjujejo izkušnje in načrtujejo razvoj tega za knjigo pomembnega identifika-
torja, ki ga sprejemajo tudi vsi knjižnični sistemi po svetu. Danes oznako ISBN
uporabljajo v 200 državah in pokrajinah, pod vodstvom Mednarodne agencije za
ISBN pa deluje že 151 nacionalnih agencij. 201 Knjižnica, 2017, 61(3), 199–211 Alenka Kanič Po svetu so nacionalne agencije organizirane na različne načine. V državah z ve-
likim knjižnim trgom praviloma delujejo samostojno ali v okviru različnih zdru-
ženj, ki se tako ali drugače vsa ukvarjajo s knjigo (npr. Bowker v ZDA, MVB Marke-
ting- und Verlagsservice des Buchhandels v Nemčiji, AFNIL Agence Francophone
pour la Numérotation Internationale du Livre v Franciji), v manjših državah z
manjšim knjižnim trgom pa so agencije praviloma del nacionalnih knjižnic (npr. Národni knihovna České republiky na Češkem ali pa Bibliothèque Nationale de
Tunisie v Tuniziji, pa tudi pri nas). Glavne naloge delovanja Mednarodne agencije
za ISBN, pa tudi nacionalnih agencij za ISBN, določa že standard in z vsako novo
izdajo so te naloge bolj trdne in zavezujoče. 3 Razvoj standarda ISBN in drugih sorodnih standardov v
povezavi s knjižničnim gradivom Mednarodnemu standardu za številčenje knjig se je leta 1975 pridružil standard
ISO 3297 (ISSN – International Standard Serial Number) za označevanje serijskih
publikacij in leta 1993 še standard ISO 10957 (ISMN – International Standard
Music Number), ki velja za glasbene tiske, sicer pa v knjižnicah in knjigotrštvu
poznamo še vrsto drugih mednarodnih identifikatorjev: poznamo še vrsto drugih mednarodnih identifikatorjev: poznamo še vrsto drugih mednarodnih identifikatorjev: –
– ISTC – International Standard Text Code (ISO 21047); –
– ISWC – International Standard Work Code (ISO 15707); –
– ISNI – International Standard Name Identifier (ISO 27729); –
– DOI – Digital Object Identifier (ISO 26324); –
– ISAN – International Standard Audivisual Number (ISO 15706:2002 in ISO
15706-2); –
– ISRC – International Standard Recording Code (ISO 3901). 4.1 ISBN in črtna koda EAN Leta 1980 je hitro in vsesplošno razširjeno računalniško branje črtnih kod prived
lo do dogovora in sklenitve pogodbe o medsebojnem sodelovanju med organi-
zacijama EAN (European Article Numbering) in UCC (Uniform Code Council), ki
sta predhodnika GS1 (Global Standard 1), na eni strani ter Mednarodno agencijo
za ISBN na drugi strani. Pri pretvorbi oznake ISBN je bila pred celotni niz desetih
števk dodana še oznaka za t. i. »deželo knjig« (angl. bookland) 978- oziroma 977-
in 979-, ki jih je velikodušno odstopil GS1. Kontrolna številka se je s tem začela
preračunavati na novo. S pretvorbo oznake ISBN v črtno kodo EAN se je tako
povečal pomen sistema ISBN kot mednarodnega sistema identifikacije znotraj
vsesplošne sheme črtnih kod, ki jih nadzoruje GS1. Sodelovanje z GS1 se je leta
2007 še poglobilo, ko je stopila v veljavo četrta izdaja standarda ISO 2108, kjer
je oznaka ISBN dosegla popolno ujemanje z GTIN (Global Trade Item Number). 202 Knjižnica, 2017, 61(3), 199–211 Petdeset let mednarodnega identifikatorja ISBN Predstavnik GS1 je član sveta direktorjev agencije. Ob tem velja poudariti, da je
bila knjiga prepoznana kot kulturna dobrina, zato je tudi GS1 ob strogem upo-
števanju uporabe oznak ISBN te tri oznake za »deželo knjig« velikodušno, brez
stroškov, odstopil v ta namen. Za enkrat imamo na voljo tri predpone 977, 978 in
979, ki bodo zadostovale za kar precej časa. Predpona 977 je namenjena ISSN-ju,
predpona 978 ISBN-ju in predpona 979-0 ISMN-ju, preostanek predpone 979 pa
ponovno ISBN-ju. Ko bodo izrabljene vse možnosti v okviru teh treh predpon, bo
treba z GS1 ponovno sesti za pogajalsko mizo. Predstavnik GS1 je član sveta direktorjev agencije. Ob tem velja poudariti, da je
bila knjiga prepoznana kot kulturna dobrina, zato je tudi GS1 ob strogem upo-
števanju uporabe oznak ISBN te tri oznake za »deželo knjig« velikodušno, brez
stroškov, odstopil v ta namen. Za enkrat imamo na voljo tri predpone 977, 978 in
979, ki bodo zadostovale za kar precej časa. Predpona 977 je namenjena ISSN-ju,
predpona 978 ISBN-ju in predpona 979-0 ISMN-ju, preostanek predpone 979 pa
ponovno ISBN-ju. Ko bodo izrabljene vse možnosti v okviru teh treh predpon, bo
treba z GS1 ponovno sesti za pogajalsko mizo. 4.2 Struktura oznake ISBN po ISO 2108:2005(E) Danes je ISBN niz 13 števk, ki so razdeljene na pet elementov, od teh so trije ele-
menti spremenljive dolžine; le prvi in zadnji element sta stalne dolžine: –
– element s predpono GS1 (»dežela knjig«); –
– element registracijske skupine (oznaka države); –
– element registranta (oznaka založnika); –
– element publikacije (oznaka naslova); –
– kontrolna številka. –
– kontrolna številka. Niz vedno uvaja akronim ISBN, v deželah, kjer ne uporabljajo latinične pisave, se
doda še ISBN v pisavi, ki je tam v uradni rabi. Zaradi prijaznosti do uporabnika so oznake ISBN zapisane z vezaji ali presledki. Npr.: ISBN 978-961-282-287-3 ali ISBN 978 961 282 287 3 Velja priporočilo, da založnik ISBN natisne na hrbet naslovne strani ali, če tam ni
prostora, na dno naslovne strani, pa tudi na spodnji del zadnje platnice skupaj
s črtno kodo in seveda na spodnji del ščitnega ovitka ali druge zunanje zaščite. ISBN 978-961-282-287-3
Slika 1: ISBN in črtna koda ISBN 978-961-282-287-3
Slika 1: ISBN in črtna koda Slika 1: ISBN in črtna koda Tudi pri elektronskih publikacijah se mora oznaka ISBN pojaviti na več različ-
nih mestih: na naslovnem zaslonu, na prvem prikazanem zaslonu (laserski disk, 203 Knjižnica, 2017, 61(3), 199–211 Alenka Kanič elektronske publikacije) ali na zaslonu, ki prikazuje naslov ali njegovo nadome-
stilo. elektronske publikacije) ali na zaslonu, ki prikazuje naslov ali njegovo nadome-
stilo. Pri vzgojnih in/ali izobraževalnih filmih, videoposnetkih in prosojnicah se mora
oznaka ISBN pojaviti v najavni/odjavni špici in na embalaži, ki je sestavni del
publikacije (npr. laserski disk, kaseta ali disketa). 4.3 Raba oznake ISBN po ISO 2108:2005(E) besedilna dela in druge ab-
straktne stvaritve intelektualne in/ali umetniške vsebine z besedilom, ki do-
bijo oznako ISTC ali ISWC za glasbena dela; 204 Knjižnica, 2017, 61(3), 199–211 Petdeset let mednarodnega identifikatorja ISBN –
– tiskano efemerno gradivo brez večje vrednosti in trajnosti, npr. propagandno
gradivo in podobno; –
– glasbeni tiski – zanje velja oznaka ISMN; –
– grafični listi in grafične mape brez naslovne strani in besedila; –
– osebni dokumenti (npr. elektronski življenjepisi ali osebne biografije);
–
– voščilnice; –
– glasbeni zvočni posnetki – oznaka ISRC velja le za zvočne posnetke, ne pa tudi
za njihove nosilce; –
– personalizirane knjige, spominski albumi in rokovniki; –
– programska oprema za kateri koli namen razen za vzgojo in izobraževanje;
–
– elektronske oglasne deske; –
– programska oprema za kateri koli namen razen za vzgojo –
– elektronska pošta in druga elektronska korespondenca; Založniki pa so izredno inventivni in vedno znova izumljajo nove proizvode, zato
so nacionalne agencije neprestano v stikih med sabo in z Mednarodno agencijo za
ISBN v Londonu ter se neprestano posvetujejo o rešitvah in usklajujejo mnenja. 4.3 Raba oznake ISBN po ISO 2108:2005(E) Z oznako ISBN so označene monografske, vsebinsko zaključene publikacije z
besedilom, pa tudi s slikami in ilustracijami. Po ISO 2108:2005(E) (2005) se te
seveda pojavljajo v najrazličnejših oblikah, najpogosteje kot: –
– tiskane knjige in brošure ter njihove druge pojavne oblike; –
– publikacije v Braillovi pisavi; –
– publikacije v Braillovi pisavi; –
– publikacije, ki jih založnik ne namerava redno dopolnjevati ali neomejeno
nadaljevati njihovega izhajanja; –
– zvočne knjige na fizičnih nosilcih ali na spletu; –
– elektronske knjige na fizičnih nosilcih ali na spletu (za prenos (angl. down-
loading) ali pretakanje (angl. streaming)); –
– elektronske knjige na fizičnih nosilcih ali na
loading) ali pretakanje (angl. streaming)); –
– digitalizirane kopije tiskanih monografskih publikacij, če so namenjene trgu;
–
– publikacije na mikrooblikah; –
– publikacije na mikrooblikah; –
– vzgojni in didaktični filmi, videoposnetki in prosojnice v vseh pojavnih obli-
kah; –
– vzgojna ali didaktična programska oprema; –
– publikacije na različnih medijih (kjer je besedilo poglavitna sestavina); –
– posamezni članki ali posamezne številke določenega kontinuiranega vira
(toda ne kontinuirani vir v celoti), če je te mogoče posebej nabaviti; –
– knjige s področja umetnosti in knjige z ilustracijami, ki imajo naslovno stran
in besedilo ob slikah; –
– vse, kar lahko obravnavamo kot zaključeno publikacijo, kjer večino predstavlja
besedilo in je namenjeno javnosti, vključuje lahko tudi gibljive slike in zvok;
–
– zemljevidi. –
– vse, kar lahko obravnavamo kot zaključeno publikacijo, kjer večino predstavlja
besedilo in je namenjeno javnosti, vključuje lahko tudi gibljive slike in zvok; –
– zemljevidi. Ob tem seveda obstaja tudi seznam založniških proizvodov, ki niso upravičene
do oznake ISBN, pač pa uporabljajo katerega od drugih mednarodnih identifika-
torjev. Ta gradiva so: Ob tem seveda obstaja tudi seznam založniških proizvodov, ki niso upravičene
do oznake ISBN, pač pa uporabljajo katerega od drugih mednarodnih identifika-
torjev. Ta gradiva so: –
– kontinuirani viri, obravnavani v celoti kot bibliografska entiteta (posamezni
deli imajo lahko oznake ISBN) – tem je namenjena oznaka ISSN; –
– intelektualna dela kot abstraktne entitete, npr. besedilna dela in druge ab-
straktne stvaritve intelektualne in/ali umetniške vsebine z besedilom, ki do-
bijo oznako ISTC ali ISWC za glasbena dela; –
– intelektualna dela kot abstraktne entitete, npr. Knjižnica, 2017, 61(3), 199–211 4.4 Prednosti oznak ISBN Tudi v knjižnicah se je ISBN odlično izkazal za: Tudi v knjižnicah se je ISBN odlično izkazal za: –
– naročila; –
– naročila; –
– prevzemanje podatkov za lastne kataloge; –
– pripravo tematskih seznamov; –
– statistične podatke o izposoji; –
– določanje nacionalne politike pravice izposojanja in avtorskih nadomes –
– medbibliotečno izposojo; –
– medbibliotečno izposojo; –
– centralne kataloge. 4.4 Prednosti oznak ISBN Prednosti oznake ISBN so na različnih področjih neizmerne, saj ISBN enkratno
in nezamenljivo identificira vsak naslov v njegovih različnih pojavnih oblikah
(izdelkih) pri enem samem založniku ali isti naslov pri različnih založnikih. ISBN
in drugi identifikacijski sistemi, ki so povezani s knjižničnim gradivom, so zlasti
uporabni v založniških hišah, distribucijskih centrih, pri prodaji na debelo in
na drobno, v centraliziranih servisih za knjižnice, v knjigarnah in knjižnicah. Prav zaradi tega, ker ISBN identificira gradivo, ki se v knjižnicah ohranja za vse
večne čase, ISBN označuje en sam izdelek in ta ISBN se ne sme nikoli porabiti v
povezavi s katerim koli drugim izdelkom. Nekatere rabe se pri vseh uporabnikih
sistema prekrivajo, nekatere pa so značilne le za določene dejavnosti. Priročnik
za uporabnike Mednarodne standardne knjižne številke (2006) natančno navaja,
kje in zakaj se oznake ISBN uporabljajo: V založniških hišah lahko ISBN uporabljajo zlasti za: V založniških hišah lahko ISBN uporabljajo zlasti za: –
– zanesljivo identifikacijo dela od rokopisa do tiskarja; –
– identifikacijo naslova v založniških katalogih in njihovih najavah; –
– navajanje v tiskanih ali elektronskih seznamih; –
– navajanje v tiskanih ali elektronskih seznamih;
–
– kontrolo zaloge; –
– delno upravljanje in nadzor avtorskih pravic in nadomestil zanje;
–
– izvajaje naročil; –
– računovodsko in blagajniško poslovanje; 205 Knjižnica, 2017, 61(3), 199–211 Alenka Kanič –
– nadzor prodaje; –
– vodenje statističnih podatkov; –
– nadzor vračil. V distribucijskih centrih in centraliziranih servisih za knjižnice ima ISBN podob-
no nalogo in se uporablja za: no nalogo in se uporablja za: –
– gradnjo bibliografskih podatkovnih zbirk; –
– vodenje podatkov o razpoložljivih naslovih; –
– elektronska naročila pri založnikih in posrednikih; –
– nadzor zaloge; –
– nadzor zaloge; –
– nadzor notranje logistike; –
– računovodsko in blagajniško poslovanje; –
– nadzor prodaje; –
– upravljanje z vračili; –
– izdelavo tematskih seznamov in katalogov. V knjigarnah ISBN služi za: V knjigarnah ISBN služi za: –
– bibliografske poizvedbe; –
– zasledovanje naslovov; –
– elektronska naročila in prednaročila; –
– upravljanje z zalogami; –
– računovodsko in blagajniško poslovanje s končnim uporabnikom; –
– računovodsko in blagajniško poslovanje s končnim uporabnikom;
– elektronsko prodajo – računovodsko in blagajniško poslovanje s končnim uporabnikom;
–
– elektronsko prodajo. –
– elektronsko prodajo. –
– elektronsko prodajo. 4.5 Metapodatki Oznaka ISBN pa ni sama sebi namen. Za oznako vedno stoji izdelek, ki ga na-
tančneje opredeljujejo določeni podatki. Poleg nekaterih osnovnih bibliografskih
podatkov, kot so naslov in avtorji, podatek o izdaji, založniku in letu izida ter
podatek o knjižni zbirki, so to še pojavna oblika izdelka (tiskana ali elektronska
oblika ter njegov fizični obseg), podatek o jeziku, državi izida, imprintu, natanč-
nem datumu izida, ISBN-ju izdelka samega in ISBN-ju s to publikacijo povezane 206 Knjižnica, 2017, 61(3), 199–211 Petdeset let mednarodnega identifikatorja ISBN druge sorodne publikacije, kot so naslovi v več zvezkih, naslovi z dodatki v obliki
rešitev vaj ali priloženega zvočnega posnetka. Agencija te podatke zbira sama
ali pa zbiranje zaupa za to delo usposobljeni ustanovi, npr. nacionalni knjižnici
(Priročnik za uporabnike mednarodne standardne knjižne številke, 2014). druge sorodne publikacije, kot so naslovi v več zvezkih, naslovi z dodatki v obliki
rešitev vaj ali priloženega zvočnega posnetka. Agencija te podatke zbira sama
ali pa zbiranje zaupa za to delo usposobljeni ustanovi, npr. nacionalni knjižnici
(Priročnik za uporabnike mednarodne standardne knjižne številke, 2014). 5 Kako pa pri nas? Leta 1988 je k sistemu pristopilo večje število držav, med njimi tudi tedanja Jugosla
vija, vlogo nacionalne agencije za ISBN pa je prevzel Jugoslovanski bibliografski
inštitut, ki je več ali manj uspešno opravljal to nalogo vse do razpada Jugoslavije. Tedaj je na pobudo slovenskih založnikov Narodna in univerzitetna knjižnica v
Ljubljani pri Mednarodni agenciji za ISBN in Centru ISSN s sedežem v Parizu spro-
žila postopek za ustanovitev slovenske nacionalne agencije za ISBN in slovenske-
ga centra za ISSN. Mednarodna agencija za ISBN, tedaj s sedežem v Berlinu, nas
je seznanila s pogoji sodelovanja, posredovati smo morali podatke o letni knjižni
produkciji, pripraviti seznam založnikov. Vse smo začeli znova, uporabili smo po-
datke iz Slovenske bibliografije, saj iz Beograda nismo dobili nič. Lastni identifi-
kator smo dobili maja 1992. Za ustanovitev nacionalne agencije za ISBN je moralo
Ministrstvo za kulturo kot naš glavni financer dati soglasje in zagotoviti finančna
sredstva za enega zaposlenega sodelavca, medtem ko je morala ustanovitev cen-
tra za ISSN podpreti vlada. Slovensko agencijo za ISBN smo dobili z majem 1992,
nekaj kasneje pa še Center za ISSN. Obema se je, tokrat na pobudo založnikov
glasbenih tiskov, v letu 1999 pridružila še agencija za ISMN. Danes vse tri agencije
složno delujejo pod okriljem Narodne in univerzitetne knjižnice v Ljubljani. Knjižnica, 2017, 61(3), 199–211 5.1 Naloge nacionalne agencije za ISBN Naloge vsake nacionalne agencije za ISBN, ki jih delno predpisuje že tudi stan-
dard ISO 2108, so po Priročniku za uporabnike mednarodne standardne številke
(2014, str. 21) naslednje: –
– zagotoviti nemoteno nudenje storitev vsem uporabnikom; –
– izdajati registrantu monografske publikacije ali založniku izpiske o dodelitvi
oznake ISBN ali oznake registranta; –
– izgrajevati, upravljati in vzdrževati registre oznak ISBN, metapodatkov ISBN
in upravnih podatkov v skladu z usmeritvami, ki jih je določila Mednarodna
agencija za ISBN; naloge v zvezi z metapodatki ISBN so lahko zaupane določe-
ni bibliografski agenciji, vendar tako, da so v skladu s temi določili; –
– popravljati netočne oznake ISBN in metapodatke ISBN, če obstajajo dokazi o
nedoslednostih ali netočnostih; 207 Knjižnica, 2017, 61(3), 199–211 Alenka Kanič –
– nuditi podatke o oznakah ISBN in z njimi povezanih metapodatkih drugim re-
gistracijskim agencijam in uporabnikom sistema ISBN v skladu z usmeritvami,
ki jih je določila Mednarodna agencija; –
– zbirati in vzdrževati statistične podatke o svojih dejavnostih v zvezi z ISBN in
o tem v dogovorjenih intervalih poročati Mednarodni agenciji za ISBN; –
– spodbujati, izobraževati in usposabljati druge deležnike pri uporabi sistema
ISBN v skladu z določili veljavne izdaje standarda ISBN; –
– slediti usmeritvam ISBN in postopkom, ki jih je postavila Mednarodna agencija
za ISBN v skladu z določili veljavne izdaje standarda ISBN. 8
Knjižnica, 2017, 61(3), 199–211 5.2 Podatkovna zbirka slovenskih založnikov Danes zajema podatkovna zbirka slovenskih založnikov 4103 članov (podatek
z dne 30. junija 2017). Njihova struktura je zelo pisana, od tistih, za katere je
to osnovna gospodarska dejavnost (467), do tistih, ki izdajajo samo občasno,
predvsem kot samozaložniki. Založniki, za katere je to gospodarska dejavnost, se
združujejo v Zbornici slovenskih knjižnih založnikov in knjigotržcev in Društvu
slovenskih založnikov. Za knjižničarje pa je beseda založnik seveda mnogo širše-
ga pomena. Danes, v 21. stoletju, je nujno redno objavljanje izsledkov in dognanj
znanstvenega in strokovnega dela na vseh področjih človekovega delovanja. Za-
radi izrednega razmaha namiznega založništva in še zlasti zelo preprostega ob-
javljanja na spletu je izdajanje publikacij postalo dostopno skorajda vsakomur,
ki malce bolje obvlada svoj osebni računalnik, pa tudi tisk večjega ali manjšega
števila izvodov je postal finančno dostopnejši. Slovenski založniki so razdeljeni v štiri skupine, kar omogoča velikim založnikom
izdajanje, zalaganje in uporabo oznak ISBN do 10.000 naslovov, malo manjšim
do 1000 naslovov, malim pa 100 in 10 naslovov, preračunano za daljše obdobje
(od 10 do 15 let). Preglednica 1: Zasedenost v registracijski skupini glede na založniške načrte založbe
Registracijska skupina
Število založnikov
00-19
6
200-599
73
6000-8999
803
90000-94999
3539
Skupaj
4304 Preglednica 1: Zasedenost v registracijski skupini glede na založniške načrte založbe Število že zasedenih identifikatorjev je večje od števila založnikov, saj ima lahko
vsak založnik tudi po več identifikatorjev, založnik lahko prehaja iz nižje v višjo 208 Knjižnica, 2017, 61(3), 199–211 Petdeset let mednarodnega identifikatorja ISBN skupino, ko enkrat porabi vse identifikatorje znotraj svojega lastnega identifi
katorja. Vse to je odvisno od njegovih založniških načrtov za daljše časovno
obdobje. Pred registracijo vsakega založnika in uvrstitev njegove založniške de-
javnosti ob bok drugim založnikom morajo založniki izpolniti vprašalnik, ki ga
najdejo na spletni strani Narodne in univerzitetne knjižnice v Ljubljani.1 skupino, ko enkrat porabi vse identifikatorje znotraj svojega lastnega identifi
katorja. Vse to je odvisno od njegovih založniških načrtov za daljše časovno
obdobje. Pred registracijo vsakega založnika in uvrstitev njegove založniške de-
javnosti ob bok drugim založnikom morajo založniki izpolniti vprašalnik, ki ga
najdejo na spletni strani Narodne in univerzitetne knjižnice v Ljubljani.1 Glede na njegove založniške načrte je uvrščen v določeno registracijsko skupino. Za samozaložnike, ki vsega skupaj izdajo samo en ali dva naslova, obstajata »dve
veliki vreči«, iz katerih se zajemajo oznake, ki so v tem primeru seveda manj po-
vedne, saj identificirajo izključno naslov in vezavo, ne pa tudi založnika. 1 Vprašalnik je dostopen na naslovu http://www.nuk.uni-lj.si/informacije/vprasalnik-isbn-ismn. 5.2 Podatkovna zbirka slovenskih založnikov S tem,
da je v Sloveniji seveda še dosti več založnikov, saj vsako leto izide okoli 500
naslovov, ki jih izdajajo v samozaložbi posamezniki kot fizične ali pravne osebe. Natančne podatke o samozaložnikih je izredno težko določiti, saj morajo posa-
mezniki zaradi davka na dodano vrednost uradno registrirati svojo dejavnost,
tam pa lahko uporabijo svoje lastno ime ali pa svojo dejavnost poimenujejo kako
drugače, tudi z uporabo izraza »založba«. Za evidenco nacionalne agencije je
ključno le število načrtovanih naslovov. Ustanovam, ki bodo v bližnji prihodnosti
poleg prvega prijavljenega naslova bolj verjetno izdale še kakšnega, je mogo-
če hitreje dodeliti samostojno oznako. Sicer pa so založniki znotraj podatkovne
zbirke ISBN, katerih založništvo ni primarna dejavnost, razvrščeni v več kategorij
(Preglednica 2). Preglednica 2: Kategorije, v katere se razvrščajo založniki (glede na njihove odgovore)
osnovne šole
98
srednje šole
74
višje in visoke šole
115
inštituti in raziskovalne organizacije
229
knjižnice
49
muzeji in galerije
88
društva
797
politične organizacije
4
verska združenja
67
vladne institucije
63
druge gospodarske organizacije
554
samozaložniki
391 Preglednica 2: Kategorije, v katere se razvrščajo založniki (glede na njihove odgovore) Da je založništvo njihova primarna dejavnost, je deklariralo 467 založnikov, vsi
drugi založniki ostajajo nerazporejeni. Nekaj pa je tudi takih (63), ki ne sodelujejo
v sistemu ISBN in smo jih kot potencialne založnike registrirali ob vzpostavitvi
podatkovne zbirke. Žal so to podatki, ki jih izpolnjevalci vprašalnika najpogosteje
izpustijo, agencija pa pri njih tudi ne vztraja. Prav tako zelo neradi izdajo svoje
založniške načrte, predvsem z izgovorom, da gre za poslovno skrivnost ali pa gre
samo za prva tipanja na knjižnem trgu. 209 Knjižnica, 2017, 61(3), 199–211 Alenka Kanič Ko založnik porabi vse oznake v svoji skupini, mu je dodeljena nova oznaka zno-
traj iste registracijske skupine ali pa mu je dodeljena višja registracijska sku-
pina, odvisno od tega, kako hitro je porabil oznake prve dodeljene skupine. V
podatkovni zbirki slovenskih založnikov je tudi nekaj takih, ki jim je bila oznaka
dodeljena še v Jugoslaviji, pa oznake niso nikoli uporabljali, nekaj založnikov je
agencija označila tudi kot neaktivne, kar pomeni, da v zadnjih petih letih niso
izdali nobenega naslova. Žal agencija tudi tega ne utegne sproti pregledovati in
dopolnjevati, še manj preverjati pri založnikih samih. Založnik preneha delati, se
preseli, zamenja se kontaktna oseba, pa o tem agencije nihče ne obvesti. 5.2 Podatkovna zbirka slovenskih založnikov Število letno podeljenih oznak ISBN posameznim naslovom niha, saj je to od-
visno od aktivnosti založniške dejavnosti same. Ob razmahu pojavljanja elek-
tronskih knjig na našem trgu je porastlo tudi število podeljenih oznak ISBN, saj
mora vsaka pojavna oblika na trgu vključno z vsakim računalniškim datotečnim
formatom imeti svojo oznako, tako kot jo imata tudi dve različni vezavi istega
naslova. Za vsemi temi oznakami ISBN stojijo metapodatki v obliki bibliografskih
zapisov v podatkovni zbirki COBIB. 0
1000
2000
3000
4000
5000
6000
7000
8000
9000
2007
2008
2009
2010
2011
2012
2013
2014
2015
2016
Slika 2: Število dodeljenih identifikatorjev v zadnjih desetih letih Slika 2: Število dodeljenih identifikatorjev v zadnjih desetih letih 6 Zaključek Zaposleni v Slovenski agenciji za ISBN smo prepričani, da se je priznani in dobro
uveljavljeni mednarodni identifikator izredno lepo vključil v slovensko knjižno
produkcijo, saj smo slovensko nacionalno agencijo ustanovili prav na pobudo
založnikov, in da ISBN slovenski založniki s pridom uporabljajo v vseh segmen-
tih poslovanja s knjigo. Z njihovo pomočjo in pod budnim očesom Slovenske
agencije za ISBN je napačne rabe v Sloveniji malo, za kar smo pri Mednarodni
agenciji pogosto pohvaljeni. Prepričani smo tudi, da je v podatkovni zbirki veliko 210 Knjižnica, 2017, 61(3), 199–211 Petdeset let mednarodnega identifikatorja ISBN zanimivih podatkov za raziskovalce slovenskega založništva, pa morda to prema-
lo poznajo, saj je podatkovna zbirka slovenskih založnikov javno dostopna le v
svojem najnujnejšem obsegu.2 zanimivih podatkov za raziskovalce slovenskega založništva, pa morda to prema-
lo poznajo, saj je podatkovna zbirka slovenskih založnikov javno dostopna le v
svojem najnujnejšem obsegu.2 Navedeni viri Gordon Foster (2017). V Wikipedia: the free Encyclopedia. Pridobljeno 11. 9. 2017 s spletne
strani: https://en.wikipedia.org/wiki/Gordon_Foster Gordon Foster (2017). V Wikipedia: the free Encyclopedia. Pridobljeno 11. 9. 2017 s spletne
strani: https://en.wikipedia.org/wiki/Gordon_Foster ISBN. (2017). V Wikipedia: the free Encyclopedia. Pridobljeno 11. 9. 2017 s spletne strani:
https://en.wikipedia.org/wiki/International_Standard_Book_Number ISBN. (2017). V Wikipedia: the free Encyclopedia. Pridobljeno 11. 9. 2017 s spletne strani:
https://en.wikipedia.org/wiki/International_Standard_Book_Number ISBN Timeline. (2015). London: International ISBN Agency. ISBN user’s manual (4th ed.). (2001). Berlin: International ISBN Agency. ISO 2108:2005(E). International standard. Information and documentation – International
Standard Book Number (ISBN). (2005). Geneva: ISO. ISO 2108:2005(E). International standard. Information and documentation – International
Standard Book Number (ISBN). (2005). Geneva: ISO. ISO 2108:2005(E). International standard. Information and documentation – International
Standard Book Number (ISBN). (2005). Geneva: ISO. Kanič, A. (2017). Morda niste vedeli: petdeset let in več mednarodnega identifikatorja
ISBN. Bukla, 13(129–130), 54. Kanič, A. (2017). Morda niste vedeli: petdeset let in več mednarodnega identifikatorja
ISBN. Bukla, 13(129–130), 54. Priročnik za uporabnike mednarodne standardne knjižne številke. (2006). Ljubljana: Naro-
dna in univerzitetna knjižnica. Priročnik za uporabnike mednarodne standardne knjižne številke. (2014). Ljubljana: Naro-
dna in univerzitetna knjižnica. Alenka Kanič Narodna in univerzitetna knjižnica, Turjaška 1, 1000 Ljubljana
e-pošta: alenka.kanic@nuk.uni-lj.si 2 Podatkovna zbirka je dostopna na naslovu https://plus.si.cobiss.net/opac7/zalisbn (COBISS+,
Druge baze podatkov, Druge specializirane baze podatkov, ZAL/ISBN – Slovenski založniki). 211 Knjižnica, 2017, 61(3), 199–211
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1 Srov. § 2 odst. 2 zákona č. 134/2016 Sb., o zadávání veřejných zakázek.
2 Internetová jazyková příručka [online]. Ústav pro jazyk český AV ČR, 2016 [cit. 11. 3. 2020]. Dostupné
z: http://prirucka.ujc.cas.cz/.
3 STAŠA, J. Správní akty. In: HENDRYCH, D. a kol. Správní právo. Obecná část. 9. vyd. Praha:
C. H. Beck, 2016, s. 140. Mezi příkladmé opravňující rozhodnutí po bok souhlasu, schválení a povolení
řadí koncesi rovněž judikatura, srov. např. usnesení rozšířeného senátu Nejvyššího správního soudu
(dále jen „NSS“) ze dne 26. 6. 2007, čj. 5 As 13/2006-46. The Concession in Czech Administrative Law The article examines standardized administrative acts called concessions. It seeks to describe
concessions as the theory of administrative law understands them, find their characteristics
and distinguish them from other administrative acts. Furthermore, the article gives examples
of concessions in positive law, particularly regarding regulation of broadcasting. Keywords: concession; permission; license; administrative act; broadcasting Klíčová slova: koncese; povolení; licence; administrativní akt; vysílání DOI: 10.14712/23366478.2020.14 DOI: 10.14712/23366478.2020.14 © 2020 The Author. 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 author and source are credited. ACTA UNIVERSITATIS CAROLINAE – IURIDICA 2 ACTA UNIVERSITATIS CAROLINAE – IURIDICA 2 PAG. 97–110 2020 KONCESE V ČESKÉM SPRÁVNÍM PRÁVU GABRIELA GÖTTELOVÁ Abstract: The Concession in Czech Administrative Law 4 STAŠA In: HENDRYCH, c. d., s. 140–141; KOPECKÝ, M. Správní právo: obecná část. Praha:
C. H. Beck, 2019, s. 161–162.
5 Příklady jsou k nalezení dále v textu; např. licence k provozování zemského analogového vysílání šíře-
ného prostřednictvím vysílačů.
6 Legislativní zkratku koncese pro státní povolení k provozování živnosti najdeme v zákoně č. 455/1991
Sb., o živnostenském podnikání (živnostenský zákon). Na udělení koncese podle živnostenského zákona
je při splnění zákonem stanovených předpokladů právní nárok, což lze dovodit jak z živnostenského
zákona (zejm. ustanovení § 53), tak i z ústavněprávního kontextu. U „koncesovaných živností“ se tak
uplatňuje povolovací, nikoli koncesní režim, jak by se mohlo zdát. Srov. STAŠA In: HENDRYCH, c. d.,
s. 140. K obdobným závěrům ohledně povahy „úředního dovolení“ určitých živnostenských činností
dospěla též prvorepubliková nauka. K tomu viz HÁCHA, E. Koncese. In: HÁCHA, E. – HOETZEL, J. –
WEYR, F. – LAŠTOVKA, K. (eds.). Slovník veřejného práva československého. Svazek II. Brno: Poly-
grafia, 1932, s. 271. 1. ÚVODEM Slovo koncese pochází z latinského slovesa concedere, které znamená
ustupovat, odcházet, přejít, postoupit, povolit, dovolit, připustit, propůjčit. Podstatné
jméno concessiō je překládáno jako dovolení, připuštění, a concessus jako ustoupení,
svolení. Tento význam si slovo ponechalo dodnes, i když to možná na první pohled není
zcela viditelné. Pojem koncese je v dnešní (právnické) mluvě používán ve vícero význa-
mech, které spolu však etymologicky souvisí; jako koncese je označován jeden z druhů
veřejných zakázek,1 územní ústupek státu2 či typizovaný správní akt. Poslednímu
uvedenému významu, tedy pojmu koncese tak, jak ho chápe správní právo a správní
věda, je věnován tento článek. i V oblasti klasifikace správních aktů je pro právní předpisy příznačná nejednotnost
terminologie a tato nejednotnost se týká také správních aktů zvaných koncese. Je tomu
tak i přes jisté tendence a snahy teorie i pozitivního práva o terminologické sjednocová-
ní, zejména co se týče aktů, jejichž obsahem je v prvé řadě něčí oprávnění; mezi takové
akty jsou tradičně řazeny souhlasy, povolení a právě koncese.3 Rozdíl mezi souhlasem,
povolením a koncesí pak nauka vidí zejména v tom, že na souhlas, popř. schválení, je
při splnění stanovených podmínek právní nárok a při jeho vydávání nemůže správní
orgán stanovit meze výkonu tohoto oprávnění. Za splnění stanovených podmínek exis- 97 tuje právní nárok rovněž na vydání povolení (dříve se používalo též označení dovolení),
ovšem v povolení mohou být stanoveny meze výkonu povolované činnosti. Koncese se
pak odlišuje tím, že na její udělení právní nárok není.4 Platné správní právo však terminologii a právní nauku často nerespektuje a pro jed-
notlivé správní akty používá nejen mnoho různých označení, ale tato mnohdy dokon-
ce ani nevypovídají o skutečné povaze příslušného aktu. Tak např. koncese může být
dikcí příslušného právního předpisu nazvána jako koncese, ale může být též schována
pod jiným označením, např. licence.5 Stejně tak správní akt nazvaný platným právem
koncese nemusí označovat to, co jako koncesi chápe teorie – v tomto ohledu lze pouká-
zat zejména na koncese ve smyslu státního povolení k provozování živnosti.6 Rovněž
Vrchní soud v Praze se k tomuto stavu vyjádřil, když konstatoval, že „je zřejmé a bylo
by možno dokumentovat, že zákonodárce obecně při své legislativní činnosti často uží-
vá promiscue pojmy jako ‚povolení, schválení, souhlas, koncese, licence‘ a málo dbá
o jejich doktrinální vymezení“.7 Není to však ani nový fenomén, ani problém (pouze)
dnešních dní. Jedná se o záležitost spíše tradiční, jak je možné vidět např. 1. ÚVODEM v Háchou
napsaném heslu „Koncese“ ve Slovníku veřejného práva československého, ve kterém
se dočteme, že „mluva zákonů a prakse právní má pro tyto správní akty bohatý rejstřík
názvů, jejichž jazykové odstíny jen zřídka napovídají rozdíly právní. (…) Výrazu konce-
se užívá se v našem právu dosti nahodile, takže někdy správní akty sobě velmi podobné
nesou názvy různé, kdežto zase akty sobě méně podobné nazvány jsou stejně. Tak mluví
se o živnostenské koncesi, ale o stavebním povolení, naproti tomu však o koncesi živ-
nostenské stejně jako o koncesi železniční, koncesi pomocných děl hornických a. j.“8 gi
7 Rozsudek Vrchního soudu v Praze ze dne 22. 9. 1995, čj. 6 A 191/94-32.
8
Á 8 HÁCHA, 1932, c. d., s. 267. ,
,
9 Tamtéž, s. 267. 2. KONCESE JAKO POJEM SPRÁVNÍHO PRÁVA Podle Háchy „koncesemi nazývá mluva zákonů některé z individuálních
aktů správních, jimiž právní řád podmiňuje určité činnosti v ten způsob, že by jinak,
tj. bez intervence aktu správního, byly činnostmi bezprávnými“.9 Hácha dále hledal
znaky, které koncesi odlišují od jiných dovolovacích správních aktů, zejména od tzv. konsensů, a blíže ji charakterizují. Klade si dvě otázky, a to (i) jaká je právní situace 98 uchazeče o úřední dovolení, zejména zdali má vůči státu právní nárok na udělení žá-
daného dovolení a zdali a do jaké míry je přípustné ke správnímu aktu připojit vedlejší
ustanovení, příjemce dovolení omezující a zatěžující, a (ii) jaká je právní situace pří-
jemce úředního dovolení, zejména zda nabývá z dovolení právního nároku vůči státu,
aby ve sféře kryté dovolením nebyl omezován veřejnou mocí, přičemž jde o otázku
odvolatelnosti a dodatečné změnitelnosti dovolení již uděleného.10 Co se týče prvního východiska, Hácha uvádí, že „jsou normy, které víží určité čin-
nosti na úřední dovolení, spojujíce s nimi, jestliže a protože byly předsevzaty bez tohoto
dovolení, následky činu bezprávného. Ale právě k činnostem téhož skutkového obsahu
vztahují se ještě jiné normy, které stanoví, že činnosti tohoto obsahu, budou-li předse-
vzaty za určitých věcných předpokladů a v určitých mezích, nebudou míti následků činu
bezprávného, čili že jsou dovoleny. Tyto normy, nazveme je normy ‚dovolující‘, bývají
někdy rozloženy ve dvojí vrstvě. Normy jedné vrstvy stanoví zásadní dovolenost určitých
činností, normy druhé vrstvy, omezujíce sféru zásadní dovolenosti, stanoví věcné před-
poklady, za kterých činnost je, resp. za kterých není dovolena.“ Uvedené je ilustrováno
příklady: tak např. některé živnostenské činnosti jsou jednou normou vázány na úřední
dovolení, zásadně jsou však tyto činnosti jinou normou dovoleny (občané mají právo
podnikat, resp. tehdejší ústavněprávní úpravou měli zaručeno právo vykonávat výdělko-
vou činnost); podle jiných norem (tady živnostenského řádu) je však tato zásadní dovo-
lenost vázána na určité předpoklady (např. spolehlivost, nezávadnost činnosti po stránce
bezpečnostní). Příslušné úřední dovolení, jímž je taková činnost podmíněna, nemá být
úřadem odepřeno, je-li činnost dovolená podle norem, které se vztahují k jejímu obsahu. Úřad vydání tohoto dovolení také nemá vázat na jiné předpoklady, než jsou ty předpo-
kládané „dovolující“ normou. Naproti tomu lze dle Háchy postavit „normy, které sice
také víží určitou činnost na dovolení úřední, nejsou však spiaty s jinými normami, které
touž činnost zásadně a za určitých věcných předpokladů označují za dovolenou. Dovo-
lovací akt tohoto druhu visí výlučně na normě, která úřad k udělení jeho zmocňuje. 10 Tamtéž, s. 270.
11 Tamtéž, s. 273.
12 Tamtéž, s. 273. 13 HOETZEL, J. Československé správní právo: část všeobecná. 2., přeprac. vyd. Praha: Melantrich, 1937,
s. 266.
14 HOETZEL, c. d., s. 266.
15 SLÁDEČEK, V. Obecné správní právo. 4., aktual. vyd. Praha: Wolters Kluwer, 2019, s. 126.
16 STAŠA In: HENDRYCH, c. d., s. 141.
17 STAŠA In: HENDRYCH, c. d., s. 133.
18 STAŠA In: HENDRYCH, c. d., s. 141.
19 KOPECKÝ, c. d., s. 162. 2. KONCESE JAKO POJEM SPRÁVNÍHO PRÁVA Ona
jest jediným jeho zdrojem, a odmyslíme-li si tuto normu, není jiné normy, jež by tu kterou
činnosti právně kryla. Byla by to vždy činnost bezprávná.“11 Příkladem je uvedeno např. omezení užívání vod a cest na tzv. usus communis, obecné užívání, resp. propůjčení
zvláštních užívacích práv stejně jako např. propůjčka horní. Není-li delegačních norem,
lze příslušné dovolení udělit jen „privilegiem“, individuálním zákonem. Tyto normy,
obsahující zmocnění dovolit činnost zásadně nedovolenou, nezakládají uchazeči nárok
na dovolení. K takovémuto dovolení mohou být připojeny jakékoliv výhrady, doložky
času, podmínky apod. Je-li však dovolení uděleno, zakládá uchazeči právní nárok vůči
moci veřejné, aby ho u výkonu tímto způsobem dovolené činnosti (dále) neomezovala. Pro tyto akty se pak dle Háchy nejlépe hodí název „koncese“, „propůjčení“, zatímco
u dříve popsaných aktů preferuje označení „povolení“, „konsens“.12
H
t
l
ýkl d
k
í h
čí á dliš
í
k t č ý h k
í d
li
j í h Hoetzel ve výkladu o koncesích začíná odlišením skutečných koncesí od policejních
povolení. Uvádí, že rozdíl bývá spatřován v tom, že policejní povolení (dovolení) ne-
poskytuje „příjemci nic nového, nýbrž pouze odstraňuje překážky, které vadí tomu, že 99 občan nemůže projevovati svou přirozenou volnost, svobodu. (…) Naproti tomu skuteč-
ná koncese (propůjčení) autorisuje občana k něčemu, co neplyne z přirozené svobody
občanské, resp. stát propůjčuje občanovi možnost užívati veřejného statku.“13 Poukazu-
je přitom na zásadní problém tohoto přístupu, resp. odlišení, spočívající v tom, že hrani-
ce mezi tím, co plyne z přirozené volnosti lidské a co nikoli, je nepřesná. Hoetzel, cituje
Merkla, který poněkud pesimisticky napsal, že „všecka snaha o systematisaci správních
aktů je málo více než neplodná hříčka“, ve svém Československém správním právu
dále odkazuje na Háchu a přiznává mu, že v článku Koncese ve Slovníku veřejného
práva československého prokázal, že hranici mezi (policejními) dovoleními a pravými
koncesemi najít lze. Hoetzel následně Háchovo slovníkové heslo shrnul a v podstatě do
své učebnice převzal.14 Dnešní odborná literatura koncesi uvádí mezi typizovanými správními akty, které se
v praxi vyskytují. Koncese jsou pojímány jako správní akty k dobru adresáta a zároveň
správní akty pozitivní, čímž se řadí především po bok souhlasu, schválení a povolení. Sládeček schvalovací akty (pod které zahrnuje souhlas a schválení), povolovací
akty a koncesi klasifikuje zejména na základě nárokovosti. 2. KONCESE JAKO POJEM SPRÁVNÍHO PRÁVA U koncese pak uvádí, že se
jedná v podstatě o povolení, ovšem s tím rozdílem, že na udělení koncese není právní
nárok – na rozdíl od povolovacích aktů, u kterých při splnění předepsaných podmínek
právní nárok na udělení je. Správní orgán má v případě koncesí možnost aplikovat
správní uvážení. Sládeček zde výslovně odkazuje na Hoetzela a jeho teze o tom, že
koncese opravňuje jejího držitele k něčemu, co neplyne z „přirozené svobody občan-
ské“, resp. že koncesí je státem občanu-koncesionáři propůjčována možnost užívat
veřejného statku.15 i Staša definuje koncesi jako správní akt, který se od souhlasu (popř. schválení) a po-
volení liší tím, že na jeho vydání, tedy na udělení koncese, není právní nárok.16 Koncese
je tedy jednostranný správní úkon (rozhodnutí), kterým vykonavatel veřejné správy
v konkrétním případě řeší právní poměry jmenovitě určených osob,17 s tím, že obsahem
tohoto správního aktu je něčí oprávnění, přičemž je věcí správního uvážení správního
orgánu, zda oprávnění přizná nebo nikoli. Zákon přitom zpravidla stanoví meze tohoto
správního uvážení.18 i Kopecký se ve své definici koncese více přibližuje k Háchovi, když začíná tím, že
koncese (propůjčení) „stanoví zvláštní právo vykonávat určitou činnost nebo užívat
veřejné statky, které by jinak z obecného práva činit vše, co není zákonem zakázáno,
nevyplývalo“. Dále shodně se Stašou uvádí, že na udělení koncese není právní nárok. Dodává také, že kritéria, kterými se správní orgán má řídit při posuzování, zda koncesi
udělit či nikoli, bývají stanovena zákonem.19 100 Průcha individuální správní akty rozlišuje mj. z pohledu jejich obsahového zamě-
ření, přičemž koncesi uvádí vedle povolení jako příklad individuálního správního aktu
oprávněnostního,20 a z hlediska členění individuálních správních aktů na konstitutivní
a deklaratorní řadí rozhodnutí o udělení koncese k aktům konstitutivním.21 Vzhledem k tomu, jakým způsobem platné právo s jednotlivými pojmy zachází,
obrací se k doktríně i soudní praxe, neboť musí „v každém konkrétním případě hodnotit,
zda zákonodárcem použitý pojem odpovídá jeho právní vědou definovanému obsahu
a jaký je popřípadě jeho skutečný smysl“.22 V odkazovaném rozsudku Vrchní soud
v Praze klasifikoval správní akty (povolení, koncese, souhlas, schválení) z hlediska
jejich legislativního vyjádření a rozsahu přezkumné pravomoci soudu. Vyšel přitom
z Háchova a Hoetzelova pojetí koncesí, odlišil je od povolení, souhlasu či schvále-
ní a výslovně k nim konstatoval, že jsou „i další činnosti, rovněž vázané na úřední
dovolení (často učiněné zvláštním zákonem), které však obecně podle svého obsahu
dovoleny nejsou nebo jsou přímo zakázány (srv. 2. KONCESE JAKO POJEM SPRÁVNÍHO PRÁVA poštovní výhradu, právo vydávat ban-
kovky, odnětí vlastnického práva). Nárok tu konstruovat nelze. Nauka tu obvykle užívá
pojmu koncese (často v rozporu s legislativní praxí, která za koncesi označuje i řadu
povolení).“23 Koncese je tedy správními soudy chápána jako správní akt, jímž zákon
podmiňuje určité činnosti tak, že bez tohoto aktu by šlo o činnosti protiprávní,24 a na
jehož udělení není právní nárok.25 21 PRŮCHA, c. d., s. 279. 26 Srov. HANDRLICA, J. Jednání soukromoprávních subjektů s důsledky ve sféře veřejného práva. In:
HANDRLICA, J. (ed.). Veřejné právo a právo soukromé: aktuální tendence a výzvy veřejnoprávní úpra-
vy plynoucí z problematiky právního dualismu. Praha: Univerzita Karlova v Praze, Právnická fakulta,
2014, s. 121.
Á ,
,
22 Rozsudek Vrchního soudu v Praze ze dne 22. 9. 1995, čj. 6 A 191/94-32.
23 Rozsudek Vrchního soudu v Praze ze dne 22. 9. 1995, čj. 6 A 191/94-32. Na toto rozhodnutí bylo
v judikatuře správních soudů dále odkazováno, srov. např. rozsudek Vrchního soudu v Praze ze dne
29. 1. 1999, čj. 6 A 57/97-28, nebo ze dne 30. 8. 1998, čj. 6 A 67/96-22.
24 Srov. usnesení zvláštního senátu zřízeného podle zákona č. 131/2002 Sb., o rozhodování některých
kompetenčních sporů, ze dne 18. 9. 2007, čj. Konf 23/2006-6. V rozhodnutí se mj. uvádí, že „rozhodnutí
o přidělení čísla je tak jistým druhem koncese (licence, povolení apod.), tj. správním aktem, jímž zákon
podmiňuje určité činnosti tak, že bez tohoto aktu by šlo o činnosti protiprávní“.
25 Výše citovaný rozsudek Vrchního soudu v Praze ze dne 22. 9. 1995, čj. 6 A 191/94-32, ale např. i Nej- 27 HÁCHA, 1932, c. d., s. 275; HOETZEL, c. d., s. 268. 20 PRŮCHA, P. Správní právo. Obecná část. 8., dopl. a aktual. vyd. (v nakl. Doplněk 3.). Brno: Doplněk,
2012, s. 280. 25 Výše citovaný rozsudek Vrchního soudu v Praze ze dne 22. 9. 1995, čj. 6 A 191/94-32, ale např. i Nej-
vyšší správní soud v rozsudku ze dne 24. 1. 1921, sp. zn. 554/21, týkajícím se koncesí k provozování
představení kinematografických. 2012, s. 280.
21 PRŮCHA, c. d., s. 279.
22 Rozsudek Vrchního soudu v Praze ze dne 22. 9. 1995, čj. 6 A 191/94-32.
23 Rozsudek Vrchního soudu v Praze ze dne 22. 9. 1995, čj. 6 A 191/94-32. Na toto rozhodnutí bylo
v judikatuře správních soudů dále odkazováno, srov. např. rozsudek Vrchního soudu v Praze ze dne
29. 1. 1999, čj. 6 A 57/97-28, nebo ze dne 30. 8. 1998, čj. 6 A 67/96-22.
24 Srov. usnesení zvláštního senátu zřízeného podle zákona č. 131/2002 Sb., o rozhodování některých
kompetenčních sporů, ze dne 18. 9. 2007, čj. Konf 23/2006-6. V rozhodnutí se mj. uvádí, že „rozhodnutí
o přidělení čísla je tak jistým druhem koncese (licence, povolení apod.), tj. správním aktem, jímž zákon
podmiňuje určité činnosti tak, že bez tohoto aktu by šlo o činnosti protiprávní“.
25 Výše citovaný rozsudek Vrchního soudu v Praze ze dne 22. 9. 1995, čj. 6 A 191/94-32, ale např. i Nej-
vyšší správní soud v rozsudku ze dne 24. 1. 1921, sp. zn. 554/21, týkajícím se koncesí k provozování
představení kinematografických.
26 Srov. HANDRLICA, J. Jednání soukromoprávních subjektů s důsledky ve sféře veřejného práva. In:
HANDRLICA, J. (ed.). Veřejné právo a právo soukromé: aktuální tendence a výzvy veřejnoprávní úpra-
vy plynoucí z problematiky právního dualismu. Praha: Univerzita Karlova v Praze, Právnická fakulta,
2014, s. 121.
27 HÁCHA, 1932, c. d., s. 275; HOETZEL, c. d., s. 268. 29. 1. 1999, čj. 6 A 57/97-28, nebo ze dne 30. 8. 1998, čj. 6 A 67/96-22.
24 Srov. usnesení zvláštního senátu zřízeného podle zákona č. 131/2002 Sb., o rozhodování některých
kompetenčních sporů, ze dne 18. 9. 2007, čj. Konf 23/2006-6. V rozhodnutí se mj. uvádí, že „rozhodnutí
o přidělení čísla je tak jistým druhem koncese (licence, povolení apod.), tj. správním aktem, jímž zákon
podmiňuje určité činnosti tak, že bez tohoto aktu by šlo o činnosti protiprávní“. 28 HÁCHA, 1932, c. d., s. 275.
29 Tamtéž.
30 Srov. § 11 zákona č. 266/1994 Sb., o drahách.
31 Srov. § 16 odst. 4 zákona č. 266/1994 Sb., o drahách.
32 Srov. § 34h zákona č. 266/1994 Sb., o drahách.
33 Srov. § 34a zákona č. 266/1994 Sb., o drahách.
34 Srov. § 25 zákona č. 266/1994 Sb., o drahách.
35 Viz zákon č. 77/2002 Sb., o akciové společnosti České dráhy, státní organizaci Správa železnic a o změ-
ně zákona č. 266/1994 Sb., o dráhách, ve znění pozdějších předpisů, a zákona č. 77/1997 Sb., o státním
podniku, ve znění pozdějších předpisů.
36 Srov. § 27 zákona č. 49/1997 Sb., o civilním letectví a o změně a doplnění zákona č. 455/1991 Sb., o živ-
nostenském podnikání (živnostenský zákon), ve znění pozdějších předpisů – zavádějící je však znění
třetího odstavce tohoto ustanovení, které říká, že povolení provozovat letiště Úřad vydá přednostně
žadateli, který prokáže, že je vlastníkem nadpoloviční většiny výměry letištních pozemků. 3. KONCESE V POZITIVNÍM SPRÁVNÍM PRÁVU Prvorepubliková právní úprava znala celou řadu aktů označovaných jako
koncese. Pro tyto akty bylo charakteristické zejména to, že dovolenost koncesované
činnosti neplynula z jiného právního předpisu. Jako příklady lze uvést např. koncesní
listinu železniční a koncesi k provozování leteckého podniku.26 Za ryzí koncesi prvo-
republiková literatura považovala dále např. povolení k výrobě, prodeji a přechovávání
radiotelegrafních a radiotelefonních zařízení.27 Ryzí koncesní princip bylo možné spat- 101 řovat též v úředním povolení k paroplavební dopravě osob a zboží na veřejných vodách,
v prohlášení podniku elektrického za všeužitečný a v povolení celních skladišť.28 V některých výše zmíněných oblastech přitom můžeme koncesní princip hledat
i dnes. Následující stránky se proto budou železnicím, letecké dopravě a zejména roz-
hlasovému a televiznímu vysílání věnovat podrobněji. 3.1 KONCESE V ŽELEZNIČNÍ A LETECKÉ DOPRAVĚ Železniční koncese za první republiky oprávněně nesla název „koncese“;
právní nárok na její udělení vylučovala jak samotná textace příslušného zákona, tak její
pojetí jako „privilegia“ a historický vývoj železničního práva.29 Dnes je k provozování
dráhy, tedy k činnostem, kterými se zabezpečuje a obsluhuje dráha a organizuje drážní
doprava, zapotřebí úředního povolení a v případě provozování dráhy celostátní nebo
regionální také osvědčení o bezpečnosti provozovatele dráhy. Toto úřední povolení vy-
dává drážní správní úřad.30 Na vydání úředního povolení není právní nárok a drážní
správní úřad v rozhodnutí o vydání úředního povolení stanoví podmínky týkající se
řádného a bezpečného provozování dráhy.31 Naproti tomu provozovat drážní dopravu
je možné na základě oprávnění k provozování drážní dopravy a v případě provozování
drážní dopravy na dráze celostátní nebo na dráze regionální také časově omezeného
osvědčení dopravce32 a přidělení kapacity dopravní cesty.33 Toto oprávnění k provozo-
vání drážní dopravy zákon nazývá licencí, kterou taktéž vydávají drážní správní úřady,
ovšem s tím, že při splnění příslušných požadavků je na její udělení nárok.34 Provozo-
vatelem železniční drážní dopravy tzv. ze zákona jsou České dráhy, a. s.35 Z nastíněného
je zřejmé, že u provozování dráhy se i v současnosti uplatňuje princip koncesní, zatímco
u provozování drážní dopravy je tento oslaben, neboť na udělení příslušného oprávnění
je při splnění zákonných požadavků právní nárok. Obdobně můžeme rozlišovat oprávnění, na jejichž udělení je právní nárok, a opráv-
nění, na jejichž udělení právní nárok není, v oblasti letecké dopravy. 37 Srov. § 58 zákona č. 49/1997 Sb., o civilním letectví.
38 Srov. § 3 odst. 3 písm. v) zákona č. 455/1991, o živnostenském podnikání (živnostenský zákon).
39 Srov. § 17 zákona č. 172/1925 Sb., o letectví.
40 HÁCHA, c. d., s. 275.
41 KRUPIČKA, M. – KŘÍŽOVÁ, D. Historie rozhlasu v kostce [online]. 25. 10. 2017 [cit. 11. 3. 2020].
Dostupné z: https://temata.rozhlas.cz/historie-rozhlasu-v-kostce-7983541.
42 Srov. § 1 odst. 1 zákona č. 60/1923 Sb., o telegrafech.
43 Srov. § 3 zákona č. 60/1923 Sb., o telegrafech.
44 ACSAY, L’. Právo elektronických komunikací v ČR. Revue pro právo a technologie [online], 2010, č. 2,
s. 33. [cit. 11. 3. 2020]. Dostupné z: https://journals.muni.cz/revue/article/view/4005.
45 Srov. § 2 zákona č. 60/1923 Sb., o telegrafech.
46 BARTOŇ, M. Stručný nástin historického vývoje právní úpravy regulace sdělovacích prostředků na
území ČR. Acta Universitatis Palackianae Olomucensis, Facultas Iuridica [online], 2003–2004, č. 5/6,
s. 19. [cit. 11. 3. 2020]. Dostupné z: http://oldwww.upol.cz/fileadmin/user_upload/Veda/AUPO/AUPO
_Iuridica_5_6.pdf. 3.1 KONCESE V ŽELEZNIČNÍ A LETECKÉ DOPRAVĚ Koncesní princip
se tak zřejmě uplatní u povolení provozovat letiště, které Úřad pro civilní letectví za spl-
nění zákonných podmínek může vydat.36 Na druhou stranu obchodní leteckou dopravu,
tedy dopravu osob, zvířat, zavazadel, pošty nebo jiného nákladu letadlem za úplatu, lze
provozovat pouze na základě licence, přičemž za zákonem stanovených podmínek Úřad 102 pro civilní letectví tuto licenci vydá; na její udělení tedy právní nárok je.37 Je případné
podotknout, že provozování obchodní letecké dopravy podle současné právní úpravy
není živností.38 Za první republiky však k živnostenskému provozu podniků, které slou-
ží letectví a nejsou provozovány státem nebo státními ústavy, tedy jak k podnikům
k provozu letišť, leteckých přístavů, míst pro vzlety a přistání, tak i k podnikům, které
provozovaly periodické lety pro dopravu osob a věcí na určitých liniích, jakož i k podni-
kům, které provozovaly jednotlivé lety ku dopravě osob a věcí jakýmkoli směrem nebo
pro takovéto lety propůjčují letadla, bylo třeba koncese.39 Uvedenou koncesi leteckých
podniků bylo možné považovat za pravou koncesi, neboť zákon provozování těchto
podniků nejen „přikazuje do akční sféry státu a státních ústavů, nýbrž právní nárok na
udělení této koncese zřetelně neguje (‚koncesi uděluje podle volného uvážení…‘)“.40 [
]
p
p
j
45 Srov. § 2 zákona č. 60/1923 Sb., o telegrafech.
Ň 3.2.1 VÝVOJ PRÁVNÍ ÚPRAVY ROZHLASOVÉHO
A POZDĚJI TÉŽ TELEVIZNÍHO VYSÍLÁNÍ Pokusy o rozhlasové vysílání na území dnešní České republiky začaly
hned po první světové válce a pravidelné rozhlasové vysílání bylo v Československu
započato dne 18. 5. 1923.41 Pro tehdejší posluchače rozhlasu byly zásadní dva zákony,
a to zákon č. 60/1923 Sb., o telegrafech, a zákon č. 9/1924 Sb., kterým se upravuje vý-
roba, prodej a přechovávání radiotelegrafních a radiotelefonních zařízení, jakož i dovoz
jejich z ciziny. Podle prvního československého zákona v této oblasti, tedy zákona č. 60/1923 Sb.,
bylo zřizovat, udržovat a provozovat telegraf výhradním právem státu,42 s tím, že stát
mohl výkon tohoto práva propůjčovat koncesí,43 čímž byl zachován předválečný model. V rakouské části habsburské monarchie totiž platil kabinetní list z 16. 1. 1847, jenž ob-
sahoval tzv. telegrafní regál, přičemž k částečné liberalizaci a zavedení možnosti získat
koncesi došlo až v roce 1905.44 Zákon č. 60/1923 Sb. používal telegraf jako legislativní
zkratku pro elektrický telegraf nebo telefon, a to drátový i bezdrátový (radiotelegraf),
jakož i elektrická návěštní zařízení; radiotelegrafem se mínil též radiotelefon.45 Telegraf
tak zahrnoval i radiofonii, tedy i rozhlas.46 Státní správu ve věcech telegrafů vykonávalo 103 především ministerstvo pošt a telegrafů; kompetence ministerstva pošt v oboru telegrafu
zahrnovala mj. zřizování rozhlasových stanic, udílení koncesí k jejich zřizování či dozor
nad výrobou, prodejem a přechováváním rozhlasových zařízení.47 Ministerstvo pošt a telegrafů v souladu s vládním nařízením č. 82/1925 Sb., kterým
se určují podmínky zřizování, udržování a provozu telegrafů, propůjčovalo koncese
zřizovati, udržovati a provozovati soukromé telegrafy. O propůjčení koncese se vy-
hotovovala koncesní listina; v koncesní listině mohly být koncesionáři uloženy ještě
zvláštní podmínky na ochránění zájmů bezpečnosti a obrany státu nad rámec podmínek
vyplývajících z nařízení.48 Koncesionář přijímací stanice, tedy rozhlasový posluchač,
byl povinen platit poštovní a telegrafní správě roční poplatky v koncesní listině stano-
vené.49 Poplatek vybíraly poštovní úřady, byl vymahatelný správní exekucí a poměrná
část tohoto poplatku se odváděla společnosti Radiojournal na úhradu správních a pro-
gramových výloh.50 Obdobná koncepce přetrvala i do doby zahájení televizního vysílá-
ní a je možné ji považovat za počátek tradování pojmu „koncesionářské poplatky“ pro
rozhlasové a televizní poplatky.51 Pro zajímavost je možné uvést, že v roce 1923 bylo
v Československu pouze 47 koncesionářů přijímacích rozhlasových stanic, o deset let
později jich však bylo již 573 109.52 Naproti tomu zákon č. 9/1924 Sb. reguloval toliko výrobu, prodej, přechovávání
a dovážení z ciziny zařízení radiotelegrafních a radiotelefonních (tj. nikoli zřizování,
udržování a provozování telegrafu, neboť to bylo upraveno zákonem č. ,
,
;
,
,
55 Srov. § 18 zákona č. 9/1924 Sb., kterým se upravuje výroba, prodej a přechovávání radiotelegrafních
a radiotelefonních zařízení, jakož i dovoz jejich z ciziny. 47 Viz zákon č. 60/1923 Sb., o telegrafech; Srov. HÁCHA, E. Rozhlas. In: HÁCHA, E. – HOETZEL, J. –
WEYR, F. – LAŠTOVKA, K. (eds.). Slovník veřejného práva československého. Svazek III. Brno: Po-
lygrafia, 1934, s. 813.
48 Srov. § 4 a § 8 vládního nařízení č. 82/1925 Sb., kterým se určují podmínky zřizování, udržování a pro-
vozu telegrafů.
49 Srov. § 7 vládního nařízení č. 82/1925 Sb., kterým se určují podmínky zřizování, udržování a provozu
telegrafů.
50 HÁCHA, 1934, c. d., s. 813.
51 POUPEROVÁ, O. Povaha rozhlasových a televizních poplatků. In: HANDRLICA (ed.), c. d., s. 180.
52 HÁCHA, 1934, c. d., s. 813.
53 Tamtéž.
54 HÁCHA, 1932, c. d., s. 275; HOETZEL, c. d., s. 268.
55 Srov. § 18 zákona č. 9/1924 Sb., kterým se upravuje výroba, prodej a přechovávání radiotelegrafních
a radiotelefonních zařízení, jakož i dovoz jejich z ciziny. 47 Viz zákon č. 60/1923 Sb., o telegrafech; Srov. HÁCHA, E. Rozhlas. In: HÁCHA, E. – HOETZEL, J. –
WEYR, F. – LAŠTOVKA, K. (eds.). Slovník veřejného práva československého. Svazek III. Brno: Po-
lygrafia, 1934, s. 813. POUPEROVÁ, O. Povaha rozhlasových a televizních poplatků. In: HANDRLICA (ed.), c. d., s. 18
2 HÁCHA, 1934, c. d., s. 813.
3 Tamtéž 50 HÁCHA, 1934, c. d., s. 813. ygi
,
,
48 Srov. § 4 a § 8 vládního nařízení č. 82/1925 Sb., kterým se určují podmínky zřizování, udržování a pro-
vozu telegrafů.
49 Srov. § 7 vládního nařízení č. 82/1925 Sb., kterým se určují podmínky zřizování, udržování a provozu 54 HÁCHA, 1932, c. d., s. 275; HOETZEL, c. d., s. 268. vozu telegrafů.
49 Srov. § 7 vládního nařízení č. 82/1925 Sb., kterým se určují podmínky zřizování, udržování a provozu
telegrafů.
50
Á
d 54 HÁCHA, 1932, c. d., s. 275; HOETZEL, c. d., s. 268.
55 3.2.1 VÝVOJ PRÁVNÍ ÚPRAVY ROZHLASOVÉHO
A POZDĚJI TÉŽ TELEVIZNÍHO VYSÍLÁNÍ 60/1923 Sb.). Podle zákona č. 9/1924 Sb. bylo k výrobě, prodeji, přechovávání jakož i dovozu z ciziny
radiotelegrafních a radiotelefonních zařízení třeba povolení, za které se platil poplatek,
přičemž nárok na udělení tohoto povolení byl výslovně vyloučen. Povolení udělovalo
ministerstvo průmyslu, obchodu a živností v dohodě s ministerstvem pošt a telegrafů
a udělovalo jej jen státním občanům s bydlištěm v Československu, kteří jsou spoleh-
liví a, v případě povolení k výrobě, mají předepsanou kvalifikaci. Zákon dále stanovil
podrobné podmínky pro udělování koncesí.53 Za zmínku jistě stojí, že Hácha i Hoetzel
povolení k výrobě, prodeji a přechovávání radiotelegrafních a radiotelefonních zařízení
podle zákona č. 9/1924 Sb. považovali za tzv. ryzí koncesi.54 Zákon č. 9/1924 Sb. také stanovil, že radiotelegrafní a radiotelefonní zařízení moh-
la být prodávána pouze osobám, které prokázaly, že jim byla udělena koncese ke
zřizování nebo k provozu radiotelegrafních a radiotelefonních stanic nebo povolení
k přechovávání radiotelegrafních a radiotelefonních zařízení.55 Zákon č. 9/1924 Sb. 104 byl zrušen zákonem č. 128/1947 Sb., jímž se upravují živnosti radiotechnická, ra-
diomechanická a obchod s radioelektrickými zařízeními; koncese k výrobě, prodeji
a přechovávání radiotelegrafních a radiotelefonních zařízení již nebyla třeba, výro-
ba a opravování radioelektrických zařízení však byly regulovány jako koncesované
živnosti a obchod s radioelektrickým zařízením jako živnost koncesovaná vázaná na
zvláštní způsobilost.56 Za první republiky byla televize v Československu teprve v počátcích technického
vývoje a byla někdy nazývána jako „obrazový rozhlas“.57 Televize je v jistém smyslu
dcerou telegrafu, telefonu a rádia;58 Československá televize, která je bytostně spjata
s počátky televizního vysílání v Československu, se zrodila v Českém rozhlase, od
kterého se osamostatnila až v roce 1957, resp. 1958.59 Téměř od samého počátku je možné pozorovat dvě souběžné linie právní úpravy
„rozhlasu“ a „televize“, a to regulaci přenosu rozhlasového a televizního vysílání a re-
gulaci rozhlasového a televizního obsahu. Po druhé světové válce mělo technologické
aspekty šíření médií v Československu tradičně na starost ministerstvo spojů jakožto
nástupce ministerstva pošt, pod které spadal rozhlas již v meziválečném období. Veš-
kerá agenda rozhlasu a televize, která se netýkala techniky a přenosu signálu, byla
v komunistickém Československu začleněna pod ministerstvo školství a osvěty jakožto
nástupce ministerstva informací a záhy přešla pod nové ministerstvo kultury. Od padesátých let se ve světě rozhlasu a televize mnohé změnilo. Jistá dvojkolej-
nost právní úpravy však přes všechny změny zůstala: od regulace obsahu rozhlasového
a televizního vysílání a obdobných nelineárních služeb, tj. p
,
,
60 V současné době existují v oblasti regulace obsahu odlišné regulační rámce pro televizní vysílání (pře-
devším zákon o provozování rozhlasového a televizního vysílání) a pro audiovizuální mediální služby
na vyžádání (především zákon o audiovizuálních mediálních službách na vyžádání). 56 Viz. zákon č. 128/1947 Sb., jímž se upravují živnosti radiotechnická, radiomechanická a obchod s radio-
elektrickými zařízeními.
Á 59 Srov. např. ŠTOLL, c. d., s. 166. 56 Viz. zákon č. 128/1947 Sb., jímž se upravují živnosti radiotechnická, radiomechanická a obchod s radio-
elektrickými zařízeními.
57 HÁCHA, 1934, c. d., s. 812.
58 ŠTOLL M 1 5 1953
zahájení televizního vysílání: zrození televizního národa Praha: Havran 2011 57 HÁCHA, 1934, c. d., s. 812. ,
,
,
58 ŠTOLL, M. 1. 5. 1953 – zahájení televizního vysílání: zrození televizního národa. Praha: Havra
s. 24. 56 Viz. zákon č. 128/1947 Sb., jímž se upravují živnosti radiotechnická, radiomechanická a obchod s radio-
elektrickými zařízeními.
57 HÁCHA, 1934, c. d., s. 812.
58 ŠTOLL, M. 1. 5. 1953 – zahájení televizního vysílání: zrození televizního národa. Praha: Havran, 2011,
s. 24.
59 Srov. např. ŠTOLL, c. d., s. 166.
60 V současné době existují v oblasti regulace obsahu odlišné regulační rámce pro televizní vysílání (pře-
devším zákon o provozování rozhlasového a televizního vysílání) a pro audiovizuální mediální služby
na vyžádání (především zákon o audiovizuálních mediálních službách na vyžádání). 3.2.2 SOUČASNÁ POVOLENÍ K PROVOZOVÁNÍ TELEVIZNÍHO
A ROZHLASOVÉHO VYSÍLÁNÍ Rámec upravující provozování rozhlasového a televizního vysílání je dán
především zákonem č. 231/2001 Sb., o provozování rozhlasového a televizního vysílání
a o změně dalších zákonů (dále také jen „zákon o provozování rozhlasového a televiz-
ního vysílání“), zákonem č. 132/2010 Sb., o audiovizuálních mediálních službách na
vyžádání a o změně některých zákonů (dále také jen „zákon o audiovizuálních medi-
álních službách na vyžádání“), a zákonem o elektronických komunikacích. Udělovat
oprávnění k provozování rozhlasového a televizního vysílání, resp. registrovat provo-
zovatele převzatého vysílání a evidovat poskytovatele audiovizuální mediální služby na
vyžádání, je svěřeno Radě pro rozhlasové a televizní vysílání (dále také jen „RRTV“),
případně RRTV ve spolupráci s Českým telekomunikačním úřadem. Současná právní
úprava přistupuje odlišně k provozování rozhlasového a televizního vysílání (dále také
jen „vysílání“), k provozování převzatého rozhlasového a televizního vysílání (dále
také jen „převzaté vysílání“) a k poskytování audiovizuálních mediálních služeb na
vyžádání. Nejpřísnější pravidla jsou stanovena pro provozování vysílání. Provozovat vysílá-
ní je možné pouze na základě zvláštních právních předpisů nebo na základě licence
udělené na základě zákona o provozování rozhlasového a televizního vysílání.61 Pro-
vozovateli vysílání tzv. ze zákona jsou Český rozhlas (na základě zákona č. 484/1991
Sb., o Českém rozhlasu) a Česká televize (na základě zákona č. 483/1991 Sb., o České
televizi). Všichni ostatní mohou provozovat vysílání pouze na základě oprávnění k pro-
vozování rozhlasového a televizního vysílání, které zákon o provozování rozhlasového
a televizního vysílání nazývá licence.62 Licenci uděluje RRTV v rámci řízení o udělení
licence (dále také jen „licenční řízení“). RRTV je oprávněna vydat licenci k vysílání
šířenému prostřednictvím a) vysílačů, b) družic a kabelových systémů, c) přenosových
systémů neuvedených v písmenech a) a b) (dále také jen „zvláštní přenosový systém“),
a to zásadně na dobu určitou. 61 Srov. § 3 odst. 1 zákona o provozování rozhlasového a televizního vysílání.
62 Srov. § 2 odst. 1 písm. y) téhož zákona.
63 Srov. § 12 téhož zákona.
64 CHALOUPKOVÁ, H. Mediální právo: komentář. Praha: C. H. Beck, 2019, s. 94.
65 Srov. § 12 – § 25 zákona o provozování rozhlasového a televizního vysílání. 3.2.1 VÝVOJ PRÁVNÍ ÚPRAVY ROZHLASOVÉHO
A POZDĚJI TÉŽ TELEVIZNÍHO VYSÍLÁNÍ audiovizuálních mediálních
služeb na vyžádání,60 je oddělena regulace přenosu audiovizuálních mediálních služeb. Přenos audiovizuálních mediálních služeb, resp. přenos informací prostřednictvím sítí
elektronických komunikací a rozhlasového a televizního vysílání upravuje především
zákon č. 127/2005 Sb., o elektronických komunikacích (dále také jen „zákon o elek-
tronických komunikacích“), který hned v § 1 odst. 2 říká, že „se nevztahuje na obsah
služeb poskytovaných prostřednictvím sítí elektronických komunikací, jako je obsah roz-
hlasového a televizního vysílání, finančních služeb a některých služeb informační spo-
lečnosti, není-li dále stanoveno jinak. Oddělením regulace přenosu od regulace obsahu
nejsou dotčeny vazby, které mezi nimi existují, zejména pro zaručení mediální plurality,
kulturní rozmanitosti a ochrany spotřebitele.“ 105 3.2.2 SOUČASNÁ POVOLENÍ K PROVOZOVÁNÍ TELEVIZNÍHO
A ROZHLASOVÉHO VYSÍLÁNÍ Nestanoví-li zákon jinak, není na udělení licence práv-
ní nárok.63 Toto se týká obecně všech licencí, tedy licencí k provozování zemského
analogového i digitálního vysílání šířeného prostřednictvím vysílačů, licencí k provo-
zovávání vysílání šířeného prostřednictvím družic, kabelových systémů a zvláštních
přenosových systémů.64 Zákon o provozování rozhlasového a televizního vysílání
však rozlišuje licenční řízení, které vyhlašuje RRTV a které je vedeno s více účastníky
(žadateli o udělení licence), a zvláštní licenční řízení zahajované z podnětu žadatele
o licenci, který je zároveň jeho jediným účastníkem.65 V tomto zvláštním licenčním
řízení jsou udělovány licence k provozování rozhlasového nebo televizního vysílání
šířeného prostřednictvím družic, kabelových systémů a zvláštních přenosových systémů
a k provozování zemského digitálního rozhlasového nebo televizního vysílání šířeného
prostřednictvím vysílačů, přičemž jsou-li splněné zákonné podmínky, má žadatel na 106 udělení licence právní nárok.66 Rozhodnutím o udělení vysílací licence zpravidla žada-
teli vzniká veřejné subjektivní právo.67 Rozhodnutí o udělení licence kromě příslušného
výroku, odůvodnění a poučení obsahuje také licenční podmínky, které stanovují další
meze vysílacího oprávnění (např. označení provozovatele vysílání, název programu,
časový a územní rozsah vysílání, dobu, na kterou je licence udělena, základní progra-
movou specifikaci apod.).68 V případě rozhodnutí o udělení licence podle ustanovení § 18 zákona o provozo-
vání rozhlasového a televizního vysílání, tedy v licenčním řízení zahájeném RRTV,
ve kterém může podat žádost o licenci více žadatelů, je rozhodováno jednak o udělení
licence jednomu ze žadatelů, jednak o zamítnutí žádostí ostatních žadatelů, a tyto
výroky jsou na sobě závislé; musí být přitom z odůvodnění rozhodnutí zřejmé a pře-
zkoumatelné, jak každé z kritérií stanovené pro udělení licence naplnil každý ze
žadatelů a proč RRTV licenci udělila, resp. neudělila.69 Ačkoliv na udělení vysílací
licence není právní nárok, není rozhodnutí RRTV vyloučeno z přezkumu ve správním
soudnictví, a ačkoliv „správní orgán rozhoduje na základě absolutně volné správní
úvahy, musí být jeho rozhodnutí přezkoumatelné a musí být zřejmé, že z mezí a hledi-
sek správního uvážení nevybočil“.70 RRTV je tedy při rozhodování o udělení licence,
na kterou žadatel nemá právní nárok, v souladu s judikaturou limitována minimálně
stanovenými procesními postupy a elementárními právními principy správního roz-
hodování.71 Provozovat převzaté vysílání je možné na základě registrace. 66 Srov. § 25 téhož zákona; CHALOUPKOVÁ, c. d., s. 9.
67 Rozsudek NSS ze dne 30. 11. 2004, čj. 3 As 24/2004-79.
68 Srov. § 18 odst. 3 a 4 zákona o provozování rozhlasového a televizního vysílání a dále též § 25 odst. 4
a 6 téhož zákona.
69 Rozsudek NSS ze dne 23. 6. 2005, čj. 7 As 10/2005-298.
70 Rozsudek NSS ze dne 30. 11. 2004, čj. 3 As 24/2004-79.
71 Rozsudek NSS ze dne 30. 11. 2004, čj. 3 As 24/2004-79.
72 Srov. § 2 odst. 1 písm. h) zákona o provozování rozhlasového a televizního vysílání.
73 Srov. § 28a téhož zákona.
74 Registrace bývá hmotněprávním předpokladem (jedním z předpokladů) jiné právní skutečnosti, s tím,
že právní skutečnost, kterou registrace podmiňuje, nastává ex lege; o registraci se vydává osvědčení,
o jejím odmítnutí správní akt. Viz STAŠA, J. Neregulativní úkony. In: HENDRYCH, c. d., s. 197.
75 Srov. § 26 odst. 2 zákona o provozování rozhlasového a televizního vysílání. 75 Srov. § 26 odst. 2 zákona o provozování rozhlasového a televizního vysílání. 73 Srov. § 28a téhož zákona. j
72 Srov. § 2 odst. 1 písm. h) zákona o provozování rozhlasového a televizního vysílání. §
74 Registrace bývá hmotněprávním předpokladem (jedním z předpokladů) jiné právní skutečnosti, s tím,
že právní skutečnost, kterou registrace podmiňuje, nastává ex lege; o registraci se vydává osvědčení,
o jejím odmítnutí správní akt. Viz STAŠA, J. Neregulativní úkony. In: HENDRYCH, c. d., s. 197.
75 Srov § 26 odst 2 zákona o provozování rozhlasového a televizního vysílání 76 CHALOUPKOVÁ, c. d., s. 119.
77 Srov. § 5 zákona o audiovizuálních mediálních službách na vyžádání.
78 CHALOUPKOVÁ, c. d., s. 210. 3.2.2 SOUČASNÁ POVOLENÍ K PROVOZOVÁNÍ TELEVIZNÍHO
A ROZHLASOVÉHO VYSÍLÁNÍ Registrací zákon
o provozování rozhlasového a televizního vysílání rozumí oprávnění k provozování
převzatého vysílání.72 RRTV registruje provozovatele převzatého vysílání prostřed-
nictvím sítě elektronických komunikací; pro provozovatele převzatého vysílání pro-
střednictvím vysílačů jsou zákonem o provozování rozhlasového a televizního vysílání
s ohledem na omezenou kapacitu kmitočtového spektra stanoveny zvláštní podmín-
ky.73 Ačkoliv by pojem registrace74 mohl navozovat odlišný dojem, registrací se dikcí
zákona o provozování rozhlasového a televizního vysílání nazývá rozhodnutí RRTV,
tedy individuální správní akt. Registraci vydává RRTV v řízení o registraci, které se
zahajuje z podnětu účastníka (přihláškou) a tento účastník je jeho jediným účastníkem. Na registraci je za splnění zákonných podmínek právní nárok.75 Registrace vzniká
dnem, kdy rozhodnutí RRTV o registraci nabylo právní moci; kladné rozhodnutí o re- 107 gistraci nabývá právní moci oznámením účastníku řízení a registrace je udělována na
dobu neurčitou.76 V případě audiovizuálních mediálních služeb na vyžádání se uplatňuje liberálnější
přístup v podobě tzv. evidence poskytovatelů audiovizuálních mediálních služeb na
vyžádání. Zákon o audiovizuálních mediálních službách na vyžádání stanovuje posky-
tovatelům těchto služeb oznamovací povinnost, s tím, že na základě řádného oznámení
RRTV zapíše příslušné údaje do Evidence poskytovatelů audiovizuálních mediálních
služeb na vyžádání, o čemž oznamovatele vyrozumí zasláním potvrzení o provedení
zápisu.77 Splní-li poskytovatel zákonné povinnosti, resp. náležitosti oznámení, má
RRTV povinnost poskytovatele audiovizuální mediální služby na vyžádání zaevidovat;
nečinnost RRTV nemá na oprávnění poskytování služby vliv a RRTV nemá možnost
stanovovat poskytovateli jakékoli podmínky poskytování služby.78 V současné úpravě rozhlasového a televizního vysílání je tedy možné za konce-
si považovat oprávnění k provozování rozhlasového a televizního vysílání (licenci),
ovšem pouze pokud jde o zemské analogové vysílání šíření prostřednictvím vysílačů. Co se týče ostatních způsobů vysílání, je na udělení příslušné licence při splnění zákon-
ných podmínek právní nárok – je tedy na místě hovořit spíše o povolení než o koncesi. V případě převzatého vysílání a audiovizuálních mediálních služeb na vyžádání je ze
shora nastíněné právní úpravy zřejmé, že o koncesním principu nemůže být vůbec
uvažováno. 4. ZÁVĚRY Koncese tak, jak ji chápe teorie správního práva, je ve své ryzí podobě
výjimečným institutem. Pojetí koncese se v teorii za posledních sto let (a snad i více)
příliš nezměnilo. Jednotliví autoři sice někdy zdůrazňují různé znaky na úkor jiných,
veskrze se však shodují na tom, že koncese je správním aktem, který svého adresáta
k něčemu opravňuje; od jiných správních aktů, jejichž obsahem je rovněž především
něčí oprávnění, se pak odlišuje zejména tím, že na její udělení neexistuje právní nárok. Tato nenárokovost v sobě skrývá další zásadní charakteristiky koncese – je jí udělováno
oprávnění k činnostem obecně nedovoleným a mohou v ní být stanoveny meze výkonu
tohoto uděleného oprávnění, resp. činnosti. Lze říci, že koncesí se uděluje oprávnění
k činnostem zásadně nedovoleným, tedy nějakým způsobem vyhrazeným státu, který
toto oprávnění „propůjčuje ze svého“ jako privilegium, pročež může též stanovit pod-
mínky výkonu dovolované činnosti. i Jak je vidět, definiční znaky koncese jakožto institutu správního práva zůstaly ply-
nutím času téměř nedotčeny a z prvorepublikových odborných textů je možné vychá-
zet i dnes. Přirozeně se však vyvíjí prostředí, ve kterém jsou koncese udělovány. Tak
se mění oblasti, ve kterých zákonodárce považuje za vhodné použít koncesní princip
namísto principu povolovacího, příp. ještě benevolentnějšího přístupu. Výčet činností, 108 které jsou „zásadně dovoleny“ jinými normami, a činností, které jsou naopak „zásadně
nedovoleny“ a oprávnění k jejich výkonu může být pouze propůjčeno, nelze stanovit
jednou provždy. Při vědomí velké zkratkovitosti: dnes ústavně zakotvené pravidlo, že
každý může činit, co není zákonem zakázáno, a nikdo nesmí být nucen činit, co zákon
neukládá, neplatilo vždy ve stejné podobě a šíři; neodmyslitelný je také ekonomický
a technický vývoj. Liberalizaci je možné vidět například na výše uvedené komerční
letecké dopravě. Ještě markantnější rozdíl lze spatřit u telekomunikací, které se od prv-
ních telegrafů a jejich přísné regulace vyvinuly až do dnešní podoby. Je však zároveň
třeba zmínit, že ne vždy stát, resp. zákonodárce na „změnu prostředí“ reaguje. Tak např. 79 Viz zákon č. 468/1991 Sb., o provozování rozhlasového a televizního vysílání (účinný do roku 2001).
80 MAISNER, M. – VANÍČEK, Z. Odpovědnost za obsah přenosu v elektronických komunikacích. Praha:
Wolters Kluwer ČR, 2012, s. 19.
81 Srov. STAŠA In: HENDRYCH, c. d., s. 141.
82 Tamtéž.
83 Viz tamtéž. Např. v případě řízení o povolení provozovat letiště posuzuje správní orgán u žadatelů
toliko zletilost, způsobilost k právním úkonům, bezúhonnost, odbornou způsobilost a právní vztah
k letišti (§ 27 zákona č. 49/1997 Sb., o civilním letectví). Srov. rozsudek NSS ze dne 16. 12. 2010, čj. 1
As 60/2010-104.
84 Srov. zejm. § 2–8 zákona č. 500/2004 Sb., správního řádu, a § 78 odst. 1 zákona č. 150/2002 Sb., soud-
ního řádu správního. Dále také např. rozsudek NSS ze dne 30. 11. 2004, čj. 3 As 24/2004-79. 79 Viz zákon č. 468/1991 Sb., o provozování rozhlasového a televizního vysílání (účinný do roku 2001).
80 MAISNER, M. – VANÍČEK, Z. Odpovědnost za obsah přenosu v elektronických komunikacích. Praha:
Wolters Kluwer ČR, 2012, s. 19.
81
Š 83 Viz tamtéž. Např. v případě řízení o povolení provozovat letiště posuzuje správní orgán u žadatelů
toliko zletilost, způsobilost k právním úkonům, bezúhonnost, odbornou způsobilost a právní vztah
k letišti (§ 27 zákona č. 49/1997 Sb., o civilním letectví). Srov. rozsudek NSS ze dne 16. 12. 2010, čj. 1
As 60/2010-104. 84 Srov. zejm. § 2–8 zákona č. 500/2004 Sb., správního řádu, a § 78 odst. 1 zákona č. 150/2002 Sb., soud-
ního řádu správního. Dále také např. rozsudek NSS ze dne 30. 11. 2004, čj. 3 As 24/2004-79. 81 Srov. STAŠA In: HENDRYCH, c. d., s. 141.
82 Tamtéž. 85 Judikatura k přezkoumatelnosti rozhodnutí správních orgánu je poměrně hojná a ustálená, viz např.
rozsudky NSS ze dne 24. 6. 2010, čj. 9 As 66/2009-46, ze dne 4. 2. 2010, čj. 7 Afs 1/2010-53, ze
dne 23. 7. 2009, čj. 9 As 71/2008-109, či rozsudek Vrchního soudu v Praze ze dne 26. 2. 1993, čj. 6
A 48/92-23. Je vhodné však v této souvislosti vzpomenout rovněž prvorepublikový rozsudek Nejvyššího
správního soudu ze dne 24. 1. 1921, sp. zn. 554/21, ve kterém se uvádí, že „(…) rozhodnutí o žádosti
za licenci kinematografickou, nejsou rozhodnutím o nějakém subjektivním právu žadatele, i pokud jde
o takovéto podniky, ponecháno jest zcela volnému uvážení úřadu, takže odepření licence není vázáno na
určité důvody a nemůže již dle své povahy zakládati porušení nějakých subjektivních práv. Se zřetelem
k tomu jest však také zcela nerozhodno, zdali a jakým způsobem úřad odepření licence odůvodnil.“ 4. ZÁVĚRY v minulosti79 koncesní princip omezoval počet provozovatelů televizního vysílání jak
v případě vysílání prostřednictvím satelitů a kabelových rozvodů, tak v případě vysíla-
čů; režim, který byl pochopitelný u kmitočtového spektra, které je ze své podstaty limi-
továno, ve svém důsledku způsobil, že Česká republika byla poslední zemí ve střední
a východní Evropě, která neprovozovala satelitní vysílání, které je stejně jako kabelové
vysílání na rozdíl od kmitočtového spektra z tohoto hlediska neomezené.80 Při rozlišování koncese od povolení v praxi někdy vznikají problémy používáním
neurčitých pojmů.81 Je však možné sledovat tendenci v pozitivním právu nahrazovat
neurčité pojmy jako např. „spolehlivost“ takovými, které nepůsobí výkladové obtíže,
tedy např. „bezúhonností“; motivací pravděpodobně není, aby bylo snadnější rozpoznat
koncese od jiných správních aktů, ale spíše omezování správního uvážení a tím (snad)
i zrychlování příslušných správních řízení. Navzdory nejrůznějším terminologickým
nedůslednostem zákonodárce se však tímto zároveň zmenšuje prostor pro pochyby, zda
je konkrétní správní akt koncesí či nikoli. Vliv na vzácnost koncesí v jejich čisté podobě má i to, že správní uvážení správ-
ního orgánu, zda koncesi udělí či neudělí, nebývá zcela neomezené. Staša s odkazem
na Háchu uvádí, že již v prvé polovině 20. století bylo výjimečné, aby správní uvá-
žení nebylo vůbec omezeno.82 I v případech, kdy je právní nárok žadatele o udělení
koncese zásadně vyloučen a správnímu orgánu je ponecháno na volném uvážení, zda
koncesi udělí či nikoli, totiž zákon většinou stanoví určitá hlediska, kterými se má
správní orgán řídit.83 Dnes je nadto správní orgán, který o udělení koncese rozhoduje,
vázán též základními zásadami činnosti správních orgánů, musí dodržet stanovený
procesní postup pro vydání rozhodnutí a nesmí překročit meze správního uvážení či
jej zneužít.84 Rozhodnutí o udělení či neudělení koncese je dále předmětem přezkumu
ve správním soudnictví, pročež musí být přezkoumatelné; musí z něj tedy být zřejmé,
podle které právní normy správní orgán rozhodl, z jakých podkladů přitom vycházel, 109 jakými úvahami se řídil apod.85 V právním státě totiž ani koncese nemůže být pro-
jevem libovůle správního orgánu, resp. státu, který „ze svého“ propůjčuje příslušné
privilegium. jakými úvahami se řídil apod.85 V právním státě totiž ani koncese nemůže být pro-
jevem libovůle správního orgánu, resp. státu, který „ze svého“ propůjčuje příslušné
privilegium. Gabriela Göttelová
Právnická fakulta Univerzity Karlovy
gabriela.gottelova@gmail.com Gabriela Göttelová
Právnická fakulta Univerzity Karlovy
gabriela.gottelova@gmail.com 110
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Formalising BPMN Service Interaction Patterns
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Lecture notes in business information processing
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To cite this version: Chiara Muzi, Luise Pufahl, Lorenzo Rossi, Mathias Weske, Francesco Tiezzi. Formalising BPMN
Service Interaction Patterns. 11th IFIP Working Conference on The Practice of Enterprise Modeling
(PoEM), Oct 2018, Vienna, Austria. pp.3-20, 10.1007/978-3-030-02302-7_1. hal-02156461 Formalising BPMN Service Interaction Patterns
Chiara Muzi, Luise Pufahl, Lorenzo Rossi, Mathias Weske, Francesco Tiezzi
To cite this version:
Chiara Muzi, Luise Pufahl, Lorenzo Rossi, Mathias Weske, Francesco Tiezzi.
Formalising BPMN
Service Interaction Patterns. 11th IFIP Working Conference on The Practice of Enterprise Modeling
(PoEM), Oct 2018, Vienna, Austria. pp.3-20, 10.1007/978-3-030-02302-7_1. hal-02156461 Formalising BPMN Service Interaction Patterns
Chiara Muzi, Luise Pufahl, Lorenzo Rossi, Mathias Weske, Francesco Tiezzi Distributed under a Creative Commons Attribution 4.0 International License Formalising BPMN Service Interaction Patterns Chiara Muzi1, Luise Pufahl2, Lorenzo Rossi1, Mathias Weske2, and Francesco Tiezzi1 Chiara Muzi1, Luise Pufahl2, Lorenzo Rossi1, Mathias Weske2, and Francesco Tie
1 School of Science and Technology, University of Camerino, Italy
{chiara.muzi,lorenzo.rossi,francesco.tiezzi}@unicam.it
2 Hasso-Plattner-Institute, University of Potsdam, Potsdam, Germany
{luise.pufahl,mathias.weske}@hpi.de 1 School of Science and Technology, University of Camerino, Italy
{chiara.muzi,lorenzo.rossi,francesco.tiezzi}@unicam.it
2 Hasso-Plattner-Institute, University of Potsdam, Potsdam, Germany
{luise.pufahl,mathias.weske}@hpi.de Abstract. Business process management is especially challenging when cross-
ing organisational boundaries. Inter-organisational business relationships are con-
sidered as a first-class citizen in BPMN collaboration diagrams, where multiple
participants interact via messages. Nevertheless, proper carrying out of such in-
teractions may be difficult due to BPMN lack of formal semantics. In particular,
no formal studies have been specifically done to cope with complex BPMN in-
teraction scenarios unified under the name of Service Interaction Patterns. In
this work the depiction of the service interaction patterns in BPMN collabora-
tion diagrams is revisited and fully formalised via a direct semantics for BPMN
multi-instance collaborations, thus leaving no room for ambiguity and validating
the BPMN semantics. To make the formalisation more accessible, a visualisation
of the patterns execution by means of a BPMN model animation tool is provided. Keywords: BPMN · Collaboration · Service Interaction Patterns · Formalisation HAL Id: hal-02156461
https://inria.hal.science/hal-02156461v1
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abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1
Introduction The effective and efficient handling of business processes is a primary goal of organ-
isations. Business Process Management (BPM) provides methods and techniques to
support these endeavors [17]. Thereby, the main artefacts are business process models
which help to document, analyse, improve, and automate organisation processes [13]. To this aim, nowadays BPMN (Business Process Model and Notation) [16] is the mod-
elling notation most widely applied in industry and academia. g
y pp
y
For conducting a successful business, an organisation does not act alone, but it is
usually involved in collaborations with other organisations. The importance of interac-
tions has been underlined by many authors [1,10,2] and a lot of effort has been done
to identify the most common interaction scenarios from a business perspective, which
have been called Service Interaction Patterns [3]. Interactions are considered as a first-
class citizen in BPMN collaboration diagrams, where multiple participants cooperate
by exchanging messages and sharing thereby data. This motivated the use of BPMN
to model service interaction patterns [17], initially defined only in terms of textual de-
scriptions. This effort provided a graphical, more intuitive, description of the patterns
and allowed to assess the suitability of BPMN to express common interaction scenar-
ios. However, a severe issue in this study is that the precise behaviour of the BPMN
models corresponding to some patterns may result unclear, in particular when multiple instances of the interacting participants are involved. This problem is mainly due to the
fact that the BPMN standard comes without a formal semantics, which is needed in
presence of tricky features, like multiple process instantiation. In this paper, we then aim at formalising the execution semantics of service interac-
tion patterns specified in BPMN. This is a particularly important challenge in the BPM
domain, as a precise semantics of the message exchanges as well as their dependencies
is a prerequisite to ensure the appropriate carrying out, in practice, of such interactions. To achieve this goal, we resort to a formal semantics for BPMN collaborations includ-
ing multiple instances introduced in [6]. The operational semantics is directly defined
on BPMN elements in terms of Labelled Transition Systems (LTSs), rather than as an
encoding into other formalisms. Specifically, for each service interaction pattern we re-
port the related BPMN collaboration model and provide its formalisation in terms of
transitions of the corresponding LTS. 1
Introduction A direct formal characterisation is crucial, as it does not leave any room for am-
biguity, and increases the potential for formal reasoning. This is especially important
when dealing with multiple instances, whose static and compact BPMN representation
hides their complex semantics. Moreover, the BPMN formalisation in [6] enables the
use of the MIDA animation tool [7] that provides a visualisation, faithfully follow-
ing the semantics, of patterns execution. This makes the behaviour of patterns easily
understandable also to an audience non-familiar with formal methods. Finally, since
the service interaction patterns have been used to evaluate different choreography lan-
guages [5], their formalisation allows to validate the BPMN semantics itself, both in
terms of the considered BPMN elements and of the expected semantic behaviour. In the remainder of this paper, we start with the introduction of a motivational ex-
ample in Sec. 2, followed by an overview of the formalisation of BPMN collaboration
diagrams in Sec. 3. In Sec. 4 we provide the representation of the service interaction
patterns in BPMN and their formalisation. We present the patterns animation in Sec. 5. Finally, related work is discussed in Sec. 6 and the paper is concluded in Sec. 7. 2
Motivating Scenario Thus, the Retailer rather
stops waiting as soon as enough responses have arrived or a given timeout is expired. This latter behaviour is rendered by the timer event attached to the sub-process, whose
activation produces the execution of task “Reduce Invoice by Missing Items”. The Re-
tailer then packs the items and passes the needed information to the Logistic Provider,
who is in charge to send the package to the Customer. When the Customer receives the
ordered items, via a receiving task, its process completes. ther to “Send Item” or not, by following one of the outgoing edges of the XOR split
gateway, according to the information in the “Stock” data object. In case the item is not
available, the Provider does not respond back to the Retailer. Thus, the Retailer rather
stops waiting as soon as enough responses have arrived or a given timeout is expired. This latter behaviour is rendered by the timer event attached to the sub-process, whose
activation produces the execution of task “Reduce Invoice by Missing Items”. The Re-
tailer then packs the items and passes the needed information to the Logistic Provider,
who is in charge to send the package to the Customer. When the Customer receives the
ordered items, via a receiving task, its process completes. In this scenario, already various interaction patterns can be observed, such as One-
to-many send/receive between the Retailer and the Item Provider(s), or the Request with
Referral between the Customer, Retailer and Logistic Provider [3]. The service interac-
tions represent different types of dynamic behaviour, ranging from simple message ex-
changes, to scenarios involving multiple participants and multiple message exchanges,
as well as routing behaviour, where information is routed to a new collaboration partner
during an interaction (e.g. from the Retailer to the Logistic Provider). As the service
interaction patterns are textually provided, a visualisation as well as a formalisation is
crucial to precisely render the message exchanges between participants, especially be-
cause multiple instances are involved. In particular, the interactions between the Retailer
sub-process and the Item Provider may result quite intricate, as both generate multiple
instances and, in addition, the sub-process is constrained by a timer event. Without a
clear understanding of the interplay between these features, formally provided by the
operational semantics, different interpretations may easily arise. 2
Motivating Scenario In this section we introduce an order fulfilment scenario to illustrate BPMN 2.0 col-
laboration diagrams and the depiction of service interaction patterns. The considered
scenario shows an interaction among a Customer, a Retailer, multiple Item Providers
and a Logistic Provider (Fig. 1). The processes of the different interacting partners are
represented inside rectangles, called pools, and their interaction is given by message
edges (dashed connectors) visualising communication flows. The order fulfilment process is started by the Customer who sends an “Order Re-
quest” to the Retailer, via a send task. The arrival of this message starts, via a message
start event, a process instance of the Retailer pool. This latter creates a list of needed
items and stores this information in the “Item” data object collection. For each item, the
Retailer sends out a request to an Item Provider and waits for the response. This interac-
tion is rendered by a multi-instance sub-process communicating with the multi-instance
pool of the Item Provider: each message of the Retailer creates a new process instance
of the Item Provider pool. Each Provider checks for items availability and decides ei- 2 Customer
Order
Management
Send Order
Receive Pack
Order Process
Completed
Retailer
Request
Received
Create Item
List
Request
Management
Request Item
Receive Item
Request
Completed
Reduce
Invoce by
Missing Items
Pack Items
Give Order to
Logistic
Provider
Request Process
Completed
Item Provider
Item
Requested
Check Item
Availability
Send Item
Item Request
Completed
Item Request
Uncompleted
Logistic
Provider
Logistic
Order Received
Send Pack
Sending Process
Completed
Order
Order Request
Item
Item Request
Sent Items
Package
Logistic Order
Stock
Is item
in stock? yes
no
Fig. 1: BPMN collaboration diagram of the Order Fulfilment Scenario. ther to “Send Item” or not, by following one of the outgoing edges of the XOR split
gateway, according to the information in the “Stock” data object. In case the item is not
available, the Provider does not respond back to the Retailer. Thus, the Retailer rather
stops waiting as soon as enough responses have arrived or a given timeout is expired. This latter behaviour is rendered by the timer event attached to the sub-process, whose
activation produces the execution of task “Reduce Invoice by Missing Items”. The Re-
tailer then packs the items and passes the needed information to the Logistic Provider,
who is in charge to send the package to the Customer. 2
Motivating Scenario When the Customer receives the
ordered items, via a receiving task, its process completes. In this scenario, already various interaction patterns can be observed, such as One- Customer
Order
Management
Send Order
Receive Pack
Order Process
Completed
Retailer
Request
Received
Create Item
List
Request
Management
Request Item
Receive Item
Request
Completed
Reduce
Invoce by
Missing Items
Pack Items
Give Order to
Logistic
Provider
Request Process
Completed
Item Provider
Item
Requested
Check Item
Availability
Send Item
Item Request
Completed
Item Request
Uncompleted
Logistic
Provider
Logistic
Order Received
Send Pack
Sending Process
Completed
Order
Order Request
Item
Item Request
Sent Items
Package
Logistic Order
Stock
Is item
in stock? yes
no
Fig. 1: BPMN collaboration diagram of the Order Fulfilment Scenario. Customer
Order
Management
Send Order
Receive Pack
Order Process
Completed
Retailer
Request
Received
Create Item
List
Request
Management
Request Item
Receive Item
Request
Completed
Reduce
Invoce by
Missing Items
Pack Items
Give Order to
Logistic
Provider
Request Process
Completed
Item Provider
Item
Requested
Check Item
Availability
Send Item
Item Request
Completed
Item Request
Uncompleted
Logistic
Provider
Logistic
Order Received
Send Pack
Sending Process
Completed
Order
Order Request
Item
Item Request
Sent Items
Package
Logistic Order
Stock
Is item
in stock? yes
no
Fig. 1: BPMN collaboration diagram of the Order Fulfilment Scenario. Fig. 1: BPMN collaboration diagram of the Order Fulfilment Scenario. g
g
ther to “Send Item” or not, by following one of the outgoing edges of the XOR split
gateway, according to the information in the “Stock” data object. In case the item is not
available, the Provider does not respond back to the Retailer. Thus, the Retailer rather
stops waiting as soon as enough responses have arrived or a given timeout is expired. This latter behaviour is rendered by the timer event attached to the sub-process, whose
activation produces the execution of task “Reduce Invoice by Missing Items”. The Re-
tailer then packs the items and passes the needed information to the Logistic Provider,
who is in charge to send the package to the Customer. When the Customer receives the
ordered items, via a receiving task, its process completes. ther to “Send Item” or not, by following one of the outgoing edges of the XOR split
gateway, according to the information in the “Stock” data object. In case the item is not
available, the Provider does not respond back to the Retailer. 3
Background Notions on the BPMN Formalisation In this section we provide an overview of the formal semantics of BPMN multi-instance
collaborations given in [6]. The formalisation relies on a textual representation of the
structure of BPMN collaboration models, defined by the Backus-Naur Form (BNF)
grammar in Fig. 2. In the proposed grammar, the non-terminal symbols C, P and A 3 C ::“ poolpp, Pq
|
miPoolpp, Pq
|
C1 ∥C2
P ::“ startpeenb, eoq | startRcvpm:˜t, eoq | endpeiq | endSndpei, m: ˜
expq | terminatepeiq
|
andSplitpei, Eoq | xorSplitpei, Gq | andJoinpEi, eoq | xorJoinpEi, eoq
|
eventBasedpei, pm1 :˜t1, eo1q, . . . , pmh :˜th, eohqq
|
taskpei, exp, A, eoq | taskRcvpei, exp, A, m:˜t, eoq | taskSndpei, exp, A, m: ˜
exp, eoq
|
interRcvpei, m:˜t, eoq | interSndpei, m: ˜
exp, eoq | P1 ∥P2
A ::“ ϵ | d.f ::“ exp, A
Fig. 2: BNF syntax of BPMN collaboration structures. Fig. 2: BNF syntax of BPMN collaboration structures. represent Collaboration Structures, Process Structures and Data Assignments, respec-
tively. The first two syntactic categories directly refer to the corresponding notions in
BPMN, while the latter refers to list of assignments used to specify updating of data
objects. The terminal symbols, denoted by the sans serif font, are the typical elements
of a BPMN model, i.e. pools, events, tasks and gateways. Intuitively, a BPMN collaboration model is rendered in this syntax as a collection
of pools, each one specifying a process. Formally, a collaboration C is a composition,
by means of the ∥operator, of pools. A pool is either of the form poolpp, Pq (for single-
instance pools), or miPoolpp, Pq (for multi-instance pools) where p is the name that
uniquely identifies the pool, and P is the enclosed process. At process level, e P E
uniquely denotes a sequence edge, while E P 2E is a set of edges. For the convenience
of the reader, ei refers to the edge incoming in an element, while eo to the outgoing
edge, and eenb to the (spurious) edge denoting the enabled status of a start event. To describe the semantics of collaboration models, we enrich the structural infor-
mation with a notion of execution state, defined by the state of each process instance
and the store of the exchanged messages. We call process configurations and collabo-
ration configurations these stateful descriptions. 3
Background Notions on the BPMN Formalisation 4 xtaskSndpei, exp1, A, m: ˜
exp, eoq, σ, αy
!m :˜v
ÝÝÝÑ
xincpdecpσ, eiq, eoq, α1y
σpeiq ą 0,
evalpexp1, α, trueq,
updpα, A, α1q,
evalp ˜
exp, α, ˜vq
pP-TaskSndq
xinterRcvpei, m:˜t, eoq, σ, αy
?m : ˜et,ϵ
ÝÝÝÝÝÑ
xincpdecpσ, eiq, eoq, αy
σpeiq ą 0,
evalp˜t , α, ˜et q
pP-InterRcvq
xxorSplitpei, tpe, expquYGq, σ, αy
ϵÝÑ
xincpdecpσ, eiq, eq, αy
σpeiq ą 0,
evalpexp, α, trueq
pP-XorSplit1q
xP1, σ, αy
ℓÝÑ xσ1, α1y
xP1 ∥P2, σ, αy
ℓÝÑ xσ1, α1y
ℓ‰ kill
pP-Int1q
ιppq “ txσ, αyu
xP, σ, αy
!m :˜v
ÝÝÝÑ xσ1, α1y
pC-Deliverq
xpoolpp, Pq, ι, δy
!m :˜v
ÝÝÝÑ xupdI pι, p, txσ1, α1yuq, addpδ, m, ˜vqy
ιppq “ txσ, αyu
xP, σ, αy
?m : ˜et,A
ÝÝÝÝÝÑ xσ1, α1y
˜v P δpmq
matchp ˜et, ˜vq “ A1
pC-Receiveq
xpoolpp, Pq, ι, δy
?m :˜v
ÝÝÝÑ xupdI pι, p, txσ1, updpα1, pA1, Aqqyuq, rmpδ, m, ˜vqy
Fig. 3: An excerpt of BPMN semantic rules. Fig. 3: An excerpt of BPMN semantic rules. We refer the interested reader to [6] for a full account of the definition of these
relations, while we report in Fig. 3, by way of example, some operational rules. Rule
P-TaskSnd is used for the execution of send tasks possibly equipped with data objects. These latter are associated to a task by means of a conditional expression, exp1, and a
list of assignments A, each of which assigns the value of an expression to a data field
(the field f of the data object named d is accessed via d.f). Sending tasks also have as
argument a pair of the form m : ˜
exp, where m is a message name and ˜
exp is a tuple of
expressions. The task is activated only when there is a token in the incoming edge of the
task (σpeiq ą 0) and the task’s guard exp1 is satisfied (evalpexp1, α, trueq). The effects
of the task execution are as follows: the marking σ of the process instance is updated
with the movement of one token from ei to eo, by means of functions dec and inc, and
the message action !m : ˜v is produced, where the message content ˜v results from the
evaluation of the expression tuple ˜
exp (evalp ˜
exp, α, ˜vq). The produced label is used to
deliver the message at the collaboration layer (see rule C-Deliver). 3
Background Notions on the BPMN Formalisation Formally, a process configuration has
the form xP, σ, αy, where: P is a process structure; σ : E Ñ N is a sequence edge state
function specifying, for each sequence edge, the current number of tokens marking it (N
is the set of natural numbers); and α : F Ñ V is the data state function assigning values
(possibly null) to data object fields (F is the set of data fields and V the set of values). A collaboration configuration has the form xC, ι, δy, where: C is a collaboration struc-
ture, ι : P Ñ 2SσˆSα is the instance state function mapping each pool name (P is the set
of pool names) to a multiset of instance states (ranged over by I and containing pairs of
the form xσ, αy), with Sσ and Sα the sets of edges and data states, and δ : M Ñ 2Vn is
a message state function specifying for each message name m P M a multiset of value
tuples representing the messages received along the message edge with the label m. The operational semantics is defined by means of a labelled transition system (LTS),
whose definition relies on an auxiliary LTS on the behaviour of processes. The latter is
a triple xP, L, Ñy where: P ranged over by xP, σ, αy is a set of process configurations,
L ranged over by ℓ, is a set of labels, and ÑĎ P ˆ L ˆ P is a transition relation. We
will write xP, σ, αy
ℓÝÑ xP, σ1, α1y to indicate that pxP, σ, αy, ℓ, xP, σ1, α1yq PÑ. Now,
the labelled transition relation on collaboration configurations formalises the message
exchange and the data update according to the process evolution. The LTS is a triple
xC, Lc, Ñcy where: C, ranged over by xC, ι, δy, is a set of collaboration configurations;
Lc, ranged over by l, is a set of labels; and ÑcĎ C ˆ Lc ˆ C is a transition relation. 3
Background Notions on the BPMN Formalisation Rule P-InterRcv
is similar, but it produces a label corresponding to the reception of a message, which
is actually consumed by rule C-Receive. Rule P-XorSplit1 is applied when a token is
available in the incoming edge of a XOR split gateway and a conditional expression
of one of its outgoing edges is evaluated to true; the rule decrements the token in the
incoming edge and increments the token in the selected outgoing edge. Finally, rule
P-Int1 deals with interleaving in a standard way for process elements. More details on
these rules are given in the next section, from time to time when they are applied. 4.1
Send Pattern Informal Description. A party sends a message to another one. This pattern can be
p
Task 1
m
e1
e2
q
Fig. 4: Send pattern. modelled as the BPMN collaboration fragment in
Fig. 4. Notably, this is only a way to model it:
the send task could be replaced by an intermediate
send event or by a message end event. However,
up to some technicalities, all cases behave in the
same way, thus we report here only one of them. Informal Description. A party sends a message to another one. This pattern can be
p
Task 1
m
e1
e2
q
Fig. 4: Send pattern. modelled as the BPMN collaboration fragment in
Fig. 4. Notably, this is only a way to model it:
the send task could be replaced by an intermediate
send event or by a message end event. However,
up to some technicalities, all cases behave in the
same way, thus we report here only one of them. Fig. 4: Send pattern. q
e1
e2
Textual
Specification. The
collaboration
fragment
in
Fig. 4
is
repre-
sented
in
the
textual
notation
as
C “ poolpp, Pq ∥poolpq, Qq
with
P
“
taskSndpe1, exp1, ϵ, m: ˜
exp2, e2q ∥P 1, where p is the sender and q a generic re-
ceiver (represented by a black-box pool in the graphical notation, whose process Q is
left unspecified in the textual one). m
p
Formal Semantics. According to the form of process P, and the current state xσ, αy of
pool p’s instance, the collaboration can evolve as follows: – Process P moves by executing Task 1. This execution step takes place by ap-
plying rule P-TaskSnd, which requires the incoming edge e1 of the task be
marked by at least one token (σpe1q ą 0), and the task’s guard exp1 be satisfied
(evalpexp1, α, trueq). The effects of the task execution are as follows: the mes-
sage action !m : ˜v is produced, where the message content ˜v results from the
evaluation of the expression tuple
˜
exp2 (evalp ˜
exp2, α, ˜vq), and the marking σ of
the process instance is updated with the movement of one token from e1 to e2,
that is σ1 “ incpdecpσ, e1q, e2q. 4
Patterns Formalisation In this section we present and formalise the Service Interaction Patterns [3] supported
by BPMN. Since BPMN is not specifically tailored to the needs of service interaction 5 patterns, the notation cannot completely support all their features. For instance, while
the informal and general description of these patterns leaves it open if in an interaction
the counter-party is known at design-time or not, in BPMN it is expected to have a
priori knowledge of the interacting partners, i.e. the target pool of a message edge
cannot be dynamically selected. On the other hand, in case a message is directed to a
multi-instance pool, BPMN supports a form of runtime binding of the message with
the correct process instance by means of the correlation mechanism [16, Sec. 8.3.2]. Moreover, it is also possible to dynamically specify other model features, such as the
number of involved participants and exchanged messages. Each pattern is presented
according to the following structure: Informal Description consists of a natural language description, and a graphical rep-
resentation in terms of a BPMN collaboration fragment. Textual Specification provides the textual notation of the BPMN collaboration model. extual Specification provides the textual notation of the BPMN collaboration model. ormal Semantics describes the operational rules applied to perform each execution
step, and shows the results in terms of the execution state functions evolution. p
Formal Semantics describes the operational rules applied to perform each execution
step, and shows the results in terms of the execution state functions evolution. p
In the following we present first those patterns concerning single transmissions,
both bilateral (Sec. 4.1-4.3) and multilateral (Sec. 4.4-4.7), and then the routing patterns
involving multiple transmissions (Sec. 4.8-4.9). 4.2
Receive Pattern Informal Description. A party receives a message from another party. This
q
e1
e2
m
p
Fig. 5: Receive pattern. pattern can be modelled as the BPMN collabora-
tion fragment shown in Fig. 5. Also here the in-
termediate receive event could be replaced, in this
case by a receive task or by a receiving start event. Textual Specification. The textual representation
of the collaboration fragment in Fig. 5 has again
the form C of the previous pattern, with Q “ interRcvpe1, m:˜t, e2q ∥Q1. Informal Description. A party receives a message from another party. This
q
e1
e2
m
p
Fig. 5: Receive pattern. pattern can be modelled as the BPMN collabora-
tion fragment shown in Fig. 5. Also here the in-
termediate receive event could be replaced, in this
case by a receive task or by a receiving start event. Textual Specification. The textual representation
of the collaboration fragment in Fig. 5 has again
the form C of the previous pattern, with Q “ interRcvpe1, m:˜t, e2q ∥Q1. Informal Description. A party receives a message from another party. This
q
e1
e2
m
pattern can be modelled as the BPMN collabora-
tion fragment shown in Fig. 5. Also here the in-
termediate receive event could be replaced, in this
case by a receive task or by a receiving start event. p
Fig. 5: Receive p
Textual Specification. The textual representation
of the collaboration fragment in Fig. 5 has again
the form C of the previous pattern, with Q “ interRcvpe1, m:˜t, e2q ∥Q1. Formal Semantics. Assuming that the intermediate receive event is enabled by a
token in e1, the process Q can perform a receiving action, that is the transition
xinterRcvpe1, m:˜t, e2q, σ, αy
?m : ˜et,ϵ
ÝÝÝÝÝÑ xincpdecpσ, e1q, e2q, αy is produced by apply-
ing rule P-InterRcv. Then, the process Q evolves by means of the interleaving rule
P-Int1. The produced label ?m: ˜et, ϵ indicates the willingness of process Q to con-
sume a message of type m matching the template ˜et. If present, the message is actually
consumed by rule C-Receive at collaboration level. 4.1
Send Pattern Therefore, the application of rule P-TaskSnd
produces the transition xtaskSndpe1, exp1, ϵ, m: ˜
exp2, e2q, σ, αy
!m :˜v
ÝÝÝÑ xσ1, αy,
where the data state α remains unchanged because no data object is connected 6 to Task 1. Hence, the overall process P can evolve according to the interleav-
ing rule P-Int1, that is xP, σ, αy
!m :˜v
ÝÝÝÑ xσ1, αy. Similarly, by applying the rule
C-Deliver, and then the interleaving rule at collaboration level, the execution
step of the overall collaboration C is represented by the transition xC, ι, δy
!m :˜v
ÝÝÝÑ
xupdI pι, p, txσ1, αyuq, addpδ, m, ˜vqy, with ιppq “ txσ, αyu. Its effects are: updating
the marking in the p’s instance (updI pι, p, txσ1, αyuq), and updating the message
state function (addpδ, m, ˜vq) by adding a value tuple ˜v to the m’s message list, in
order to be subsequently consumed by the receiving participant q. to Task 1. Hence, the overall process P can evolve according to the interleav-
ing rule P-Int1, that is xP, σ, αy
!m :˜v
ÝÝÝÑ xσ1, αy. Similarly, by applying the rule
C-Deliver, and then the interleaving rule at collaboration level, the execution step of the overall collaboration C is represented by the transition xC, ι, δy
!m :˜v
ÝÝÝÑ
xupdI pι, p, txσ1, αyuq, addpδ, m, ˜vqy, with ιppq “ txσ, αyu. Its effects are: updating
the marking in the p’s instance (updI pι, p, txσ1, αyuq), and updating the message
state function (addpδ, m, ˜vq) by adding a value tuple ˜v to the m’s message list, in
order to be subsequently consumed by the receiving participant q. step of the overall collaboration C is represented by the transition xC, ι, δy
!m : v
ÝÝÝÑ
xupdI pι, p, txσ1, αyuq, addpδ, m, ˜vqy, with ιppq “ txσ, αyu. Its effects are: updating
the marking in the p’s instance (updI pι, p, txσ1, αyuq), and updating the message
state function (addpδ, m, ˜vq) by adding a value tuple ˜v to the m’s message list, in
order to be subsequently consumed by the receiving participant q. q
y
y
g p
p
– Process P moves by executing an (unspecified) activity of P 1. 4.2
Receive Pattern Indeed, this rule requires that there
is a message in the m’s message queue (˜v P δpmq) that matches the template ˜et of the
receiving event (matchp ˜et, ˜vq “ A); the assignments A produced by this matching are
then applied to the data state α of the q’s instance, and the message is removed from
the queue (rmpδ, m, ˜vq). 4.1
Send Pattern Thus, we have a
transition xP 1, σ, αy
ℓÝÑ xσ1, α1y, from which P can evolve by means of the sym-
metric rule of P-Int1, and the overall collaboration can then evolve accordingly. This execution step, anyway, is not relevant for the pattern semantics, and hence is
not discussed in more detail. – Process Q moves by executing an (unspecified) activity. Again this execution step
is not relevant for the pattern semantics. p
Task 1
e1
e2 Relying on asynchronous communication, we are able to formalise an unreliable
and non-guaranteed delivery. The sending action in fact just updates the message state
function by adding a message, without requiring this to be received. m
q 4.3
Send/Receive Pattern Informal Description. Two parties, p and q, engage in two causally related interactions. In the first interaction, p sends a message (the request) to q, while in the second one p
receives a message (the response) from q. 7 p
Task 1
m
Task 1
q
Task 3
m1
m2
e1
e2
e2
e1
e3
e4
e5
e6
1
2
q
Fig. 6: Send/Receive pattern. This pattern can be modelled by
combining the Send and the Receive
patterns, as shown in Fig. 6. Textual Specification. The textual
representation of the collaboration
fragment in Fig. 6 has again the form
C of the previous patterns, with
P “ taskSndpe1, exp1, ϵ, m1: ˜
exp2, e2q ∥interRcvpe2, m2: ˜t1, e3q ∥P 1
Q “ interRcvpe4, m1: ˜t2, e5q ∥taskSndpe5, exp3, ϵ, m2: ˜
exp4, e6q ∥Q1 Task 1
e1
e2
This pattern can be modelled by
combining the Send and the Receive
patterns, as shown in Fig. 6. m
q
Textual Specification. The textual
representation of the collaboration
fragment in Fig. 6 has again the form
C of the previous patterns, with P “ taskSndpe1, exp1, ϵ, m1: ˜
exp2, e2q ∥interRcvpe2, m2: ˜t1, e3q ∥P 1
Q “ interRcvpe4, m1: ˜t2, e5q ∥taskSndpe5, exp3, ϵ, m2: ˜
exp4, e6q ∥Q1 m
p
Formal Semantics. The execution steps of this pattern are realised by combining
the semantic rules for the Send and Receive patterns. In detail: let us suppose that
there is a token in the incoming edge of Task 1 (σpe1q ą 0) and the other pre-
conditions of rule P-TaskSnd are satisfied; by applying this rule we have that
xtaskSndpe1, exp1, ϵ, m1: ˜
exp2, e2q, σ, αy
!m1 :˜v
ÝÝÝÑ xincpdecpσ, e1q, e2q, αy. Then, P
evolves by performing a sending action, by means of the interleaving rule P-Int1, that
is xP, σ, αy
!m1 :˜v
ÝÝÝÑ xσ1, αy. At the collaboration layer, by applying rule C-Deliver, the
message m1 is delivered to q. Now, on the receiving party, assuming that there is a to-
ken on e4 and that the template ˜t2 evaluates to ˜
et2, by applying rules P-InterRcv and
P-Int1, we have xQ, σ2, α2y
?m1 : ˜
et2,ϵ
ÝÝÝÝÝÝÑ xincpdecpσ2, e4q, e5q, α2y. The observed label
indicates the willingness to receive a message of type m1. 4.3
Send/Receive Pattern Thus, at collaboration level,
rule C-Receive can be applied to allow process Q to actually consume the sent request
message. Now, Task 3 is enabled and, by proceeding in a specular way, Q can send the
response message m2 and P can consume it. m
p
Formal Semantics. The execution steps of this pattern are realised by combining
the semantic rules for the Send and Receive patterns. In detail: let us suppose that
there is a token in the incoming edge of Task 1 (σpe1q ą 0) and the other pre-
conditions of rule P-TaskSnd are satisfied; by applying this rule we have that
xtaskSndpe1, exp1, ϵ, m1: ˜
exp2, e2q, σ, αy
!m1 :˜v
ÝÝÝÑ xincpdecpσ, e1q, e2q, αy. Then, P
evolves by performing a sending action, by means of the interleaving rule P-Int1, that
is xP, σ, αy
!m1 :˜v
ÝÝÝÑ xσ1, αy. At the collaboration layer, by applying rule C-Deliver, the
message m1 is delivered to q. Now, on the receiving party, assuming that there is a to-
ken on e4 and that the template ˜t2 evaluates to ˜
et2, by applying rules P-InterRcv and 4.4
Racing Incoming Messages Pattern Informal Description. A party expects to receive one among a set of messages. These
messages may be structurally different (i.e. different types) and may come from differ-
ent categories of partners. The way a message is processed depends on its type and/or
the category of partner from which it comes. This pattern can be modelled in BPMN by using in the receiving participant an
event-based gateway connected to receiving events. Messages can be expected from
one participant (Fig. 7) or they can arrive from different participants (Fig. 8). Textual Specification. Let us first consider the case in which messages arrive from one
participant (Fig. 7). In the textual notation the diagram is rendered as the collaboration
of the usual form C, with P “ xorSplitpe1, tpe2, exp1q, pe3, exp2quq ∥taskSndpe2, exp3, ϵ, m1: ˜
exp4, e4q ∥
taskSndpe3, exp5, ϵ, m2: ˜
exp6, e5q ∥P 1
Q
B
dp 6 p 1 ˜
7q p 2 ˜
8qq ∥Q1 Q “ eventBasedpe6, pm1:˜t1, e7q, pm2:˜t2, e8qq ∥Q1 Q “ eventBasedpe6, pm1:˜t1, e7q, pm2:˜t2, e8qq ∥Q1 Q “ eventBasedpe6, pm1:˜t1, e7q, pm2:˜t2, e8qq ∥Q1 The case in which messages arrive from two different participants (Fig. 8) is ren-
dered in the textual notation as C “ poolpp, P 2q ∥poolpr, Rq ∥poolpq, Qq, where 8 P
P
p
Task 1
Task 2
q
e1
e2
e3
e4
e5
e6
e7
e8
m1
m2
A
m
e1
e2
P
R
Task A
B
e1
e2
e3
e4
A
N
e2
e1
e(n-1)
m1
Q
Fig. 7: Racing incoming messages (a). q
e6
e7
e8
m1
m2
p
r
Fig. 8: Racing incoming messages (b). process Q is as the above one, while P 2 and R are left unspecified (because they are
included in black-box pools). p
Task 1
Task 2
q
e1
e2
e3
e4
e5
e6
e7
e8
m1
m2
Fig. 7: Racing incoming messages (a). P
A
m
e1
e2
Q
q
e6
e7
e8
m1
m2
p
r
Fig. 8: Racing incoming messages (b). Fig. 8: Racing incoming messages (b). Fig. 7: Racing incoming messages (a). P
P
R
T
k A
B
e1
e2
e3
e4
A
N
e2
e1
e(n-1)
m1
process Q is as the above one, while P 2 and R are left unspecified (because they are
included in black-box pools). Qk
Q1
Q
e5
e6
e7
m1
m2
e(k-1)
ek
m
Formal Semantics. 4.4
Racing Incoming Messages Pattern Let us start with the case in which messages arrive from a single
participant, and assume that a token is available in the incoming edge of the XOR split
gateway of P (σpe1q ą 0) and the conditional expression exp1 is evaluated to true
(evalpexp1, α, trueq). Thus, rule P-XorSplit1 can be applied and the token is moved
to the edge e2, hence enabling Task 1. Formally, this step corresponds to the transition
xP, σ, αy ϵÝÑ xincpdecpσ, e1q, e2q, αy, where label ϵ denotes the movement of the token
internally to the process. The next step corresponds to the execution of Task 1, which
is as in the case of the Send pattern. Once the message m1 has been sent (hence, there
exists a ˜v such that ˜v P δpm1q), and assuming that there is a token in e6 (σpe6q ą 0),
the event-based gateway can evolve by applying the corresponding rule. This corre-
sponds to the transition xeventBasedpe6, pm1 :˜t1, e7q, pm2:˜t2, e8qq, σ1, α1y
?m1 : ˜
et1,ϵ
ÝÝÝÝÝÝÑ
xincpdecpσ1, e6q, e7q, α1y, with template ˜et1 matching the message ˜v. The rule moves
the token from the incoming edge to the outgoing edge corresponding to the received
message. The produced label enables the application of rule C-Receive at collaboration
level, which takes care of consuming the message ˜v of type m1 in δ. The case where
message m2 is selected to be sent is similar. In the scenario shown in Fig. 8, even if the transitions produced by the collaboration
have the same labels, the pattern semantics is quite different. In fact, in the previous
case the organisation p internally decides which message will be sent and only one
message will be delivered and consumed, while in this case the organisations p and r
act independently from each other and it may occur that both m1 and m2 are sent to q. In such a case, one of the two messages will be consumed, depending on their arrival
time, and the other message will be pending forever. 4.5
One-To-Many Send Pattern Informal Description. A party sends messages to several parties. All messages have the
same type (although their contents may be different). The number of parties to whom
the message is sent may or may not be known at design time. In BPMN, this pattern can be modelled as the collaboration fragment in Fig. 9,
where each party is represented as an instance of a multi-instance pool and a message
is sent to each process instance via a sequential multi-instance send task. From now on, when a message is sent/received to/by several parties, we will
model these parties as a multiple instance pool. This is the most interesting 9 among various interpretations which are not considered in this work (e.g., repre-
senting multiple receiving parties as different single-instance pools). We can have
p
A
B
e4
e5
Task 1
m
e1
e2
that the number of sent messages is either known
at design time (by setting the LoopCardinality at-
tribute of the send task) or it is read from a data
object during the process execution. p
Task 1
m
e1
e2
q
Fig. 9: One-To-Many-Send. e7
e8
m1
m2
e2
e1
e(n 1)
q
Fig. 9: One-To-Many-Send. Textual Specification. Here, to keep the pattern
formalisation more manageable, the sequential
multi-instance task is rendered as a macro. The macro encloses the task in a FOR-
loop expressed by means of a pair of XOR join and split gateways, and an additional
data object c1 for the loop counter. In the textual notation we have C “ poolpp, Pq ∥
miPoolpq, Qq, where process Q is left unspecified and in P the attribute LoopCardinal-
ity is set to n:
P
A
e1
e2 Fig. 9: One-To-Many-Send. Fig. 9: One-To-Many-Send. P
m1
r
P “ xorJoinpte1, e13u, e11q ∥taskSndpe11, c1.c ‰ null, c1.c :“ c1.c ` 1, m: ˜
exp1, e12q
∥xorSplitpe12, tpe13, c1.c ď nq, pe2, defaultquq ∥P 1
e7
m Qk
Q1
e3
e3
m1
m2
m
Formal semantics. The execution steps are realised as in the previous cases, by repeat-
edly applying the semantic rules of the XOR gateway and the send task. It is worth
noticing that at each application of rule P-TaskSnd the field c of the data object c1 is
updated with the assignment c1.c :“ c1.c ` 1. At the end of the pattern execution, the
message list δpmq contains n sent messages. 4.5
One-To-Many Send Pattern Q
e3
e4
e5 4.6
One-From-Many Receive Pattern
P Informal Description. A party receives several logically related messages arising from
autonomous events occurring at different parties. The arrival of messages must be
timely so that they can be correlated as a single logical request. The interaction may
complete successfully or not depending on the messages gathered. In this pattern the
receiver does not know the number of messages that will arrive, and stops waiting as
soon as a certain number of messages have arrived or a timeout occurs. Q
C
m1
m2
e4
e5 g
This pattern can be modelled as the collaboration fragment shown in Fig 10. q
Task 1
e1
e3
e2
e4
m
p
Fig. 10: One-From-Many-Receive. q
Task 1
e1
e3
e2
e4
m
p
Fig. 10: One-From-Many-Receive. Textual Specification. Also in this case, to sim-
plify the formal treatment, we rely on a macro
for the multi-instance receive task with a timer. In particular, the multi-instance behaviour is rep-
resented by enclosing the receive task in a FOR-
loop (as for the sequential multi-instance task). The timer attached to the receive task is instead
abstracted via a non-deterministic choice, by resorting to a race condition. In detail, the
receiving party q will get, via an event-based gateway, either a message from a sending
party (i.e., an instance of p) or a time-out message from a specific pool t representing
the timer. In the textual notation we have C “ miPoolpp, Pq ∥poolpq, Qq ∥poolpt, Tq,
where Fig. 10: One-From-Many-Receive. 10 Q “ taskSndpe1, exp1, ϵ, mstartTimer : ˜
exp2, e1q ∥xorJoinpte1, evu, e2q ∥
eventBasedpe2, pm:˜t1, e3q, pmtimeout :˜t2, e3qq ∥
taskpe3, c1.c ‰ null, c1.c :“ c1.c ` 1, eivq ∥
xorSplitpeiv, tpev, c1.c ď nq, pe2, defaultquq ∥xorJoinpte2, e3u, e4q ∥Q1
T “ startRcvpmstartTimer : ˜t3, e5q ∥taskSndpe5, exp3, ϵ, mtimeout : ˜
exp4, e6q ∥endpe6q Formal Semantics. Once a token arrives at e1 in the process Q, a mstartTimer message
is sent to the pool t by means of the send task, in order to activate an instance of the
timer process T. This instance will perform a send task, delivering a message mtimeout,
to signal that the timeout is expired, and then it terminates. As effect of the execution of
the send task in Q, a token is moved in e1, which enables the looping behaviour regulated
by the XOR gateways. 4.6
One-From-Many Receive Pattern
P At each iteration, the event-based gateway consumes either a
message m or mtimeout; in the former case the non-communicating task increments
the loop counter and the execution of another interaction is evaluated (by means of the
XOR split conditions), while in the latter case the edge e3 is followed and the pattern
execution completes. 4.7
One-To-Many Send/Receive Pattern Informal Description. A party sends a request to several other parties. Responses are
expected within a given time-frame. However, some responses may not arrive within
the time-frame and some parties may even not respond at all. This pattern can be rendered as the collaboration fragment in Fig. 11. A practical
use of this pattern is shown in the scenario in Fig. 1. q
p
Task 1
Task 2
Task 3
m1
m2
e2
e3
e4
e8
e9
e1
e5
e7
e6
Fig. 11: One-To-Many Send/Receive. Textual
Specification. This
pat-
tern relies on a multi-instance sub-
process with a specific form, i.e. it
is characterised by a sequence of a
send task and a receive task, pro-
ceeded and followed by a start and
an end event, respectively. As usual,
to simplify the formal treatment we
resort to a macro. In this case, it con-
sists of a sequential send task followed by a multi-instance receive task with a timer. We have C “ poolpp, Pq ∥miPoolpq, Qq, where process P is rendered in terms of
macros as already shown in the previous patterns (hence, for the sake of presentation,
its specification is omitted), while process Q is as follows: q
p
Task 1
Task 2
Task 3
m1
m2
e2
e3
e4
e8
e9
e1
e5
e7
e6
Fig. 11: One-To-Many Send/Receive. Textual
Specification. This
pat-
tern relies on a multi-instance sub-
process with a specific form, i.e. it
is characterised by a sequence of a
send task and a receive task, pro-
ceeded and followed by a start and
an end event, respectively. As usual,
to simplify the formal treatment we
resort to a macro. In this case, it con-
sists of a sequential send task followed by a multi-instance receive task with a timer. We have C “ poolpp, Pq ∥miPoolpq, Qq, where process P is rendered in terms of
macros as already shown in the previous patterns (hence, for the sake of presentation,
its specification is omitted), while process Q is as follows: q
p
Task 1
Task 2
Task 3
m1
m2
e2
e3
e4
e8
e9
e1
e5
e7
e6
Fig. 11: One-To-Many Send/Receive. Fig. 11: One-To-Many Send/Receive. Fig. 11: One-To-Many Send/Receive. resort to a macro. In this case, it con-
sists of a sequential send task followed by a multi-instance receive task with a timer. 4.7
One-To-Many Send/Receive Pattern Now, the correlation takes place according
to the data states, which we assume to be as follows: α1pd.fq “ α2pd.fq “ “foo”,
α1pd.idq “ 7, and α2pd.idq “ 5. Therefore, the first message is delivered to the second
instance, updating α2 with the assignment d.code “9876, while the second message is
delivered to the first instance, updating α1 with the assignment d.code “1234. 4.8
Request with Referral Pattern In the textual specification we have C
“
poolpp, Pq
∥
poolpq, Qq ∥poolpr, Rq, where: P “ startpeenb, e1q ∥taskSndpe1, exp1, A1, m1: ˜
exp2, e2q ∥interRcvpe2, m2: ˜t1, e3q ∥
taskSndpe3, exp3, A2, m3: ˜
exp4, e4q ∥interRcvpe4, m4: ˜t2, e5q ∥endpe5q and Q and R are defined in a similar way. and Q and R are defined in a similar way. Formal Semantics. The execution steps and their results are simply realised by applying
the semantic rules for the different BPMN elements, as already shown for the previous
patterns. It is up to the message sent by pool p to pool r to specify in its content the
reference to pool q, whose process waits for the routed message. 4.7
One-To-Many Send/Receive Pattern We have C “ poolpp, Pq ∥miPoolpq, Qq, where process P is rendered in terms of
macros as already shown in the previous patterns (hence, for the sake of presentation,
its specification is omitted), while process Q is as follows: Q “ interRcvpe7, m1: ˜t1, e8q ∥taskSndpe8, exp, A, m2: ˜
exp, e9q ∥Q1 Q “ interRcvpe7, m1: ˜t1, e8q ∥taskSndpe8, exp, A, m2: ˜
exp, e9q ∥Q1 Formal Semantics. In this pattern we have that process P sends out, by means of rule
P-TaskSnd, several messages of type m1 that need to be properly correlated with the
correct process instance of Q. The content of the messages themselves provides the
correlation information. For example, let us assume that two messages of type m1 are
sent to q, and that consist of three fields, say x“foo”, 5, 1234y and x“foo”, 7, 9876y. Also, let us consider the case where there are two receiving instances, i.e. ιpqq “ Formal Semantics. In this pattern we have that process P sends out, by means of rule
P-TaskSnd, several messages of type m1 that need to be properly correlated with the
correct process instance of Q. The content of the messages themselves provides the
correlation information. For example, let us assume that two messages of type m1 are
sent to q, and that consist of three fields, say x“foo”, 5, 1234y and x“foo”, 7, 9876y. Also, let us consider the case where there are two receiving instances, i.e. ιpqq “ 11 txσ1, α1y, xσ2, α2yu, and that template ˜t1 of the intermediate receiving event is defined
as xd.f, d.id, ?d.codey, meaning that the fields f and id of the data object d identify cor-
relation data while code is a formal field. Now, the correlation takes place according
to the data states, which we assume to be as follows: α1pd.fq “ α2pd.fq “ “foo”,
α1pd.idq “ 7, and α2pd.idq “ 5. Therefore, the first message is delivered to the second
instance, updating α2 with the assignment d.code “9876, while the second message is
delivered to the first instance, updating α1 with the assignment d.code “1234. txσ1, α1y, xσ2, α2yu, and that template ˜t1 of the intermediate receiving event is defined
as xd.f, d.id, ?d.codey, meaning that the fields f and id of the data object d identify cor-
relation data while code is a formal field. 4.8
Request with Referral Pattern r
q
p
Task 1
Task 3
Task 4
Task 5
e1
e2
e3
e4
e5
e6
e7
e8
e9
e10
e11
m1
m2
m3
m4
m5
Task 2
Fig. 12: Request with Referral pattern. 1 r
q
p
Task 1
Task 3
Task 4
Task 5
e1
e2
e3
e4
e5
e6
e7
e8
e9
e10
e11
m1
m2
m3
m4
m5
Task 2
Fig. 12: Request with Referral pattern. Informal Description. A party p
sends a request to another party
q indicating that any follow-up
should be sent to another party
r. An example of a BPMN col-
laboration involving the request
with referral pattern is shown in
Fig. 12, and also in the com-
munication between the Retailer
and the Logistic Provider in Fig. 1. r
q
p
Task 1
Task 3
Task 4
Task 5
e1
e2
e3
e4
e5
e6
e7
e8
e9
e10
e11
m1
m2
m3
m4
m5
Task 2
Fig. 12: Request with Referral pattern. Informal Description. A party p
sends a request to another party
q indicating that any follow-up
should be sent to another party
r. An example of a BPMN col-
laboration involving the request
with referral pattern is shown in
Fig. 12, and also in the com-
munication between the Retailer
and the Logistic Provider in Fig. 1. Textual Specification. In the textual specification we have C
“
poolpp, Pq
∥
poolpq, Qq ∥poolpr, Rq, where:
P “ startpeenb, e1q ∥taskSndpe1, exp1, A1, m1: ˜
exp2, e2q ∥interRcvpe2, m2: ˜t1, e3q ∥
taskSndpe3, exp3, A2, m3: ˜
exp4, e4q ∥interRcvpe4, m4: ˜t2, e5q ∥endpe5q r
q
p
Task 1
Task 3
Task 4
Task 5
e1
e2
e3
e4
e5
e6
e7
e8
e9
e10
e11
m1
m2
m3
m4
m5
Task 2
Fig. 12: Request with Referral pattern. 1 Informal Description. A party p
sends a request to another party
q indicating that any follow-up
should be sent to another party
r. An example of a BPMN col-
laboration involving the request
with referral pattern is shown in
Fig. 12, and also in the com-
munication between the Retailer
and the Logistic Provider in Fig. 1. Fig. 12: Request with Referral pattern. Textual Specification. In the textual specification we have C
“
poolpp, Pq
∥
poolpq, Qq ∥poolpr, Rq, where: Textual Specification. while P and Q are defined in a similar way. while P and Q are defined in a similar way. Formal Semantics. Similar to the previous pattern, the formal semantics of this pattern
is determined by the application of rules for sending and receiving message already
described, except for the AND split gateway that simply consumes a token in e7 and,
simultaneously, produces one token in e8 and one token in e9. 3 http://pros.unicam.it/mida 5
Patterns Animation via MIDA The MIDA (Multiple Instances and Data Animator) tool3 is a web application writ-
ten in JavaScript, based on the Camunda bpmn.io modeller. MIDA, whose graphical
interface is shown in Fig. 14, is an animator of collaboration models that can involve
multiple instances and data objects. MIDA animates process models by means of the
visualisation of tokens flow and data evolution. To correctly enact the collaboration be-
haviour, the implementation of the tool relies on the formalisation presented in Sec. 3. The core feature of MIDA
is the model animation. It re-
sults helpful both in educational
contexts, for explaining the be-
haviour of BPMN elements, and
in practical modelling activities,
for debugging errors. In fact, de-
signers can achieve a precise un-
derstanding of the behaviour of
processes and collaborations by
means of the visualisation of the Fig. 14: MIDA tool interface. The core feature of MIDA
is the model animation. It re-
sults helpful both in educational
contexts, for explaining the be-
haviour of BPMN elements, and
in practical modelling activities,
for debugging errors. In fact, de-
signers can achieve a precise un-
derstanding of the behaviour of
processes and collaborations by
means of the visualisation of the model execution. Fig. 14: MIDA tool interface. The core feature of MIDA
is the model animation. It re-
sults helpful both in educational
contexts, for explaining the be-
haviour of BPMN elements, and
in practical modelling activities,
for debugging errors. In fact, de-
signers can achieve a precise un-
derstanding of the behaviour of
processes and collaborations by
means of the visualisation of the model execution. Fig. 14: MIDA tool interface. Fig. 14: MIDA tool interface. We have exploited MIDA to model and animate the interaction patterns presented
in Sec. 4, providing an intuitive knowledge of their behaviour. We have also used
MIDA to animate the motivating example in Fig. 1, thus showing how the tool sup-
ports the study of more intricate scenarios resulting from the combination of var-
ious patterns. These animations are available from http://pros.unicam.it/
service-interaction-patterns/. 4.9
Relayed Request Pattern r
q
p
Task 1
Task 2
Task 3
Task 4
e1
e2
e3
e4
e5
e6
e8
e7
e9
e10
e11
m1
m2
m3
m4
Fig. 13: An example of Relayed Request pattern. Informal Description. A party
p makes a request to party q,
which delegates the request pro-
cessing to another party r. This
latter party interacts with party
p while party q observes a view
of the interactions. This pattern can be rendered
as the collaboration fragment in
Fig. 13. Fig. 13: An example of Relayed Request pattern. 12 Textual Specification. In the textual notation the pattern is rendered as follows: C “
poolpp, Pq ∥poolpq, Qq ∥poolpr, Rq, where: Textual Specification. In the textual notation the pattern is rendered as follows: C “
poolpp, Pq ∥poolpq, Qq ∥poolpr, Rq, where: R “ startRcvpm2: ˜t1, e7q ∥andSplitpe7, te8, e9uq ∥
taskSndpe8, exp1, ϵ, m4: ˜
exp2, e10q ∥taskSndpe9, exp3, ϵ, m3: ˜
exp4, e11q ∥R1 6
Related Work The most common interaction scenarios from a business perspective, named Service
Interaction Patterns, have been described in [3,17]. However, they lack of visualisation
as well as formal semantics. Since then, effort has been devoted to visualise [5,1,9] and 13 formalise these patterns [10,2,15], as shown in Table 1. This provides a comparison
among the state-of-the-art approaches dealing with service interaction patterns with
respect to: (i) the language used for patterns specification, (ii) the main contribution
of the work and (iii) its limitations. In the following, these works are compared to the
contribution of this paper. Paper Year
Language
Contribution
Limitations
[2]
2005
ASM
Formalisation and extension
No models analysis
[10]
2006
π-calculus
Formalisation
Ambiguities
[15]
2007
CPN
Formalisation and extension
No correlation
[9]
2008
BPMN 2.0
iBPMN: extension for interaction modelling
No formalisation
[1]
2009
Open Nets
Overview of services domain challenges
No formalisation
[5]
2014
BPMN 2.0
Extension of BPMN supported patterns
No formalisation
Table 1: Review on the Service Interaction Patterns literature. Considering the patterns specified in BPMN, relevant works are [5,9]. Campagna et
al. [5] discuss BPMN 2.0 support for the service interaction patterns and propose a set
of enhancements to broaden it. However, they do not formalise these patterns and thus,
they do not provide formal validation of the proposed solutions. Decker and Barros [9]
introduce iBPMN, a set of extensions to the BPMN standard for interaction modelling. They show that most service interaction patterns can be expressed using iBPMN and
present an algorithm for deriving interface behaviour models from simple interaction
models. However, they do not aim at providing a formal characterisation of the proposed
extensions. Both the above works are more interested in overcoming BPMN lacks for
supporting interaction patterns rather than clarifying the semantics of the supported
patterns, which is instead a major challenge when using BPMN collaborations [8]. Abstract State Machines (ASM) [4], π-calculus [14] and Petri Nets [12] have been
proposed as a solid ground to formalise service interaction patterns. The first formal-
ization of the patterns was given by Barros and Börger [2] proposing a compositional
framework and interaction flows. They provide ASM for eight service interaction sce-
narios and illustrate how, by combinations and refinements of them, one can define
arbitrarily complex interaction patterns. The ASMs offer an implementation draft of
the patterns, but are less suited for the analysis of collaborations. 6
Related Work Decker and Puhlmann
[10] provide a formalisation of service interactions via π-calculus as a first step to anal-
yse collaborations. However, their work shows still some ambiguities. For instance, the
Racing Incoming Messages pattern allows to receive multiple messages at once, but the
work does not clarify how one among the competing messages is chosen for consump-
tion. Moreover, the authors refer to a synchronous communication model, not compliant
with the BPMN standard. Mulyar et al. [15] formalise the semantics of the interaction
patterns by means of Coloured Petri Nets (CPN). Moreover, they extend the scope of
the original service interaction patterns by describing various pattern variants. How-
ever, even if Petri Nets provide support for multiple instance patterns, process instances
are characterised by their identities, rather than by the values of their data, which are
necessary for correlation [11]. Finally, van der Aalst et al. [1] provide an overview of
the challenges in the domain of service interaction patterns and they propose to use
open nets as a formal framework for addressing these challenges. However, they do not 14 aim at formalising the interaction patterns, of which they only provide a brief descrip-
tion. Differently from the mentioned works, we focus on BPMN by directly defining
the semantics of the supported patterns, thus avoiding the mapping to other formalisms
equipped with their own semantics. 7
Concluding Remarks In this work we focus on service interaction patterns, visualising them in BPMN col-
laborations and providing a comprehensive formalisation by means of a direct formal
semantics for BPMN collaboration diagrams. This allows to validate the semantics in
[6], as we show it is suitable to cover the interaction patterns expressed in BPMN. Fur-
ther, the animation tool MIDA has been exploited to model and animate the BPMN
collaborations, allowing an intuitive understanding of the patterns execution. As a future work, we plan to investigate the formalisation of new BPMN interaction
patterns where data objects play a more central role. References 1. van der Aalst, W.M., Mooij, A.J., Stahl, C., Wolf, K.: Service interaction: Patterns, formal-
ization, and analysis. In: Formal Methods for Web Services. pp. 42–88. Springer (2009) y
pp
p
g
(
)
2. Barros, A., Börger, E.: A compositional framework for service interaction patterns and inter-
action flows. In: Formal Engineering Methods. pp. 5–35. Springer (2005) 3. Barros, A., Dumas, M., Ter Hofstede, A.H.: Service interaction patterns. In: Business Process
Management, pp. 302–318. Springer (2005) 4. Börger, E., Thalheim, B.: Modeling Workflows, Interaction Patterns, Web Services and Busi-
ness Processes: The ASM-Based Approach. In: ASM, B and Z, pp. 24–38. Springer (2008) 5. Campagna, D., Kavka, C., Onesti, L.: Enhancing BPMN 2.0 support for service interaction
patterns. In: Software Engineering and Applications (ICSOFT-EA) (2014) 6. Corradini, F., Muzi, C., Re, B., Tiezzi, F., Rossi, L.: Animating multiple instances in bpmn
collaborations: from formal semantics to tool support. In: BPM 2018. LNCS, Springer
(2018), to appear 7. Corradini, F., Muzi, C., Re, B., Tiezzi, F., Rossi, L.: Mida: Multiple instances and data ani-
mator. In: BPM 2018 (Demo). LNCS, Springer (2018), to appear 8. Cortes-Cornax, M., Dupuy-Chessa, S., Rieu, D.: Choreographies in BPMN 2.0: new chal-
lenges and open questions. In: ZEUS. vol. 847, pp. 50–57. CEUR-WS.org (2012) 9. Decker, G., Barros, A.: Interaction modeling using BPMN. LNCS 4928, 208–219 (2008) ,
,
,
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g
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(
)
10. Decker, G., Puhlmann, F., Weske, M.: Formalizing service interactions. In: International
Conference on Business Process Management. pp. 414–419. Springer (2006) 11. Decker, G., Weske, M.: Instance isolation analysis for service-oriented architectures. In:
SCC. vol. 1, pp. 249–256. IEEE (2008) pp
12. Dijkman, R.M., Dumas, M., Ouyang, C.: Semantics and analysis of business process models
in BPMN. Information and Software Technology 50(12), 1281–1294 (2008) 13. Dumas, M., La Rosa, M., Mendling, J., Reijers, H.A.: Fundamentals of business process
management. Springer (2013) 14. Milner, R.: A Calculus of Communicating Systems. Springer, Secaucus, NJ, USA (1980) 15. Mulyar, N., van der Aalst, W.M., Aldred, L., Russell, N.: Service interaction patterns: A
configurable framework. management 34, 29 (2007) g
g
16. OMG: Business Process Model and Notation (BPMN V 2.0) (2011) 17. Weske, M.: Business Process Management. Springer (2012) 15
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Intellectual support for the analysis of the implementation of innovative development programs of the regional agro-industrial cluster
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IOP Conference Series: Earth and Environmental Science IOP Conference Series: Earth and Environmental Science You may also like
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al. - PAPER • OPEN ACCESS Intellectual support for the analysis of the
implementation of innovative development
programs of the regional agro-industrial cluster View the article online for updates and enhancements. This content was downloaded from IP address 188.170.217.49 on 09/12/2021 at 13:21 This content was downloaded from IP address 188.170.217.49 on 09/12/2021 at 13:21 This content was downloaded from IP address 188.170.217.49 on 09/12/2021 at 13:21 AGRITECH-V-2021
IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
IOP Publishing
doi:10.1088/1755-1315/839/2/022068 AGRITECH V 2021
IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
IOP Publishing
doi:10.1088/1755-1315/839/2/022068 onf. Series: Earth and Environmental Science 839 (2021) 022068
doi:10.1088/1755-1315/839/2/0220 V A Lomazov1, A V Lomazov2, O A Ivashchuk2, O S Akupiyan1 and
E V Nesterova2 1 Belgorod State Agricultural University named after V.Gorin, 1, Vavilova st.,
Mayskiy, Belgorod region, 308503, Russia
2 Belgorod State National Research University, 85, Pobedy st., Belgorod, 308015,
Russia Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd
1 E-mail: vlomazov@yandex.ru Abstract. The paper is devoted to the problems of using modern digital technologies to manage
the implementation of programs for the development of the regional economy’s agro-industrial
cluster. An approach based on the use of intelligent (knowledge-oriented) information systems
for analyzing the progress of program implementation is proposed. As a model for representing
knowledge about the subject area, it is proposed to use the apparatus of the linguistic variable
theory and fuzzy production rules, which makes it possible to take into account the high level of
uncertainty inherent in the agricultural sector of the economy. The inference engine included in
the information system is based on explicitly interpreted Mamdani's procedure of fuzzy logical
inference, which makes it possible to form explanations of the course of reasoning. The
developed structure of the intelligent information system is a concretization and extension of the
traditional one, taking into account the reflection of the specifics of the tasks of managing the
implementation of programs for the development of the agro-industrial cluster. The preliminary
results of the experimental operation of the research prototype of the developed system can serve
as confirmation of the effectiveness of the proposed design solutions. Intellectual support for the analysis of the implementation of
innovative development programs of the regional agro-
industrial cluster V A Lomazov1, A V Lomazov2, O A Ivashchuk2, O S Akupiyan1 and
E V Nesterova2
1 Belgorod State Agricultural University named after V.Gorin, 1, Vavilova st.,
Mayskiy, Belgorod region, 308503, Russia
2 Belgorod State National Research University, 85, Pobedy st., Belgorod, 308015,
Russia 1. Introduction Improving the functioning of the agricultural sector of the national economy is impossible without
permanent attention of society and the state, interested not only in profitability of agribusiness, but also
in ensuring integrated sustainable development of rural areas [1], including, in particular, issues of not
only employment, but also improving the quality of life in rural areas. the general population [2];
introduction of knowledge intensive high-performance technologies [3]; environmental friendliness of
both production processes and agricultural products [4]. The use of an innovative approach to solving
these problems is associated not only with positive aspects [5-7], but also with risks (caused, in
particular, by insufficient scientific study and absence of experience in using the latest agro-industrial
technologies). This leads to the need for thorough working-out and monitoring of the implementation
of projects and programs for the development of agro-industrial systems. The purpose of this study is to
develop a toolkit for intellectual support for the analysis of the implementation of programs for the
development of a regional agro-industrial cluster. The toolkit should be able to take into account the 1 AGRITECH-V-2021 AGRITECH-V-2021
IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
IOP Publishing
doi:10.1088/1755-1315/839/2/022068 IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
g
doi:10.1088/1755-1315/839/2/022068 onf. Series: Earth and Environmental Science 839 (2021) 022068
doi:10.1088/1755-1315/839/2/02206 high level of uncertainty characteristic of innovative agricultural production. The models and methods
of artificial intelligence that underlie the developed information and analytical system (used for
processing expert knowledge) make it possible to reduce the time for making and increase the scientific
validity of managerial decisions with possible correction of projects (programs). high level of uncertainty characteristic of innovative agricultural production. The models and methods
of artificial intelligence that underlie the developed information and analytical system (used for
processing expert knowledge) make it possible to reduce the time for making and increase the scientific
validity of managerial decisions with possible correction of projects (programs). thods 2. Materials and methods Within the framework of the indicative approach, we will assume that the implementation of the program
is associated with the achievement of social, environmental, economic, agro-technological and
scientific-innovative indicators of the regional agro-industrial cluster at the specified time points t1, t2,...,
tn values, not worse than the normative ones. Formally this can be written in the form of a system of
restrictions (inequalities): IndSoc -j (ti) ≤ NormIndSoc –j (ti),
IndSoc +j (ti) NormIndSoc +j (ti)
IndEcol -j (ti) ≤ NormIndEcol –j (ti),
IndEcol +j (ti) NormIndEcol +j (ti)
IndEcon -j (ti) ≤ NormIndEcon –j (ti),
IndEcon +j (ti) NormIndEcon +j (ti)
(1)
IndTech -j (ti) ≤ NormIndTech –j (ti),
IndTech +j (ti) NormIndTech +j (ti)
IndIn -j (ti) ≤ NormIndIn –j (ti),
IndIn +j (ti) NormIndIn +j (ti) IndSoc -j (ti) ≤ NormIndSoc –j (ti),
IndSoc +j (ti) NormIndSoc +j (ti)
IndEcol -j (ti) ≤ NormIndEcol –j (ti),
IndEcol +j (ti) NormIndEcol +j (ti)
IndEcon -j (ti) ≤ NormIndEcon –j (ti),
IndEcon +j (ti) NormIndEcon +j (ti)
(1)
IndTech -j (ti) ≤ NormIndTech –j (ti),
IndTech +j (ti) NormIndTech +j (ti)
IndIn -j (ti) ≤ NormIndIn –j (ti),
IndIn +j (ti) NormIndIn +j (ti) (1) where IndSoc(ti), IndEcol(ti), IndEcon(ti), IndTech(ti), IndIn(ti) are measured in time ti, and
NormIndSoc(ti), NormIndEcol(ti), NormIndEcon(ti), NormIndTech(ti), NormIndIn(ti) are the normative
target values of social, environmental, economic, agrotechnological, and scientific – innovative
indicators set by the program for these time points. The upper index + ( – ) means that the indicator in
question belongs to a group of positive (negative) indicators, for which the improvement is associated
with an increase (decrease) in the value of the indicator. In the future (to avoid unnecessary
cumbersomeness), without limiting generality, all indicators will be considered positive, which will
allow us not to specify this index. The lower index j is the number of the indicator under consideration
in the corresponding list of indicators INDSoc, INDEcol, INDEcon, INDTech, INDIn. p
g
For each inequality considered, we introduce a binary indicator that reflects its implementation and
takes the values 1 (the constraint is met) or 0 (the constraint is not met). Thus, the progress of the regional
agro-industrial cluster development program at time ti is described by the tuple. (2) FIndTech(ti), FIndTechj (ti)[0, 1], where the conjunction () and disjunction () operations included in formula (4) are replaced by the
min and max functions (in accordance with [8]). However, there are still questions of interpretation of
the fuzzy values of FIndTechj (ti) indicators and ways of determining them, which makes the use of
model descriptions of the theory of linguistic variables more reasonable [9]. where the conjunction () and disjunction () operations included in formula (4) are replaced by the
min and max functions (in accordance with [8]). However, there are still questions of interpretation of
the fuzzy values of FIndTechj (ti) indicators and ways of determining them, which makes the use of
model descriptions of the theory of linguistic variables more reasonable [9]. p
y
g
he formal linguistic description of the FIndTech, FIndTechj indicators is represented as tuples (6) LingFIndTech, UnIndTech, TbaseIndTech, GIndTech, MIndTech>
(6) <LingFIndTech, UnIndTech, TbaseIndTech, GIndTech, MIndTech>
(6) ndTechj, UnIndTechj, TbaseIndTech, GIndTechj, MIndTechj>,
j =1, 2,…, k <LingFIndTechj, UnIndTechj, TbaseIndTech, GIndTechj, MIndTechj>,
j =1, 2,…, k where LingFIndTech, LingFIndTechj – names of linguistic variables; UnIndTech, UnIndTechj –
universal sets of linguistic variables containing all possible measured numeric values of IndTech,
IndTechj, (as a percentage of the normative numeric values of NormIndTech, NormIndTechj);
TbaseIndTech = {low, medium, high} – the general base term set for the values under consideration;
MIndTechj – syntactic rules that allow generate term names from the names of the TbaseIndTech
elements (which leads to the formation of the TIndTech term set); semantic rules that establish
correspondences between terms from TIndTech and fuzzy subsets of UnIndTech, UnIndTechj (j =1, 2,...,
k). The membership functions of these sets can have, in particular, a trapezoidal type, as shown in figure
1. Figure 1. Graphs of membership functions low (IndTech), medium(IndTech), high(IndTech) of fuzzy
subsets of UnIndTech that define the semantics of the terms low, medium, high. Figure 1. Graphs of membership functions low (IndTech), medium(IndTech), high(IndTech) of fuzzy
subsets of UnIndTech that define the semantics of the terms low, medium, high. AGRITECH-V-2021
IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
IOP Publishing
doi:10.1088/1755-1315/839/2/022068 AGRITECH-V-2021 IOP Publishing AGRITECH-V-2021
IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
IOP Publishing
doi:10.1088/1755-1315/839/2/022068 IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
doi:10.1088/1755-1315/839/2/022068 For a more detailed analysis, formula (4) can be generalized to the fuzzy case, in which the indicators
included in this formula can take intermediate values in range of 0 and 1, which reflects the degree of
fulfillment of the corresponding restrictions (the degree of truth of the statements about the fulfillment
of the restrictions), i.e. FIndTech(ti), FIndTechj (ti)[0, 1],
j =1, 2,…, k
(5) (5) FIND(ti) =< FINDSoc(ti), FINDEcol(ti), FINDEcon(ti), FINDTech(ti), FINDIn(ti) >
(2) each component of which reflects the fulfillment of the corresponding groups of inequalities from the
system of constraints (1). each component of which reflects the fulfillment of the corresponding groups of inequalities from the
system of constraints (1). Let us consider more thoroughly the agrotechnological aspects of the program (corresponding
indicators and limitations), bearing in mind that the analysis for the rest of the program indicators'
complexes is carried out in the same way. We represent FINDTech(ti) as a binary vector. FINDTech(ti) = < FIndTech1 (ti), FIndTech2 (ti),…, FIndTechk (ti)>
(3)
FIndTechj (ti){0, 1},
j =1, 2,…, k (3) (3) FIndTechj (ti){0, 1}, We will assume that the INDTech list contains subgroups of" duplicate " indicators INDTech1,
INDTech2,...., INDTechp , such that in the framework of the program implementation analysis, it is
sufficient to achieve the normative value of at least one of the indicators of the subgroup (the remaining
indicators may not even be measured). Then the binary FIndTech indicator is calculated using the logical
formula: FIndTech(ti) = ⋀
( ⋁
FIndTechj(ti)
IndTechj∈INDTechs
)
p
s=1
(4) (4) where, if the value 1 (true) means the execution of a set of agrotechnological indicators at time ti, and 0
(false) – non-execution, , – the signs of the conjunction and disjunction operations. 2 FIndTech(ti), FIndTechj (ti)[0, 1], The relationship between the UnIndTech indicator, which reflects the overall degree of compliance
with a group of agrotechnological standards, and the degree of compliance with individual FIndTechj
standards (j =1, 2,..., k) is set by fuzzy production rules of the form: if Flow(FIndTech1, FIndTech2,…, FIndTechk) then (FIndTech – low)
if Fmedium(FIndTech1, FIndTech2,…, FIndTechk) then (FIndTech – medium) (7)
if Fhigh (FIndTech1, FIndTech2,…, FIndTechk) then (FIndTech – high) if Flow(FIndTech1, FIndTech2,…, FIndTechk) then (FIndTech – low)
if Fmedium(FIndTech1, FIndTech2,…, FIndTechk) then (FIndTech – medium) (7)
if Fhigh (FIndTech1, FIndTech2,…, FIndTechk) then (FIndTech – high) (7) (7) where Flow, Fmedium, Fhigh are linguistic expressions relative to the correspondences of numeric values of
FIndTechj (j =1, 2,..., k) to terms from TIndTech. For example, where Flow, Fmedium, Fhigh are linguistic expressions relative to the correspondences of numeric values of
FIndTechj (j =1, 2,..., k) to terms from TIndTech. For example, where Flow, Fmedium, Fhigh are linguistic expressions relative to the correspondences of numeric values of
FIndTechj (j =1, 2,..., k) to terms from TIndTech. For example, Flow = (FIndTech1 – low) (FIndTech2 – low) ((FIndTech3 – low) (FIndTech3 – medium)) Flow = (FIndTech1 – low) (FIndTech2 – low) ((FIndTech3 – low) (FIndTech3 – medium)) Flow = (FIndTech1 – low) (FIndTech2 – low) ((FIndTech3 – low) (FIndTech3 – medium)) 3 AGRITECH-V-2021 AGRITECH-V-2021
IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
IOP Publishing
doi:10.1088/1755-1315/839/2/022068 IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
g
doi:10.1088/1755-1315/839/2/022068 Expressions Flow, Medium, High are set by experts according to their experience and knowledge of
the subject area. The analysis of the implementation of the development programs of the regional agro-industrial
cluster at the time ti in terms of achieving the required values of social (FINDSoc(ti)), environmental
(FINDEcol(ti)), economic (FINDEcon(ti)) and scientific and innovative (FINDIn(ti)) indicators is
performed in the same way. AGRITECH-V-2021
IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
IOP Publishing
doi:10.1088/1755-1315/839/2/022068 AGRITECH-V-2021
IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
IOP Publishing
doi:10.1088/1755-1315/839/2/022068 of the results generated by a special component (Explain component), which can serve as an additional
check of the correctness of the system and increases the confidence in the recommendations developed. of the results generated by a special component (Explain component), which can serve as an additional
check of the correctness of the system and increases the confidence in the recommendations developed. 4. Conclusion The application of the program approach to solving the problems of sustainable development of the
agro-industrial cluster of the regional economy is due to the high requirements for the management of
social, environmental, economic, agrotechnological and scientific-innovative processes within the
framework of the implementation of public-private partnership programs. The necessary level of
management cannot be achieved without the extensive use of modern digital intelligent technologies. The paper considers the task of developing tools for intellectual support of the analysis of the
implementation of regional agro-industrial cluster development programs, taking into account the high
level of uncertainty characteristic of innovative agricultural production. The success of the individual
stages of the program is proposed to be evaluated by the degree of achievement of normative targets,
for which the apparatus of representing knowledge about the subject area in the form of linguistic
variables describing the indicators and fuzzy production rules defining the relationships between the
indicators is used. The use of the interpreted Mamdani fuzzy inference procedure makes it possible to
form explanations of the course of reasoning when obtaining (logical inference) values of indicators that
cannot be directly measured. The proposed design solutions are used in the development of an intelligent information system for
analyzing the implementation of programs. The preliminary results of the pilot operation of the research
prototype of the developed system can serve as a confirmation of the effectiveness of its use in managing
the development programs of the regional agro-industrial cluster. It is proposed to develop further
research in this direction, supplementing the system functionality with the possibilities of modifying
programs based on expert judgments, as well as models and methods of evolutionary synthesis of
discrete systems with a given behavior [11, 12], which will allow for timely support for the adjustment
of regional agro-industrial policy when the conditions for its implementation change. 3. Results and discussion The main result of the work is building of an information analysis system for implementation of regional
agro-industrial development programs, the structure of which is shown in figure 2. Figure 2. Structure of the information and analytical system for analyzing the implementation of
regional agro-industrial cluster development programs. Figure 2. Structure of the information and analytical system for analyzing the implementation of
regional agro-industrial cluster development programs. The management of the operation of the information and analytical system is carried out by the
administrator, whose functional responsibilities include entering the normative data of the program
which are set during its initial development (adjustment) and data from monitoring for its
implementation, for which a special component of the system (Component data entry) is provided. Separate storage of these types of data in different databases (Base normative data and Base monitoring
data) allows to use various formats of information representation and to differentiate access to ensure
the necessary level of data security. Users of the system (Expert, Cognitologist, Analyst) interact with it in a limited subset of natural
language (reflecting the specifics of the program) using the Linguistic processor. Expert and
Cognitologist perform the filling of knowledge bases (Linguistic variable base and Fuzzy production
rule base), the division of which reflects the specifics of the methodological apparatus used (and the
subject area) and is another feature of the developed system. Processing of knowledge and facts (data) based on the Mamdani fuzzy inference algorithm [10] is
performed (as part of solving the analysis problem) by the Inference engine. After which its result (in
the form of an assessment of the program progress and recommendations for its possible correction) is
transmitted to the working memory area (Bulletin board) and then (through the Linguistic processor) is
transmitted to the Analyst user who set the task. On a special request, the Analyst can get the justification 4 AGRITECH-V-2021
IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
IOP Publishing
doi:10.1088/1755-1315/839/2/022068 selection of the models of interacting processes on the basis of expert assessments
International Journal of Applied Engineering Research 11 (3) 1867-73 AGRITECH-V-2021
IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
IOP Publishing
doi:10.1088/1755-1315/839/2/022068
selection of the models of interacting processes on the basis of expert assessments
International Journal of Applied Engineering Research 11 (3) 1867-73 [12] Petrosov D A, Lomazov V A, Lomazova V I and Glushak A V 2018 Applications of parallel
computations in the problems of structural-parametric synthesis of discrete systems based on
evolution methods Journal of Advanced Research in Dynamical and Control Systems 10(10)
1840-6 AGRITECH-V-2021
IOP Conf. Series: Earth and Environmental Science 839 (2021) 022068
IOP Publishing
doi:10.1088/1755-1315/839/2/022068 References [1]
Blewitt J 2017 Understanding Sustainable Development (London: Routledge) [1]
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development in the context of technological and ecology problems Journal of Critical Reviews
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saving waste-free production as an innovative method of improving enterprises' business
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York: CRC Press) [7]
Sunggyu L S and Shah Y T 2012 Biofuels and Bioenergy: Processes and Technologies (New
York: CRC Press) [8]
Russell S J and Norvig P 2020 Artificial Intelligence Artificial Intelligence: A Modern Approach
(New Jersey: Prentice Hall) [9]
Zadeh L A 2001 The role of soft computing and fuzzy logic in the understanding, design and
development of information, intelligent systems News of Artificial Intelligence 2-3 7 [10] Mamdani E H 1974 Applications of fuzzy algorithms for control of simple dynamic plant
Proceedings of the Institution of Electrical Engineers 121(12) 1585-8 g
f
f
g
(
)
azov V A, Petrosov D A, Dobrunova A I, Lomazova V I and Matorin S I 2016 Evolutionary [11] Lomazov V A, Petrosov D A, Dobrunova A I, Lomazova V I and Matorin S I 2016 Evol 5 AGRITECH-V-2021
IOP Conf. selection of the models of interacting processes on the basis of expert assessments
International Journal of Applied Engineering Research 11 (3) 1867-73
[12] Petrosov D A, Lomazov V A, Lomazova V I and Glushak A V 2018 Applications of parallel
computations in the problems of structural-parametric synthesis of discrete systems based on References Series: Earth and Environmental Science 839 (2021) 022068
IOP Publishing
doi:10.1088/1755-1315/839/2/022068
selection of the models of interacting processes on the basis of expert assessments
International Journal of Applied Engineering Research 11 (3) 1867-73
[12] Petrosov D A, Lomazov V A, Lomazova V I and Glushak A V 2018 Applications of parallel
computations in the problems of structural-parametric synthesis of discrete systems based on
evolution methods Journal of Advanced Research in Dynamical and Control Systems 10(10)
1840-6 [12] Petrosov D A, Lomazov V A, Lomazova V I and Glushak A V 2018 Applications of parallel
computations in the problems of structural-parametric synthesis of discrete systems based on
evolution methods Journal of Advanced Research in Dynamical and Control Systems 10(10)
1840-6 6
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https://clok.uclan.ac.uk/46155/8/Children%20%20%20Society%20-%202023%20-%20Graham%20-%20Child%E2%80%90safe%20organizations%20and%20the%20ethics%20of%20empowered%20inclusion.pdf
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<scp>Child‐safe</scp> organizations and the ethics of empowered inclusion
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It is advisable to refer to the publisher’s version if you intend to cite from the work.
https://doi.org/10.1111/chso.12732 It is advisable to refer to the publisher’s version if you intend to cite from the work. https://doi.org/10.1111/chso.12732 For information about Research at UCLan please go to http://www.uclan.ac.uk/research/ Central Lancashire Online Knowledge (CLoK)
Title
Child-Safe Organisations and the Ethics of Empowered Inclusion
Type
Article
URL
https://clok.uclan.ac.uk/46155/
DOI
https://doi.org/10.1111/chso.12732
Date
2023
Citation
Graham, Anne, Canosa, Antonia, Wall, John and Thomas, Nigel Patrick
(2023) Child-Safe Organisations and the Ethics of Empowered Inclusion.
Children & Society. ISSN 0951-0605
Creators
Graham, Anne, Canosa, Antonia, Wall, John and Thomas, Nigel Patrick Central Lancashire Online Knowledge (CLoK)
Title
Child-Safe Organisations and the Ethics of Empowered Inclusion
Type
Article
URL
https://clok.uclan.ac.uk/46155/
DOI
https://doi.org/10.1111/chso.12732
Date
2023
Citation
Graham, Anne, Canosa, Antonia, Wall, John and Thomas, Nigel Patrick
(2023) Child-Safe Organisations and the Ethics of Empowered Inclusion. Children & Society. ISSN 0951-0605
Creators
Graham, Anne, Canosa, Antonia, Wall, John and Thomas, Nigel Patrick Central Lancashire Online Knowledge (CLoK) Central Lancashire Online Knowledge (CLoK) Title
Child-Safe Organisations and the Ethics of Empowered Inclusion
Type
Article
URL
https://clok.uclan.ac.uk/46155/
DOI
https://doi.org/10.1111/chso.12732
Date
2023
Citation
Graham, Anne, Canosa, Antonia, Wall, John and Thomas, Nigel Patrick
(2023) Child-Safe Organisations and the Ethics of Empowered Inclusion. Children & Society. ISSN 0951-0605
Creators
Graham, Anne, Canosa, Antonia, Wall, John and Thomas, Nigel Patrick O R I G I N A L A R T I C L E Anne Graham1 | Antonia Canosa1 | John Wall2 |
Patrick Thomas3 Anne Graham1 | Antonia Canosa1 | John Wall2 |
Patrick Thomas3 1Centre for Children and Young People,
Faculty of Health, Southern Cross
University, Coolangatta, New South
Wales, Australia
2Childism Institute, Rutgers University
Camden, Camden, New Jersey, USA
3Centre for Children and Young People's
Participation, School of Social Work,
University of Central Lancashire, Preston,
Lancashire, UK 1Centre for Children and Young People,
Faculty of Health, Southern Cross
University, Coolangatta, New South
Wales, Australia
2Childism Institute, Rutgers University
Camden, Camden, New Jersey, USA
3Centre for Children and Young People's
Participation, School of Social Work,
University of Central Lancashire, Preston,
Lancashire, UK Abstract Abstract
The emergence of the ‘child-safe’ organization requires
close attention to practices that contribute to children's
wellbeing and safety. Based on data collected in schools,
residential care and disability services, this article argues
for a more nuanced understanding of the ethical frame-
works informing practice in these settings. Findings
suggest both young people and adults predominantly
describe ethical practice in terms of intersubjective
relations. This ethical relationality is understood, less
in terms of vertical responsibilities of care (largely the
domain of adults and shaped by institutional norms),
and more in terms of vertical and horizontal (inter-
personal) relations, giving way to more empowered
inclusion. Correspondence
Anne Graham, Southern Cross University,
Locked Mail Bag 4, Coolangatta, QLD
4225, Australia. E
il
h
@
d Funding information
Australian Research Council Discovery,
Grant/Award Number: DP180100465 Funding information
Australian Research Council Discovery,
Grant/Award Number: DP180100465 Funding information K E Y W O R D S
childism, ethical practice, ethical theory, ethics, relational ethics K E Y W O R D S
childism, ethical practice, ethical theory, ethics, relational ethics For information about Research at UCLan please go to http://www.uclan.ac.uk/research/ All outputs in CLoK are protected by Intellectual Property Rights law, including Copyright law. Copyright, IPR and Moral Rights for the works on this site are retained by the individual authors
and/or other copyright owners. Terms and conditions for use of this material are defined in the
http://clok.uclan.ac.uk/policies/ DOI: 10.1111/chso.12732
Accepted: 27 March 2023 Accepted: 27 March 2023 DOI: 10.1111/chso.12732 m https://onlinelibrary.wiley.com/doi/10.1111/chso.12732 by Test, Wiley Online Library on [19/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley While some of these findings have been reported elsewhere (Graham, Canosa, et al., 2022;
Robinson et al., 2022), this paper gives critical consideration to the significance of applying an
ethical lens in the analysis of practices seeking to improve children's safety. It focuses on the
following central question: How do children and practitioners in different institutional settings
understand and experience ‘ethical’ practice with regard to children's safety and wellbeing and what
do they perceive to be the challenges, barriers and enablers? In doing so, the discussion not only
provides a nuanced insight into the ethical frameworks informing child safe practice but also
draws on these to illuminate ethical aspects of the practices identified in the fieldwork. We employ the theory of practice architectures, hereafter TPA (Kemmis et al., 2014) to support
this analysis. TPA provides a site ontological view of what ethical practice is, how such practice is
mediated, and how practices relate to each other. It is an analytical resource for revealing the ways
practices are enabled and constrained by the conditions in which they occur, and a transformational
resource for finding ways to change practices where current practices are untoward. Practitioners'
agency is understood to be enabled and constrained within organizational, cultural, institutional,
and knowledge-producing practices, by material and economic conditions, by relations of power
and systems developed over time (Kemmis et al., 2014). Practice traditions develop over time,
grounded in tacit knowledge and coming to be recognized as ‘the way we do things around here’. The way practices happen is always shaped by the conditions of a particular site at a particular
point in time; they do not unfold from ‘predetermined scripts’ (Kemmis et al., 2014, p. 33). In Australia, the term ‘child safe, child friendly’ was first promoted by the NSW Commis-
sion of Children and Young People in the early 2000s. The inclusion of discourses surrounding
‘child safe/friendly’ was a reminder to organizations that their task is not just risk detection and
mitigation, but one of providing protective and responsive environments (Smallbone, 2017). It
was not until the Royal Commission into Institutional Responses to Child Sexual Abuse (here-
after ‘Royal Commission’); however, that Australian organizations working with children were
required to implement standards laid out in the National Principles for Child Safe Organizations
(2018). BACKGROUND The emergence of ‘child safety’ as a national policy priority in Australia, as in numerous other
international jurisdictions, has been accompanied by a plethora of policies, principles, frameworks
and guidelines across all sectors with responsibility for the wellbeing, care, safety and support of
children and young people. To date, however, little research has explored the ethical dimensions
of practices intended to ensure improved safety. This article presents findings from the qualita-
tive phase (Phase 2) of a mixed method Australian Research Council Discovery (ARC-D) project, Child Soc. 2023;00:1–18. wileyonlinelibrary.com/journal/chso
1
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and repro-
duction in any medium, provided the original work is properly cited. © 2023 The Authors. Children & Society published by National Children's Bureau and John Wiley & Sons Ltd. wileyonlinelibrary.com/journal/chso Child Soc. 2023;00:1–18. 1 GRAHAM et al. 2 which involved interviews and focus groups with staff and young people in schools, residen-
tial care and disability services in three Australian states (New South Wales, Victoria and South
Australia). The overall study employed a framework based on relational ethics, recognition theory
and the theory of practice architectures, to explore the ethical dimension of practices within these
three sector contexts that are intended to support safety and wellbeing (Phase 2) and to identify
which of these are positively associated with recognition, safety and wellbeing (Phase 3). Our
endeavours were certainly inspired by important and timely legislative and policy developments
seeking to change and improve practice, largely aided by compliance obligations. However, the
interest of our inquiry was in looking beyond whether such practice might be safe, to whether it
is also ethical. That is, we sought to understand what it means for children and young people not
only to be protected but also to be recognized, empowered and included. m https://onlinelibrary.wiley.com/doi/10.1111/chso.12732 by Test, Wiley Online Library on [19/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley These National Principles are underpinned by a ‘child-rights approach to build capacity
and to deliver child safety and wellbeing in organisations, families and communities’ (Australian
Human Rights Commission, 2018). The remit of these principles is to support and guide organi-
zations to create a culture, adopt strategies and take action to promote child safety and wellbeing
(see Figure 1 below). Framed at a high level given diverse organizational contexts, sizes and
capacities where these are now being implemented, these principles reflect ten elements consid-
ered central to ensuring an organization is safe for children. A number of these National Principles arguably have an ethical emphasis. One example is the
principle of recognizing children's rights: ‘children and young people are informed about their
rights, participate in decisions affecting them and are taken seriously’. On the ground, though, 3 GRAHAM et al. 3 F I G U R E 1
Wheel of child safety. Source: The National Principles for Child Safe Organizations, 2018. F I G U R E 1
Wheel of child safety. Source: The National Principles for Child Safe Organizations, 2018. R E 1
Wheel of child safety. Source: The National Principles for Child Safe Organizations, 2018. the associated practices, largely informed by ethical decision-making, are usually complex with
no clear-cut steps. This means that ethical decision-making as a ‘craft’ may not be easily acquired
(Pawlukewicz & Ondrus, 2013). Furthermore, the institutional context and practice environ-
ments in which one operates often give rise to a range of ethical questions. the associated practices, largely informed by ethical decision-making, are usually complex with
no clear-cut steps. This means that ethical decision-making as a ‘craft’ may not be easily acquired
(Pawlukewicz & Ondrus, 2013). Furthermore, the institutional context and practice environ-
ments in which one operates often give rise to a range of ethical questions. While these aims for child safe and child friendly practices are an important development, it is
unclear whether they actually make contact with children's and practitioners' experiences of the
ethical issues encountered in every day routine settings. General principles may or may not oper-
ationalize within particular and different organizational contexts. Furthermore, the principles
themselves might not capture the full range of ethical concerns at play. For example, the idea that
children and young people “actively participate in the organisation” could potentially obscure
children's tokenism, silencing, or lack of real power to influence outcomes. m https://onlinelibrary.wiley.com/doi/10.1111/chso.12732 by Test, Wiley Online Library on [19/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley Promoting “equity
and diversity,” while laudable, could end up prioritizing certain diversities over others. As a result,
our study aims to unpack the underlying ethical questions inherent in issues of child safety in
order to understand both the possibilities and the barriers to their implementation in practice. CONSIDERING SAFETY THROUGH AN ETHICS LENS Ethics is commonly defined as ‘the branch of philosophy concerned with living a good life, being
a good person, and doing the right thing’ (Freeman, 2000, p. 30). As a discipline, it involves both
the study of our values and their justification as well as the actual values and standards of conduct 10990860, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/chso.12732 by Test, Wiley Online Library on [19/04/2023]. See the Terms and C GRAHAM et al. 4 by which we live (Freeman, 2000). Ethical theory, on the other hand, brings together a collection
of ethical concepts and principles ‘into a coherent whole in order to answer an ethical question or
solve an ethical problem’ (Mizzoni, 2010, p. 5). It seeks to develop guidelines that could be useful
in addressing difficult or controversial social issues. Theories can change over time and develop
into new strands which are encompassed by a particular ‘ethical tradition,’ traditions that some-
times conflict but can also be complementary Goodwin and Rossow-Kimball (2012). m https://onlinelibrary.wiley.com/doi/10.1111/chso.12732 by Test, Wiley Online Library on [19/04/2023]. See the Terms and Conditions (https://onlinelibrar For this study, we recognized that several ethical theories potentially help to shed light on
practices in organizations seeking to uphold children's safety and wellbeing. Figure 2 provides
an overview of what are commonly understood in philosophical ethics to constitute the four
major contemporary types of ethical theory: consequentialist ethics, deontological ethics, virtue
ethics and relational ethics. These are not the only possibilities and each contains many internal
variations. However, as the primary possibilities informing ethical theory today, they provide
a valuable starting framework for analysis. They can be understood as providing four different
theoretical lenses or tools for examining the underlying ethical dimensions of practice. In a consequentialist ethics framework, moral practices are evaluated in terms of how well they
advance good consequences or outcomes. Consequentialism is usually thought to promote the
greatest happiness for the greatest number of people (Mill, 1998). That is, what is most impor tant in
ethical action is whether it advances the well-being, or reduces the suffering, of anyone it impacts. This approach can be termed ‘teleological’ in that it is focused on teloi or ends, aims, consequences
of actions. ‘Rightness or wrongness depends on the consequences of personal actions … what is
right produces good consequences’ (Brockett & Hiemstra, 2004, p. 21). For example, will chil-
dren having a say in designing school curricula improve educational outcomes? Different versions
of consequentialism might focus on maximizing each involved individual's self-interest (ethical
egoism) or the larger well-being of the group overall (utilitarianism). In any case, consequentialist
ethics focuses on the question of what course of action produces the best overall outcome. F I G U R E 2
Ethical frameworks. Source: Authors. F I G U R E 2
Ethical frameworks. Source: Authors. F I G U R E 2
Ethical frameworks. Source: Authors. 5 GRAHAM et al. In contrast, deontological theories evaluate ethical practices in terms of how well they adhere
to duty (deon) or obligation. They hold that ‘consequences should not be viewed as a criterion for
determining what is right’, but rather ‘fulfilling personal duty, regardless of the consequences’ is key
(Brockett & Hiemstra, 2004, p. 21). Based on the principles of Immanuel Kant, this approach places
importance on following what is implicitly just, rational, or, as Kant puts it, universalizable. Others
should be treated as you would like to be treated: as respected and dignified human beings. wiley.com/doi/10.1111/chso.12732 by Test, Wiley Online Library on [19/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditi Virtue ethics, differently again, frames moral questions around the goodness of a person's
moral character. Character refers here to one's roles and responsibilities as a human person
among others and in relation to wider culture, traditions, and community (Thiroux &
Krasemann, 2007). Virtue ethics derives from Aristotle's Nicomachean Ethics, according to
which, ‘actions are right when they stem from good character, or the disposition to be just,
benevolent, or courageous in situations where someone is in need of help’ (Goodwin &
Rossow-Kimball, 2012, p. 298). The ‘goodness of an act is determined not by its consequence
or adherence to established rules but primarily by the qualities of the agent performing the act’
(Freeman, 2000, p. 89). For example, would children participating in designing school curric-
ula help them exercise good character and play an appropriate role in the school community? Virtue ethics prioritizes the question of whether a person is acting with good or bad character. p
q
p
g
g
Finally, in a relational ethics framework, moral practices are evaluated in terms of how
well they care for and enhance the relationships and responsibilities that connect persons to
one another. Following the work of Carol Gilligan (1982), Noddings builds on the notion of an
ethic of care to propose a ‘relational ethics’ which is centred more upon the ‘moral quality of
relationships than upon individuals’ (Liaschenko & Peter, 2003, p. 36). From a relational ethics
framework, ‘relationships are built upon the principles of engagement, embodiment, mutual
respect, and environment,’ the latter of which is construed as a ‘relational space’ (Cloutier
et al., 2015, p. 768). Good moral practices are not most fundamentally about individual inter-
ests, duties, or character, but instead about relationships of care, recognition, inclusion, and
mutual responsiveness. For example, would children's involvement in develop ing school
curricula enhance or diminish relations of care and responsibility among students and with
teachers? Relational ethics locates the basis of moral life in the quality of human connections. In this paper, the practices identified in the study (see Graham, Canosa, et al., 2022; Robinson
et al., 2022) will be critically examined in light of these ethical frameworks. Each framework
or theory provides a useful lens for understanding ethical practice. None captures everything
of relevance. However, they can be used to understand the ethical complexities involved in the
actual practices and thinking of children, young people, and staff themselves. wiley.com/doi/10.1111/chso.12732 by Test, Wiley Online Library on [19/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditi And they can
therefore also help to illuminate what might in reality contribute in organizational settings to
child safety and well-being. m https://onlinelibrary.wiley.com/doi/10.1111/chso.12732 by Test, Wiley Online Library on [19/04/2023]. See the Terms and Conditions (https://onlinelibrar For
example, would children having a say in curriculum design better respect their dignity and capaci-
ties? Deontological theories are often referred to as ‘rule-based thinking’ and inform the universal
rules and laws that govern our respect for ourselves and others (Brockett & Hiemstra, 2004, p. 21). Focus groups with children and young people Consistent with the guidelines from previous studies, focus groups were conducted with young
people of similar ages (Gibson, 2007) and were predominantly facilitated in schools. In total
9 focus groups were carried out across the five schools. Generally, no more than 7 students
participated in each focus group. In total, 58 students participated in focus groups across the
three states. A total of two focus groups were also conducted in the disability sector (total of
8 participants) and one in the residential care sector (total of 3 participants). Two researchers
co-facilitated the 60-minute focus groups and invited children and young people to reflect on
the practices that helped, or did not help, in feeling happy, safe and well in the specific organ-
izational context (mapping activity 1). Subsequently, participants were asked to reflect on the
prac tices they viewed as having an ‘ethical’ dimension, discussing their views on the word ‘ethi-
cal’ (mapping activity 2). The children and young people then suggested changes to practices
that might support them to feel happy, safe and well (mapping activity 3). The use of post-it notes
assisted in capturing participants' views which were then visualized on whiteboards or large
sheets of paper. The wording of the interview questions for young people was developed in close
consultation with our Young People's Advisory Group (YPAG), a group of six young people aged
between 12 and 18 years, some of whom identified with disability. Site selection and sampling strategies Young people, staff and leaders were recruited from three sector contexts (government, Catho-
lic and independent schools, residential care and disability services) across two metropolitan GRAHAM et al. 6 (Melbourne and Adelaide) and two regional areas in Australia (Northern NSW and Limestone
Coast, SA). The selection of contexts was intended to provide access to a diverse sample of partic-
ipants in different areas of geographical remoteness (as determined by the Australian Bureau
of Statistics), a factor considered important given regionality and rurality in Australia has been
linked to staff shortages and less access to resourcing, such as professional development oppor-
tunities (Cosgrave et al., 2019; Cuervo & Acquaro, 2018). Diversity was also sought in terms of
size of organizations, socioeconomic status, and cultural characteristics of participants. Due to
the COVID-19 pandemic, the number of organizations accessed was lower than anticipated with
some of the interviews and focus groups facilitated online via Zoom. Recruitment in the disabil-
ity sector was particularly challenging given recent systemic changes to the mode of delivery of
services following the National Disability Insurance Scheme (NDIS) in Australia. A total of 11
organizations participated in the qualitative phase of the research, involving 85 young people
(aged 12–18 years) and 33 adult staff members (a mix of leaders, managers and frontline workers). (Melbourne and Adelaide) and two regional areas in Australia (Northern NSW and Limestone
Coast, SA). The selection of contexts was intended to provide access to a diverse sample of partic-
ipants in different areas of geographical remoteness (as determined by the Australian Bureau
of Statistics), a factor considered important given regionality and rurality in Australia has been
linked to staff shortages and less access to resourcing, such as professional development oppor-
tunities (Cosgrave et al., 2019; Cuervo & Acquaro, 2018). Diversity was also sought in terms of
size of organizations, socioeconomic status, and cultural characteristics of participants. Due to
the COVID-19 pandemic, the number of organizations accessed was lower than anticipated with
some of the interviews and focus groups facilitated online via Zoom. Recruitment in the disabil-
ity sector was particularly challenging given recent systemic changes to the mode of delivery of
services following the National Disability Insurance Scheme (NDIS) in Australia. A total of 11
organizations participated in the qualitative phase of the research, involving 85 young people
(aged 12–18 years) and 33 adult staff members (a mix of leaders, managers and frontline workers). Interviews with staff and leaders Interviews with staff and leaders were undertaken in schools (n = 15), disability services (n = 9)
and residential care services (n = 9) in the three geographical locations. One leader (principal or
deputy principal in schools and managers in other settings) and two other staff members were
invited to participate in the interviews. The semi-structured interviews each took between 40 and
60 min, and participants were asked to describe the setting they worked in, their role and level
of experience. Following this, and similar to the young people's focus groups, staff were asked to
reflect on the practices that support the safety and wellbeing of children and young people. Staff
were then asked to identify and elaborate on the ethical dimensions of these practices and how
things could be improved in their organization. Interviews with children and young people In the disability (n = 6) and residential care settings (n = 10), children and young people were
interviewed one-on-one to accommodate their individual needs and ensure confidentiality and
anonymity. The interviews followed a similar format to the focus groups with the mapping activ-
ities adapted for comprehension and interest. After introducing the research team and reiterating
our ethical responsibilities, participants were asked to identify their personal interests to create
an environment of trust and put each young person at ease. The interview then moved on to
cover the three areas mentioned above: (1) practices that help/do not help in feeling happy, safe
and well, including gentle probing about the role that support workers play and ways they influ-
enced practices in specific sites; (2) the ethical dimensions of practices; and (3) how practices
might be improved. 7 GRAHAM et al. Data analysis With participants' consent the interviews and focus groups were audio recorded and subse-
quently transcribed, coded and analysed using QSR NVivo12, a qualitative data management
and analysis software (Bazeley & Jackson, 2013). TPA was employed as an analytical tool to
explore young people's lived experiences of the practices that support their safety and wellbeing
in the three organizational contexts (‘zooming in’), and the ethical dimensions of such practices
to understand how these are enabled and constrained by the conditions at each site (‘zoom-
ing out’; Kemmis et al., 2014; Nicolini, 2012). Using a TPA lens in coding the data allowed us
to move beyond just identifying these practices at a microlevel to simultaneously reflecting on
conditions that often constrain practices in these contexts. The ethical practices identified below
acknowledge that people encounter each other in intersubjective spaces and that these spaces are
pre-configured by site specific conditions. These conditions may be cultural-discursive (‘sayings’),
material-economic (‘doings’) and social-political (‘relatings’; Kemmis et al., 2014). Data analysis involved comparing and reviewing the text assigned to each theme/node. Reflective field notes were also analysed and particularly useful for young people with higher
support needs who were unable to fully engage in the interview process. Visual representations
such as mind maps were also helpful for organizing and refining themes and seeking feedback
from the YPAG members. Institutional ethics approval was obtained from the lead University's Human Research Ethics
Committee (approval number: ECN-19-047) and relevant government and non-government
school systems (approval numbers: 2019-759 078; 2019-0611). Parental informed consent was
also obtained where possible. In the residential care setting, blanket approval was provided from
the relevant Government Department in each State in addition to young people's individual
informed consent. The core ethical principles of the International Ethical Research Involving
Children (ERIC) Charter and Guidance (Graham et al., 2013) were employed to guide the
research processes. FINDINGS Children, young people and staff in schools, residential care services and disability services
identified several practices that contribute to child safety and wellbeing in these institutional
contexts. When viewed through a lens of ‘what is the most ‘right’ thing to do?’ particular prac- GRAHAM et al. 8
F I G U R E 3
Coding density for young people data in the three sectors. GRAHAM et al. 8 8 F I G U R E 3
Coding density for young people data in the three sectors. tices emerged which participants identified as having an ethical dimension. These were consist-
ent across all three sectors and included: (i) building positive relationships with peers and staff;
(ii) Having a say and influencing practice; (iii) being treated equally, fairly and inclusively; and
(iv) building an authentic child-centred organizational culture. Given our interest in profiling
children's voices, the findings are presented according to coding density of the young people
data (see Figure 3) with discussion of the similarities and differences with the adult data in the
three sectors (see Figure 4). In addition, the TPA lens allowed for analysis of site-based condi-
tions that enable and constrain these practices. At the end of the findings section, we discuss
how these practices are broadly aligned with the ethical frameworks previously identified. tices emerged which participants identified as having an ethical dimension. These were consist-
ent across all three sectors and included: (i) building positive relationships with peers and staff;
(ii) Having a say and influencing practice; (iii) being treated equally, fairly and inclusively; and
(iv) building an authentic child-centred organizational culture. Given our interest in profiling
children's voices, the findings are presented according to coding density of the young people
data (see Figure 3) with discussion of the similarities and differences with the adult data in the
three sectors (see Figure 4). In addition, the TPA lens allowed for analysis of site-based condi-
tions that enable and constrain these practices. At the end of the findings section, we discuss
how these practices are broadly aligned with the ethical frameworks previously identified. Building positive relationship with peers and staff Building positive relationships with peers and staff was the most prominent theme in the young
people data across all sectors (see Figure 3). Trust and respect were central issues, with young people
identifying staff who were encouraging and took the time to get to know them as being more success-
ful in building positive relationships. These relationships were primarily described in recognition
terms, aligned with being cared for, respected and valued. Participants in one secondary school focus
group described such trust in terms of staff knowing them and being there to support them: ‘My teachers encourage me to do my best. My teacher notices when I'm unhappy
and makes me feel good’ (Mapping activity 1, 13–15 years, focus group secondary
school B1). This was particularly important for children and young people in the residential care sector who
identified positive relationships with peers and staff as far more important than any other practices
(see Figure 3). They often commented on the ideal worker being someone who is not there ‘because GRAHAM et al. 9
F I G U R E 4
Coding density for adult data in the three sectors. 9 GRAHAM et al. 9 F I G U R E 4
Coding density for adult data in the three sectors. it's a job but because they want to help and they want to give the kid a life, that they enjoy making
the kid feel safe, loved, and cared for. And that they won't go until that kid is stable’ (16 year old boy,
residential care service A). Likewise, young people in the disability sector described feeling safe and
well when support workers were genuinely warm and empathic in their interactions with young
people: ‘I don't know how to describe it much. He [support worker] makes me happy. He enjoys
working with us…He is like a nice guy; likes hanging out with us’ (Oliver, 13–15 years, interview,
disability service A). In addition, young people in disability services also raised the importance of
peer relationships more so than in other contexts. The solidarity they felt from spending time with
peers who had similar lived experiences was very important in building trust and feeling safe. Having a say and influencing practice These staff reported having to deprioritize young people's wishes in order to keep
them safe, as the following view underlines: ‘When it comes down to a child's safety, all of those ethical dilemmas that are in your
mind, safety needs to trump [them]’ (Leader, residential care service B). Building positive relationship with peers and staff While adults provided similar conceptualisations of the practices that supported positive rela-
tionships with young people in their care, they often discussed some of the barriers and chal-
lenges experienced due to compliance, administrative and systemic obligations (see Figure 4). Across all three sectors staff often felt ‘overworked’ and ‘overburdened’ and unable to nurture
positive relationships with young people: Realistically with all these other outcomes and everything on top along with your
assessment, your reporting, and just all those compliance issues, and sometimes you
get bogged down in that and forget about the little person on the other side.’
(Interview, leader primary school A) (Interview, leader primary school A) ‘What I find isn't helpful is the absolute administrative burden …You don't really
have a great relationship with the kids or their families. There's a trade-off.’
(Interview, leader 2 residential care service A) ‘We come into someone's life, and we want to connect people to services, whether
they're mainstream or funded support. You come into someone's life, and you build 10 GRAHAM et al. up a relationship of trust. And that takes a long, long time…but we don't have a heap
of time or a heap of funding to be able to do that.’ up a relationship of trust. And that takes a long, long time…but we don't have a heap
of time or a heap of funding to be able to do that.’ (Interview, staff 2 disability service B) Such experiences evidenced the tensions identified by staff across all three sectors as they sought
to deliver on their compliance and administrative obligations, which most regarded as something
of a fait accompli, while acknowledging the impact of diminished time for developing meaning-
ful relationships with young people. Having a say and influencing practice Having a say and influencing decisions was the second most important theme emerging from the
young people data across all three sectors but particularly for young people in the residential care
sector (see theme ‘voice & agency’ in Figure 3). In residential care, this was discussed in terms
of balancing young people's needs for autonomy and independence and their need for safety. In
schools, students highlighted the importance of having the opportunity to regularly put forward
ideas and work toward shared goals, articulated by one young person in this way: ‘In one of my classes, one of the teachers helped us, we made our own four class
rules, and we all had the input on what those rules would be… I've really enjoyed that
because most of the class works well now because we all know what the rules are
because we made them.’ (Cheetah, 13–15 years, focus group secondary school A1). Young people in the disability sector also discussed the importance of having a say, particularly in
day-to-day decisions regarding the activities they were involved in (i.e., cooking classes, excursions
and planned games). However, young people in this sector also spoke of the difficulties in influenc-
ing practice. For example, several young people highlighted that staff turnover and the instability in
care received was destabilizing. This was a systemic problem which they felt they could not change. Young people in the disability sector also discussed the importance of having a say, particularly in
day-to-day decisions regarding the activities they were involved in (i.e., cooking classes, excursions
and planned games). However, young people in this sector also spoke of the difficulties in influenc-
ing practice. For example, several young people highlighted that staff turnover and the instability in
care received was destabilizing. This was a systemic problem which they felt they could not change. Staff identified ‘listening’ and valuing young people's opinion as a critical dimension of ethi
cal practice contributing to their wellbeing and safety. While staff in the three sectors identified
mechanisms to elicit young people's views, they seldom discussed how these views were acted
upon. Staff in the residential care sector, in particular, often highlighted the tricky balancing ac
of supporting children's and young people's autonomy while also looking out for their best inter
ests and safety. Being treated equally, fairly and inclusively Being treated equally, fairly and inclusively was a practice identified as having an ethical dimen-
sion and raised more often in conversation with students and staff in schools. As the views of
primary school students in the following mapping activity suggest, teachers' attitudes and the 11 GRAHAM et al. way they were able to create a welcoming and inclusive environment was viewed as foundational
in creating the cultural conditions for children to be well and feel safe: way they were able to create a welcoming and inclusive environment was viewed as foundational
in creating the cultural conditions for children to be well and feel safe: ‘Teachers care for everybody, no one is left out. You are respected, included, cared
for, loved by everyone. Everybody needs to be included no matter how you look/are.’
(Mapping activity 2, 11-12 years, focus group secondary school A) Conversely, bias and discrimination—enacted by peers and/or staff—were identified as a barrier
in achieving the fairness and inclusivity necessary for a felt sense of safety and wellbeing. While
issues connected with being treated unfairly or inequitably were not articulated openly by young
people with disability, the interviews and reflective notes suggest that these practices were
distressing for them. Two examples in the disability sector show how young people were excluded
from accessing the service because they had behaviour outbursts in response to changes to rules
which restricted what they had previously been able to do during their time with the service. While less prominent in the staff data across the three sectors, treating young people equally,
fairly and inclusively was identified as an ethical practice (see Figure 4). In schools this practice
was discussed in terms of clear, open and transparent communication with students and respect
for their dignity. As one Principal in a primary school suggests, students feel as though they are
treated equally, fairly and inclusively when teachers genuinely value them: ‘I think it's around
making sure that the kids know that we are here for them, we do value them, we care for them,
and we respect them, so that's just not in words, it's in how we treat them, it's in whatever we
do with them so that they can see it in action all the time’ (Interview, leader primary school A). Being treated equally, fairly and inclusively In the disability sector, staff and leaders argued that providing the spaces necessary for young
people with a lived experience of a disability to come together and feel as though they belong was
fundamental in contributing to a culture of equality, fairness and inclusiveness. Practitioners in
the disability sector also discussed how they often take on an advocacy role to ensure children
with disability are not treated or spoken to unfairly, often by their own families. In the residential
care sector, practices which were identified as being ‘ethical’ and promoting equality, fairness and
inclusiveness included among others inclusive language and modelling these behaviours with
the young people in their care. As the following residential care worker argued, inclusive and
non-discriminatory language was important to promote the safety and wellbeing of young people: ‘Having a zero tolerance for discrimination. I feel like LGBQTI+ is quite a
big thing so having gender-inclusive language is a really important thing and
culturally-appropriate language. So we role model it. Sometimes they'll say some-
thing racist and you'll challenge them on what they just said because they can't even
say why they think it. So I think it's important to challenge them as well.’ (Staff 1, residential care service B) (Staff 1, residential care service B) Building an authentic child-centred organizational culture I think that's how they get
an idea of what a person is like.’ (Interview, staff 1 residential care service C) (Interview, staff 1 residential care service C) In the disability sector, probationary periods were often implemented to test the suitability of
practitioners to work in child-centred and ethical ways: ‘We learn pretty fast if there are people
that aren't actually really here for the children or for the support needs of the clients, so they tend
to be taken out on probation because we don't want that here’ (Interview, staff 1 disability service
A). The building of authentic child-centred organizational cultures was thus inextricably linked
to, or bundled with, ‘relational’ practices and the personal qualities of staff. This suggests that
‘child-centredness’ is strongly related to practitioners' ethics of care, as well as their character,
and their commitment to promoting children's wellbeing and safety. Building an authentic child-centred organizational culture While organizational culture was talked about more often among staff and leaders in the three
sectors, young people referred to the kind of environment conducive to feeling happy, safe and
well. Such an environment was a place where children and young people were placed at the
‘centre’ and where ‘relationships’ with staff and peers were based on mutual trust and respect,
‘voice and agency’, ‘fairness, equality and inclusivity.’ For staff and leaders, organizational culture was the most prominent theme across the three
sectors (see Figure 4). They referred to an authentic child-centred organizational culture as one 12 GRAHAM et al. with a shared ethos that values children and young people as individuals with rights and dignity. Staff also talked about the importance of a child-centred culture being led ‘from the top’ and staff
supported in their role to place children's needs at the centre of practices: ‘I just think in terms of
what I was saying before about the culture of the school and the type of school you want to be,
and I think it stems from our leadership team’ (Interview, teacher primary school A). with a shared ethos that values children and young people as individuals with rights and dignity. Staff also talked about the importance of a child-centred culture being led ‘from the top’ and staff
supported in their role to place children's needs at the centre of practices: ‘I just think in terms of
what I was saying before about the culture of the school and the type of school you want to be,
and I think it stems from our leadership team’ (Interview, teacher primary school A). In the disability and residential care sectors, particularly, mechanisms such as recruitment,
induction and professional development were seen as important in promoting a shared under-
standing of the organizational ethos and in equipping staff to deal with ethically challenging situ-
ations in their everyday practice. In one residential care service, ethical dilemmas were presented
to potential employees from the outset to gauge their suitability for the job: ‘When we do the interview to get the job, they try and understand your ethics. What
would happen to you if a kid said that Jesus isn't real? How would you feel about
that? And then it's like, well, this is how I'd cope with it. DISCUSSION Ethical theory
Practices
Practice elements
Consequentialist
Children and young people are benefitted and
not harmed
• Prioritization of good outcomes
• Measures toward safety and well-being
• Effective compliance imperatives
• Consequences are consistent and just
Deontological
Children and young people are treated equally
and with dignity
• Inclusive and welcoming environment
• Lack of bias and unfairness
• Respect for children's dignity and
humanity
• Valuing of diversity and difference
Virtue
Children and young people exhibit, develop,
and are treated with good character
• Value placed on strong moral compass
• Clear roles and responsibilities
• Relatability
• Self-reflectiveness
Relational
Children and young people build and take part
in positive relationships
• Mutual care, responsiveness, and trust
• Positive and open connections
• Strong communication
• Advocacy and support TA B L E 1
Practices that align with the ethical frameworks. Notably, however, staff are much less concerned than children with issues of equality, fairness,
and inclusiveness. Notably, however, staff are much less concerned than children with issues of equality, fairness,
and inclusiveness. It appears that virtue ethics plays the least important role among ethical frameworks for all
groups involved. It does appear in preferences among both young people and adults for strong
moral character in staff. It is also arguably evident in the way children and young people refer to
staff and practitioners who are more ‘relatable’ and thus better suited to working effectively with
them. However, neither young people nor staff refer to moral character or community responsi-
bilities as central aspects of ethical practice. Finally, the most prominent ethical framework among children and young people, as well
as being an important framework for adults as well, is relational ethics. Both groups view young
people's safety and well-being as strongly dependent on the quality of child-adult connections,
care, and trust. Across all sectors of schools, residential care services, and disability services,
children and young people, in particular, stress the importance of being cared for and supported
by means of strong intersubjective relations. Staff and leaders similarly view children's posi-
tive growth as grounded in an ethical relation of mutual trust. Across schools, residential care
services, and disability services, all groups view child safety and wellbeing as strongly dependent
on the quality of child-adult connections, care and trust. DISCUSSION We return now to consider whether and how the practices identified by the young people and
staff in this research resonate with the different ethical frameworks discussed earlier, these being
consequentialist, deontological, virtue, and relational (Table 1 below). Consequentialist ethics plays a significantly more important role for adults than it does for
children and young people. It is most evident in adults' desires for a child-centred organiza-
tional culture that places a high value on achieving the best possible outcomes for children and
young people. This is clear also in adults' expressions of concern for young people's avoidance
of outcomes that are unsafe or harmful. Adults also emphasize adhering to administrative and
compliance imperatives as key to keeping children safe. Children and young people themselves,
while concerned to do well and avoid harm, tend not to prioritize this type of ethical language. In contrast, a deontological ethical framework is prominent among young people and adults
both, though interestingly for different reasons. Children and young people place a greater empha-
sis than staff in the three sectors on the importance of being included, welcomed and treated
equally without bias or discrimination. They also value respect for their diversity and difference
and ‘doing what is right.’ Staff, in contrast, emphasize the importance of being supported in criti-
cally reflecting on ethically challenging situations, given there is often more than one ‘right way’
to do things. Albeit significantly less than children, they do value children's voices and agency. 13 GRAHAM et al. TA B L E 1
Practices that align with the ethical frameworks. DISCUSSION Inquiries into historic child abuse have, over many years, drawn
attention to the crucial importance of listening to children in preventing and detecting abuse
(e.g., Levy & Kahan, 1991; Moore et al., 2016, 2017; Waterhouse, 2000); however, safety is not
merely a matter of protection from abuse. It also embraces a wide range of harms, including in
relation to mental health, if children are not listened to and/or are unable to identify a trusted
adult who can help facilitate their voice and agency. Our research suggests that promoting chil-
dren's agency and protecting children from harm are two expressions of an underlying ethical
value of treating children with equal recognition and respect. Consistent with our earlier research, young people and staff place high value on relationships
of mutual recognition (Graham, Anderson, et al., 2022). The term “recognition” could be said to
capture the strong overlap in our findings between relational and deontological ethics. Recogni-
tion of the importance and value of others is a way of building strong and caring relationships,
but it also calls for respecting each other's experiences, voices, and agency (Thomas, 2012). The
findings in this study suggest, then, that mutual recognition involves care, respect and valuing
of one another's dignity, made possible through strong ethical relationships. This association
with relational ethics is further investigated in Phase 3 of the research where we quantitatively
test which of these practices are positively associated with Honneth's (1995) three modes of
recognition—love, rights and esteem (translated as being cared for, respected and valued). One could also frame this combination of relational and agentic values with the notion
of children's empowered inclusion. This term has been used in childhood studies research to
describe an ethics of “recognizing [persons'] deep interdependency by taking steps to empower
them actively” (Josefsson & Wall, 2020, p. 1052). Inclusion is a relational term arising out of
feminist ethics and refers to the ethical responsibility to overcome the marginalization of histor-
ically suppressed experiences by opening up new ways for those experiences to gain agency and
be voiced (Young 2000). The notion of empowered inclusion recognizes, in addition, that chil-
dren and other marginalized groups are best included, not just as individual agents, but inter-
dependently, that is, as members of mutually supporting ethical relationships. In this way, the
concept of mutual recognition may actually be complicated by consideration of the experiences
specifically of children. DISCUSSION Such findings underline the importance of using a range of ethical frameworks to interpret
how children, young people and adults understand and experience ethical practice. Clearly, the
participants are not philosophers who have adopted one ethical framework which they adhere to
or are consistently guided by; rather their practices reflect different ethical angles, often bundled
together, to address particular issues. Relational ethics is by far the most discernible ethical
approach in the way that both children and adults place great value on connections of trust and
care. At the same time, consequentialist ethics emerges in the way adults seek the best possible
outcomes for children and young people by promoting a child-centred organizational culture. Deontological ethics is evident particularly in children and young people's desire to be treated
equally and inclusively and in adults' need for support in critically reflecting on ethically chal- 14 GRAHAM et al. lenging situations. And virtue ethics, while the least visible of the frameworks, emerged in the
way both young people and adults place importance on the strong moral character of staff in
working effectively with children and young people. lenging situations. And virtue ethics, while the least visible of the frameworks, emerged in the
way both young people and adults place importance on the strong moral character of staff in
working effectively with children and young people. In terms of the implications of this research for improving policy and practice, the find-
ings give further weight to the growing evidence around the importance of relationships for
ethical practice—something that has been advocated repeatedly in social work (see Ingram &
Smith, 2018), in education (e.g., Bingham, 2001) and in relation to services for disabled children
(Jacobs et al., 2021). These authors point to the ways in which relationship work can be impeded
by organizational imperatives, as well as by overly goal-oriented assessments. Our research
suggests that the most important ethical practice for children's safety and well-being is the culti-
vation of caring, responsive, and mutual relationships between children and adults. Another implication of these findings for further consideration is the importance children
and young people place on having a sense of voice and agency, as we also identified in previous
research (Graham et al., 2018). CONCLUSION Informed by theories of ethics and practice architectures, this research explored the practices
that contribute to children's wellbeing and safety in schools, residential care and disability
services. There is much to be learned from the different perspectives of young people and adults
as expressed in our study. While the sector-specific findings are reported in-depth elsewhere
(Graham, Canosa, et al., 2022; Robinson et al., 2022), this article provides a more nuanced insight
into the different ethical dimensions of practice in these settings. The broadest conclusion that can be drawn from this study is that the participants are most
likely to link children's safety and well-being to relational ethics, followed closely by deontolog-
ical ethics. They are least likely to draw upon consequentialist or virtue ethics. These findings
suggest that children's safety and well-being are viewed as primarily dependent on an ethics of
what could be called mutual recognition or empowered inclusion. Consistent with previous find-
ings from this research, an ethics of recognition is important for fostering deep connections of
care that are also just and equal. As we see here, these connections also foster children's own
power and agency. To put it differently, both the children and the adults in our study see chil-
dren's safety and well-being as rooted in their empowered inclusion. At the same time, although they concurred on the importance of equal and caring relation-
ships, children talked much more about voice and agency, while adults emphasized organiza-
tional culture (even more than relationships). We suggest that one important learning here is
the inherent value of including different perspectives. It is not that young people are right to
emphasize voice and agency, or that adult staff are wrong in paying more attention to organiza-
tional culture; it is rather that each group has access to information and understanding from their
positioning and lived experience which can potentially be shared to give a more complete and
rounded picture. In other words, while it is instructive to look at these different aspects of ethical
practice as distinct, this should not obscure the extent to which they are actually interwoven. Strong relationships are attentive to children's (indeed everyone's) views, equally inclusive, and
consequently fully child-centred. The findings suggest that both children and adults predominantly describe ethical practice,
then, in terms of the quality and equality of intersubjective relations. DISCUSSION It is understood as intentional and dynamic. That is, mutual recognition
involves adult caregivers actively making efforts to empower children's voices so that children
themselves are able fully and equally to impact child-adult relationships. Or, to put it as a general
or universal ethics of empowered inclusion: Marginalized groups call for mutually interdepend-
ent relations that empower their otherwise normatively excluded voices. GRAHAM et al. 15 Children in care settings reveal in this way an important ethical truth for us all: That humans
are thoroughly interdependent beings who deserve both to be included in impacting, and to be
empowered by, their relations to one another. Each of us is both vulnerable to systemic harm
and capable of exercising agency in relation to others. What is more, for children as much as
for adults, this empowered inclusion is intersectional. Marginalized persons are marginalized
in many different possible ways that interact with each other, not only by age but also by gender,
race, class, sexuality, disability, and much else. Honneth's concept of recognition gets at this point
by naming what is to be recognized as not just a self but also an “other.” Selves are both agents
and recipients of diverse systemics of othering. As children especially make clear, this means that
mutual recognition is a dynamic and interdependent process of simultaneously including and
empowering one another. It is beyond the scope of this article to discuss in depth the challenges of
making this concept real, in particular with groups of children who are marginalized in multiple
ways, or in different cultural contexts; but a fundamental starting point must be to heed the call
of Josefsson and Wall (2020: 1054), that ‘institutions need to become more actively self-critical’. AUTHOR CONTRIBUTIONS All authors contributed to the study conception and design. Material preparation, data collection
and analysis were led by Prof Anne Graham and Dr Antonia Canosa. The first draft of the manu-
script was written by Prof Anne Graham, and all authors commented on previous versions of the
manuscript. All authors read and approved the final manuscript. CONCLUSION This ethical relationality is
understood, less in terms of vertical responsibilities of care and protection (largely the domain GRAHAM et al. 16 of adults and shaped by institutional norms), and more in terms of simultaneously vertical and
horizontal interdependence. Both the children and the adults in our research appear to agree
that children's safety and well-being is principally a matter of children's mutual recognition and
hence of empowered inclusion. of adults and shaped by institutional norms), and more in terms of simultaneously vertical and
horizontal interdependence. Both the children and the adults in our research appear to agree
that children's safety and well-being is principally a matter of children's mutual recognition and
hence of empowered inclusion. ACKNOWLEDGEMENTS This research was funded by the Australian Research Council Discovery Grant (DP180100465). We are grateful to all the young people and staff who gave so freely of their time to participate in
this research. We also acknowledge the contributions of the research team, along with the Young
People Advisory Group (YPAG) and adult Project Advisory Group (PAG), and thank them for
their guidance throughout the research. Open access publishing facilitated by Southern Cross
University, as part of the Wiley - Southern Cross University agreement via the Council of Austral-
ian University Librarians. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available on request from the corresponding
author. The data are not publicly available due to privacy or ethical restrictions. CONFLICT OF INTEREST STATEMENT The authors declared no potential conflicts of interest with respect to the research, authorship,
and/or publication of this article. ORCID Anne Graham
https://orcid.org/0000-0002-9308-8536
Antonia Canosa
https://orcid.org/0000-0002-3422-9095
John Wall
https://orcid.org/0000-0001-5297-0357
Patrick Thomas
https://orcid.org/0000-0002-5310-9144 ETHICS STATEMENT The ethical aspects of this research were approved by the Southern Cross University Human
Research Ethics Committee (approval number: ECN-19-047) and relevant government
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search.ebscohost.com/login.aspx?direct=true&db=psyh&AN=2013-10492-001&site=ehost-live ch.ebscohost.com/login.aspx?direct=true&db=psyh&AN=2013-10492-001&site=ehost-live Graham, A., Anderson, D., Truscott, J., Simmons, C., Thomas, N. P., Cashmore, J., & Bessell, S. (2022). REFERENCES Exploring
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(ERIC): Compendium. Retrieved from http://childethics.com/ aham, A., Powell, M. A., Taylor, N., Anderson, D. L., & Fitzgerald, R. (2013). Ethical research involv (ERIC): Compendium. Retrieved from http://childethics.com/ Graham, A., Truscott, J., Simmons, C., Anderson, D., & Thomas, N. (2018). Exploring student participation across
different arenas of school life. British Educational Research Journal, 44(6), 1029–1046. Honneth, A. (1995). The struggle for recognition: The moral grammar of social conflicts. Polity Press Ingram, R., & Smith, M. (2018). Relationship-based practice: Emergent themes in social work literature (p. 41). Insti-
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abuse and harm in residential care:“It's got to be better than home”. Children and Youth Services Review, 81,
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tutional Responses to Child Sexual Abuse. Nicolini, D. (2012). Practice theory, work and organisation: An introduction. Oxford University Press. Pawlukewicz, J., & Ondrus, S. (2013). Ethical dilemmas: The use of applied scenarios in the helping professions. Journal of Social Work Values and Ethics, 10(1), 2–12. 18 GRAHAM et al. 18 Robinson, S., Graham, A., Canosa, A., Moore, T., Taylor, N., & Boyle, T. (2022). Ethical practice in disability
services: Views of young people and staff. Ethics and Social Welfare, 16(4) 412–431. https://doi.org/10.1080/
17496535.2022.2072519 Smallbone, S. (2017). The impact of Australia's Royal Commission on child- and youth- serving organisations. Child Abuse and Neglect, 74, 99–102. iroux, J. P., & Krasemann, K. W. (2007). Ethics: Theory and practice (9th ed.). Pearson Prentice Hall Thomas, N. (2012). Love, rights and solidarity: Studying children’s participation using Honneth’s theory of recog-
nition. Childhood, 19(4), 453–466. Waterhouse, R. (2000). Lost in care: Report of the Tribunal of Inquiry into the abuse of children in care in the former
county council areas of Gwynedd and Clwyd since 1974. Stationery Office. How to cite this article: Graham, A., Canosa, A., Wall, J., & Thomas, P. (2023). Child-
safe organizations and the ethics of empowered inclusion. Children & Society, 00, 1–18.
https://doi.org/10.1111/chso.12732 AUTHOR BIOGRAPHIES Anne Graham is Professor of Childhood Studies and Director of the Centre for Children and
Young People, Faculty of Health, Southern Cross University, Australia. Anne is nationally and
internationally known for her research around children and young people's rights and wellbe-
ing. She has published widely in areas related to social and emotional wellbeing, participation
rights and practices, adapting to loss and grief, and ethical research. Dr Antonia Canosa is a social anthropologist and Research Fellow on the ‘Ethical Practice
Involving Children’ (EPIC) research project at the Centre for Children and Young People,
Faculty of Health, Southern Cross University, Australia. Antonia's work focuses on children's
rights, participation and wellbeing in schools and the community context. Her research has
been published in highly ranked international journals and presented at national and inter-
national conferences. John Wall is Professor of Philosophy, Religion, and Childhood Studies, as well as Director of
the Childism Institute, at Rutgers University Camden, USA. He is a theoretical ethicist who
works in political philosophy, post-structuralism, and children's rights. John is internationally
known for advancing children's rights and the field of childhood studies including in his latest
book Give Children the Vote: On Democratizing Democracy. Nigel Patrick Thomas is Professor Emeritus of Childhood and Youth at the University of
Central Lancashire in the U.K. and founder of The Centre for Children and Young People's
Participation in the School of Social Work, Care and Community. Patrick's research embraces
child welfare, children's rights and theories of childhood, with a particular focus on children
and young people's participation. He is internationally known for his work on children and
young people's participation, and featured in the book Key Thinkers in Childhood Studies. ry for rules of use; OA articles are governed by the applicable Creative Commons License How to cite this article: Graham, A., Canosa, A., Wall, J., & Thomas, P. (2023). Child-
safe organizations and the ethics of empowered inclusion. Children & Society, 00, 1–18. https://doi.org/10.1111/chso.12732 verned by the applicable Creative Commons License
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https://eprints.whiterose.ac.uk/193986/3/Metaphilosophy%20-%202022%20-%20Habgood%E2%80%90Coote%20-%20Can%20a%20good%20philosophical%20contribution%20be%20made%20just%20by%20asking%20a%20question%201.pdf
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Can a good philosophical contribution be made just by asking a question?<sup>1</sup>
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Metaphilosophy
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DOI: 10.1111/meta.12599 DOI: 10.1111/meta.12599 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 Metaphilosophy. 2022;1–1. Joshua Habgood-Coote1 1School of Philosophy, Religion, and History of Science, University of Leeds, United Kingdom
2Faculty of Theology and Religion and Faculty of Philosophy, University of Oxford, United Kingdom
3Department of Philosophy, Western Washington University, Washington, USA Metaphilosophy. 2022;1–1.
wileyonlinelibrary.com/journal/meta
1
1The authors are listed in alphabetical order. Joshua Habgood-Coote and Dennis Whitcomb conceptualised this paper. All three authors contrib-
uted to writing it and signed off on its final version. Joshua Habgood-Coote's research on this project received funding from the European
Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement no. 818633).
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.
© 2022 The Author. Metaphilosophy published by Metaphilosophy LLC and John Wiley & Sons Ltd. wileyonlinelibrary.com/journal/meta Correspondence p
Dennis Whitcomb, Department of Philosophy, Western Washington University, 516 High St., Bellingham, WA 98255, USA
Email: whitcod@wwu.edu © 2022 The Author. Metaphilosophy published by Metaphilosophy LLC and John Wiley & Sons Ltd. Metaphilosophy. 2022;1–1. 1
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A new coactivator complex required for retinoic acid-dependent regulation of embryonic symmetry
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ARTICLE ARTICLE Results
d
fi Identification of the WHHERE complex. In order to understand
the mechanism of action of Rere in the RA pathway controlling
somite symmetry, we first set out to identify Rere-interacting
proteins in the mouse mesoderm. To that end, we generated a
transgenic mouse line, which allows the conditional expression
of a tagged version of Rere containing two HA epitopes at the
C-terminal end of the protein (Rere-HA). A Rere-HA construct
preceded by a LoxP-STOP-LoxP cassette was introduced into the
Rosa26 locus by homologous recombination in mouse embryonic
stem (ES) cells. We then used these cells to generate a Rosa26-
LoxP-STOP-LoxP-Rere-HA
mouse
line
(RS-Rere-HA
line). Whereas Rere mutants (Rereom/om) die around E9.5 with defects
in forebrain, heart and a right side specific delay in somite
formation8, 9, expression of at least one Rere-HA allele in the
mutant Rereom/om background led to morphologically normal
embryos (Supplementary Fig. 1a–c). Therefore, the tagged
Rere-HA protein is functional in vivo. WHHERE complex members act as coactivators of RA sig-
naling. To test the coactivator properties of WHHERE on RA
signaling, we transfected NIH3T3 fibroblast cells with expression
vectors coding for either Rere, Wdr5, Hdac1, or Hdac2, together
with a RA reporter containing the well-characterized retinoic acid
response element (RARE) of the known RA target retinoic Acid
Receptor beta (Rarβ)17, driving luciferase expression. In the
presence of RA, overexpression of each of the four WHHERE
complex proteins increased RA reporter expression (Fig. 2a). This
activation was not observed in the absence of RA (Supplementary
Fig. 3a). Also overexpression of the WHHERE complex compo-
nents did not activate either a minimal or a SV40 promoter
driving luciferase supporting the specificity of the RA response
(Supplementary Fig. 3b, c). Furthermore, co-transfection of
Hdac1 and Hdac2 cooperated to activate RA signaling (Fig. 2b). Conversely, treatment of fibroblast cultures with siRNAs against
Rere, Hdac1, or Wdr5 in the presence of RA decreased RARE-
Luciferase reporter activity (Fig. 2c and Supplementary Fig. 3d–f). Treatment with Hdac2 siRNA led to an increase of RARE-
Luciferase reporter activity (Fig. 2c and Supplementary Fig. 3g),
which might be explained by a stabilization of Hdac1 (due to the
decrease of Hdac2), potentially resulting in an increase in RA To direct expression of Rere-HA to the mesoderm, RS-Rere-
HA mice were crossed to the T-Cre mouse line10. The WHHERE coactivator complex is required for
retinoic acid-dependent regulation of embryonic
symmetry Gonçalo C. Vilhais-Neto1,2, Marjorie Fournier1, Jean-Luc Plassat1, Mihaela E. Sardiu2, Anita Saraf2,
Jean-Marie Garnier1, Mitsuji Maruhashi1,2, Laurence Florens2, Michael P. Washburn
2,3
& Olivier Pourquié
1,2,4,5,6 Gonçalo C. Vilhais-Neto1,2, Marjorie Fournier1, Jean-Luc Plassat1, Mihaela E. Sardiu2, Anita Saraf2,
Jean-Marie Garnier1, Mitsuji Maruhashi1,2, Laurence Florens2, Michael P. Washburn
2,3
& Olivier Pourquié
1,2,4,5,6 Bilateral symmetry is a striking feature of the vertebrate body plan organization. Vertebral
precursors, called somites, provide one of the best illustrations of embryonic symmetry. Maintenance of somitogenesis symmetry requires retinoic acid (RA) and its coactivator
Rere/Atrophin2. Here, using a proteomic approach we identify a protein complex, containing
Wdr5, Hdac1, Hdac2 and Rere (named WHHERE), which regulates RA signaling and controls
embryonic symmetry. We demonstrate that Wdr5, Hdac1, and Hdac2 are required for RA
signaling in vitro and in vivo. Mouse mutants for Wdr5 and Hdac1 exhibit asymmetrical somite
formation characteristic of RA-deficiency. We also identify the Rere-binding histone
methyltransferase Ehmt2/G9a, as a RA coactivator controlling somite symmetry. Upon RA
treatment, WHHERE and Ehmt2 become enriched at RA target genes to promote RNA
polymerase II recruitment. Our work identifies a protein complex linking key epigenetic
regulators acting in the molecular control of embryonic bilateral symmetry. 1 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS (UMR 7104), Inserm U964, Université de Strasbourg, Illkirch F-67400, France. 2 Stowers Institute for Medical Research, Kansas City, MO 64110, USA. 3 Department of Pathology and Laboratory Medicine, University of Kansas Medical
Center, Kansas City, KS 66160, USA. 4 Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA. 5 Howard Hughes Medical Institute, Kansas City, MO 64110, USA. 6 Department of Genetics, Harvard Medical School and Department of Pathology, Brigham
and Women’s Hospital, 60 Fenwood Road, Boston, MA 02115, USA. Correspondence and requests for materials should be addressed to
O.P. (email: pourquie@genetics.med.harvard.edu) 1 NATURE COMMUNICATIONS| 8: 728
| DOI: 10.1038/s41467-017-00593-6| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 and Hdac2 as well as a previously unreported partner, Wdr513–16
(Fig. 1b, Supplementary Figs. 2a and 9). To estimate relative
protein levels, we compared the NSAF values for each protein12. NSAF for Rere, Hdac1, and Hdac2 were similar while it was three
times higher for Wdr5 (Fig. 1b). These results suggest that the
proteins Rere, Hdac1, Hdac2, and Wdr5 can interact in the
mesoderm. The WHHERE coactivator complex is required for
retinoic acid-dependent regulation of embryonic
symmetry p
g
g
Here, we identify and characterize biochemically a previously
undescribed retinoic coactivator complex, containing the proteins
Wdr5, Hdac1, Hdac2, and Rere (the WHHERE complex). We
demonstrate that mouse mutants of the WHHERE complex
members down-regulate RA signaling and exhibit somite bilateral
symmetry defects. We report that the WHHERE complex also
binds the histone methyltransferase Ehmt2/G9a. Null mouse
mutants for Ehmt2 also exhibit RA downregulation and somite
symmetry defects indicating that it also acts as an activator of RA
signaling. We show that Ehmt2 and the WHHERE complex bind
to the promoter of RA targets and serve to recruit PolII to trigger
gene activation. The WHHERE coactivator complex is required for
retinoic acid-dependent regulation of embryonic
symmetry When the RA ligand binds to RAR, the
corepressors are replaced by a set of coactivators including
histone acetyltransferases, contributing to active transcription of
RA target genes5, 6. In the absence of RA signaling in the mouse
embryo, somite formation becomes asymmetrical, showing a
significant delay on the right side7. A similar somite desynchro-
nization phenotype is also observed in mutants for the protein
Rere (or Atrophin2) which acts as a coactivator for RA signalling8. In vitro characterization of the WHHERE complex. To validate
the identification of Wdr5 as an interacting partner of Rere,
Hdac1, and Hdac2, we co-expressed tagged versions of the four
proteins (Rere-Flag, Flag-Hdac1, Flag-Hdac2, and HA-Wdr5)
using
a
baculovirus-insect
cell
expression
system. After
Flag immunoprecipitation of Rere, Hdac1 and Hdac2, HA-Wdr5
was detected in the eluates, as confirmed by LC−MS/MS analysis
of the Coomassie stained gel bands (Fig. 1c, Supplementary
Figs. 2b and 7). The four proteins still co-purified together at
high
salt
concentration
(500 mM
KCl)
suggesting
the
existence of a stable protein complex comprising Rere, Hdac1,
Hdac2, and Wdr5 (Fig. 1c and Supplementary Fig. 7). By
co-immunoprecipitation of baculovirus-expressed Rere-Flag and
HA-Wdr5, we could demonstrate that Rere binds directly to
Wdr5 (Fig. 1d and Supplementary Fig. 7). Wdr5 does not bind
directly to Hdac1 or Hdac2 in high or low salt wash conditions
(Supplementary Figs. 2c and 9). Wdr5 binding to Hdac1 and
Hdac2 could only be detected when Rere is also co-expressed
(Supplementary Figs. 2c and 9), suggesting that Rere acts as a
scaffolding
component
binding
Hdac1/Hdac2
and
Wdr5. To
analyze
whether
the
four
co-immunopurified
proteins
form a stable protein complex, we carried out gel filtration
chromatography followed by western blot and mass spectrometry
analysis. All four proteins co-eluted together in a fraction cor-
responding to a high molecular weight complex of 0.5–0.6 MDa
(Figs. 1e, f, Supplementary Figs. 2d, e and 7). This molecular
weight is consistent with the abundance predicted by the NSAF
values of the proteomic analysis. Additionally, Hdac1, Hdac2, and
Wdr5
co-immunoprecipitated
with
the
endogenous
Rere
in NIH3T3 cells further supporting the existence of such a
protein complex (Fig. 1g and Supplementary Fig. 7). Altogether,
these results suggest that Wdr5, Hdac1, Hdac2 and Rere can
form a stable protein complex, which we called the WHHERE
complex. The WHHERE coactivator complex is required for
retinoic acid-dependent regulation of embryonic
symmetry T T
he development of bilaterally symmetrical structures, such
as limbs or somites takes place concomitantly with the
asymmetric formation of internal organs such as heart, gut,
and liver. Whereas the pathway responsible for establishing
left–right identity in the embryo begins to be well understood1,
little is known about the mechanisms controlling embryonic
symmetry. Retinoic acid (RA) is a derivative of vitamin A,
signaling
via
a
heterodimeric
RAR/RXR
nuclear
receptor
transcription factor2–4. In the absence of RA, the heterodimer
binds
target
genes
together
with
the
SMRT
and
NCoR
corepressor complexes and histone deacetylases such as Hdac3 to
silence gene expression. When the RA ligand binds to RAR, the
corepressors are replaced by a set of coactivators including
histone acetyltransferases, contributing to active transcription of
RA target genes5, 6. In the absence of RA signaling in the mouse
embryo, somite formation becomes asymmetrical, showing a
significant delay on the right side7. A similar somite desynchro-
nization phenotype is also observed in mutants for the protein
Rere (or Atrophin2) which acts as a coactivator for RA signalling8. Here, we identify and characterize biochemically a previously
undescribed retinoic coactivator complex, containing the proteins
Wdr5, Hdac1, Hdac2, and Rere (the WHHERE complex). We
demonstrate that mouse mutants of the WHHERE complex
members down-regulate RA signaling and exhibit somite bilateral
symmetry defects. We report that the WHHERE complex also
binds the histone methyltransferase Ehmt2/G9a. Null mouse
mutants for Ehmt2 also exhibit RA downregulation and somite
symmetry defects indicating that it also acts as an activator of RA
signaling. We show that Ehmt2 and the WHHERE complex bind
to the promoter of RA targets and serve to recruit PolII to trigger
gene activation. T
he development of bilaterally symmetrical structures, such
as limbs or somites takes place concomitantly with the
asymmetric formation of internal organs such as heart, gut,
and liver. Whereas the pathway responsible for establishing
left–right identity in the embryo begins to be well understood1,
little is known about the mechanisms controlling embryonic
symmetry. Retinoic acid (RA) is a derivative of vitamin A,
signaling
via
a
heterodimeric
RAR/RXR
nuclear
receptor
transcription factor2–4. In the absence of RA, the heterodimer
binds
target
genes
together
with
the
SMRT
and
NCoR
corepressor complexes and histone deacetylases such as Hdac3 to
silence gene expression. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 Peptide
count
Spectral
count
Sequence
coverage
Average
NSAF
Rere
41
1131
39.79
0.0021
Wdr5
17
847
76.05
0.0068
Hdac1
21
334
62.03
0.0016
Hdac2
23
421
69.06
0.0020
High salt
Low salt
Rere
Wdr5
Hdac1
Hdac2
0.000 0.005 0.037
NSAF
36
WHHERE
salt wash
150 mM
500 mM
250
130
95
72
55
(kDa)
Rere-flag
Flag-Hdac1/2
HA-Wdr5
Gel filtration profile
(superose 6)
1.0
1.4
Elution volume (ml)
Protein absorbance
(A280 nm)
0
WHHERE
(0.5–0.6 MDa)
1.2
1.6
Vo
50
A9
A11
B1
B3
Elution fractions
A8
A9 A10 A11 A12 B1
B2
250
Wdr5
Rere
Hdac2
Hdac1
65
65
36
Rere-flag
HA-Wdr5
Rere-flag
HA-Wdr5
+
+
+
+
+
–
Western blot
flag/HA
Identification of Rere
binding partners
Rosa
Rere-HA
HA immunopurification
in high and low
salt conditions
Proteolytic
digestion
2D LC–MS/MS
MudPIT
Intensity
m/z
Embryos expressing
Rere-HA in
paraxial mesoderm
a
Input IgG Rere
IP NIH3T3
Rere
Hdac2
Wdr5
Hdac1
250
(kDa)
72
72
36
T
Cre
Protein
name
(kDa)
Control
IP: Flag
Input
b
c
d
e
f
g
Fig. 1 Proteomic identification of the WHHERE protein complex containing Wdr5, Hdac1, Hdac2, and Rere. a Schematic workflow for the proteomic strategy
used to identify Rere-associated proteins. b Hierarchical clustering of the NSAF values showing Wdr5, Hdac1, and Hdac2 clustering with Rere identified by
MudPIT. Columns correspond to different immunoprecipitations conditions. Table showing the peptide count, spectral count, sequence coverage (%) and
average NSAF values for Rere, Wdr5, Hdac1, and Hdac2. c SDS-PAGE gel stained with Coomassie blue after Flag immunopurification at low (150 mM KCl)
and high (500 mM KCl) salt wash of the recombinant WHHERE complex from baculoviruses infected cells co-expressing Rere-Flag, Flag-Hdac1,
Flag-Hdac2, and HA-Wdr5. Identification of the different components was confirmed by LC–MS/MS analysis of the corresponding gel bands. d Flag
immunoprecipitation from extracts of cells infected with baculoviruses expressing HA-Wdr5 and Rere-Flag. Flag and HA western blot. The three bands
recognized by the anti-Flag antibodies correspond to Rere and its degradation products. e Gel filtration chromatography profile of the WHHERE complex
purified from baculoviruses infected cells co-expressing Rere-Flag, Flag-Hdac1, Flag-Hdac2, and HA-Wdr5. f Immunoblot analysis for Rere, Hdac1, Hdac2,
and Wdr5 showing the gel filtration chromatography elution fractions from A8 to B2. g Rere immunoprecipitation from NIH3T3 cells identifying the
endogenous WHHERE complex components. Results
d
fi We prepared
whole
cell
protein
extracts
from
~ 600
RS-Rere-HA;T-Cre
embryos, and performed affinity purification using anti-HA
antibodies under high or low salt conditions (Fig. 1a). To identify
the immunoprecipitated proteins, eluted fractions were submitted
to mass spectrometry analysis using the multidimensional protein
identification technology (MudPIT)11. A set of 105 common
proteins was found between the different immunopurification
conditions (Supplementary Figs. 1d, 6 and Supplementary Data 1). Hierarchical clustering analysis of the 105 proteins based on
the
normalized
spectral
abundance
factor
(NSAF)12
in
the different immunoprecipitation conditions identified three
abundant proteins tightly clustering with Rere (Supplementary
Fig. 1e, f). These include Rere’s known binding partners Hdac1 NATURE COMMUNICATIONS| 8: 728
| DOI: 10.1038/s41467-017-00593-6| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 Rere, Hdac1, Hdac2, and Wdr5 were detected after western blot analysis of Rere immunoprecipitation eluates
and compared to a negative control immunoprecipitation (IgG) and input extract Peptide
count
Spectral
count
Sequence
coverage
Average
NSAF
Rere
41
1131
39.79
0.0021
Wdr5
17
847
76.05
0.0068
Hdac1
21
334
62.03
0.0016
Hdac2
23
421
69.06
0.0020
High salt
Low salt
Rere
Wdr5
Hdac1
Hdac2
0.000 0.005 0.037
NSAF
S
Protein
name
b 36
WHHERE
salt wash
150 mM
500 mM
250
130
95
72
55
(kDa)
Rere-flag
Flag-Hdac1/2
HA-Wdr5
Identification of Rere
binding partners
Rosa
Rere-HA
HA immunopurification
in high and low
salt conditions
Proteolytic
digestion
2D LC–MS/MS
MudPIT
Intensity
m/z
Embryos expressing
Rere-HA in
paraxial mesoderm
a
T
Cre
c a 36
WHHERE
salt wash
150 mM
500 mM
250
130
95
72
55
(kDa)
Rere-flag
Flag-Hdac1/2
HA-Wdr5
c c c Input IgG Rere
IP NIH3T3
Rere
Hdac2
Wdr5
Hdac1
250
(kDa)
72
72
36
g g g Gel filtration profile
(superose 6)
1.0
1.4
Elution volume (ml)
Protein absorbance
(A280 nm)
0
WHHERE
(0.5–0.6 MDa)
1.2
1.6
Vo
50
A9
A11
B1
B3
e Rere-flag
HA-Wdr5
Rere-flag
HA-Wdr5
+
+
+
+
+
–
Western blot
flag/HA
Control
IP: Flag
Input
d Gel filtration profile
(superose 6)
1.0
1.4
Elution volume (ml)
Protein absorbance
(A280 nm)
0
WHHERE
(0.5–0.6 MDa)
1.2
1.6
Vo
50
A9
A11
B1
B3
Rere-flag
HA-Wdr5
Rere-flag
HA-Wdr5
+
+
+
+
+
–
Western blot
flag/HA
Control
IP: Flag
Input
d
e d Elution fractions
A8
A9 A10 A11 A12 B1
B2
250
Wdr5
Rere
Hdac2
Hdac1
65
65
36
(kDa)
f f Fig. 1 Proteomic identification of the WHHERE protein complex containing Wdr5, Hdac1, Hdac2, and Rere. a Schematic workflow for the proteomic strategy
used to identify Rere-associated proteins. b Hierarchical clustering of the NSAF values showing Wdr5, Hdac1, and Hdac2 clustering with Rere identified by
MudPIT. Columns correspond to different immunoprecipitations conditions. Table showing the peptide count, spectral count, sequence coverage (%) and
average NSAF values for Rere, Wdr5, Hdac1, and Hdac2. c SDS-PAGE gel stained with Coomassie blue after Flag immunopurification at low (150 mM KCl)
and high (500 mM KCl) salt wash of the recombinant WHHERE complex from baculoviruses infected cells co-expressing Rere-Flag, Flag-Hdac1,
Flag-Hdac2, and HA-Wdr5. Identification of the different components was confirmed by LC–MS/MS analysis of the corresponding gel bands. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 d Flag
immunoprecipitation from extracts of cells infected with baculoviruses expressing HA-Wdr5 and Rere-Flag. Flag and HA western blot. The three bands
recognized by the anti-Flag antibodies correspond to Rere and its degradation products. e Gel filtration chromatography profile of the WHHERE complex
purified from baculoviruses infected cells co-expressing Rere-Flag, Flag-Hdac1, Flag-Hdac2, and HA-Wdr5. f Immunoblot analysis for Rere, Hdac1, Hdac2,
and Wdr5 showing the gel filtration chromatography elution fractions from A8 to B2. g Rere immunoprecipitation from NIH3T3 cells identifying the
endogenous WHHERE complex components. Rere, Hdac1, Hdac2, and Wdr5 were detected after western blot analysis of Rere immunoprecipitation eluates
and compared to a negative control immunoprecipitation (IgG) and input extract the RA pathway in NIH3T3 cells. Moreover, activation of RA
signaling by the WHHERE complex depends on Hdac1 and
Hdac2 deacetylase activity. signalling18, 19. Consistent with this possibility, the double
knockdown of Hdac1 and Hdac2 further decreased RA signaling
compared to Hdac1 siRNA alone (Fig. 2d). Furthermore, Hdac1
or Hdac1/Hdac2 depletion reduced RA activation mediated by
Rere and Wdr5 (Fig. 2e, f). Inhibition of deacetylase enzymatic
activity with a range of chemical inhibitors decreased RA signa-
ling (Fig. 2g)20. In line with this, overexpression of Rere or Wdr5
did not lead to a significant increase in RA signaling when cells
were treated with the HDAC inhibitors Trichostatin A (TSA) or
sodium butyrate (SB) (Fig. 2h, i). Hdac1 and Hdac2 have been
shown to bind Rere N-terminal region9, 13, 14. In the presence of
RA, overexpression of the N-terminal region of Rere (N-Rere)
strongly increased RA signaling whereas no activation could
be seen with Rere C-terminal region (Rere C) (Fig. 2j). The
activation by N-Rere was dependent on deacetylase activity since
TSA or SB treatment strongly decreased N-Rere-dependent RA
signaling (Fig. 2k). Overall, these results show that the WHHERE
complex proteins Rere, Wdr5, Hdac1, and Hdac2 act to activate signalling18, 19. Consistent with this possibility, the double
knockdown of Hdac1 and Hdac2 further decreased RA signaling
compared to Hdac1 siRNA alone (Fig. 2d). Furthermore, Hdac1
or Hdac1/Hdac2 depletion reduced RA activation mediated by
Rere and Wdr5 (Fig. 2e, f). Inhibition of deacetylase enzymatic
activity with a range of chemical inhibitors decreased RA signa-
ling (Fig. 2g)20. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 In line with this, overexpression of Rere or Wdr5
did not lead to a significant increase in RA signaling when cells
were treated with the HDAC inhibitors Trichostatin A (TSA) or
sodium butyrate (SB) (Fig. 2h, i). Hdac1 and Hdac2 have been
shown to bind Rere N-terminal region9, 13, 14. In the presence of
RA, overexpression of the N-terminal region of Rere (N-Rere)
strongly increased RA signaling whereas no activation could
be seen with Rere C-terminal region (Rere C) (Fig. 2j). The
activation by N-Rere was dependent on deacetylase activity since
TSA or SB treatment strongly decreased N-Rere-dependent RA
signaling (Fig. 2k). Overall, these results show that the WHHERE
complex proteins Rere, Wdr5, Hdac1, and Hdac2 act to activate WHHERE regulates RA signaling and somite bilateral sym-
metry. To analyze WHHERE-dependent RA regulation in vivo,
we characterized the phenotype of mouse embryos mutant for the
different components of the complex. Hdac1 mutants exhibit a
variety of developmental defects similar to Rereom/om embryos21. We introduced the RARE-LacZ reporter22 in Hdac1-null embryos
and observed a strong downregulation of LacZ expression, similar
to that seen in Rere mutants (Fig. 3a–c). Hdac2 mutants do not
show developmental defects and can survive until the perinatal
period21. To analyze Wdr5 function in vivo we first set out to
generate a conditional knockout mouse line by introducing
LoxP sites flanking exons 2 to exon 4 (Supplementary Fig. 4a). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 No heterozygous embryos were recovered at E8.5 after crossing NATURE COMMUNICATIONS| 8: 728
| DOI: 10.1038/s41467-017-00593-6| www.nature.com/naturecommunications 3 Fold activation
0
1
2
3
4
5
6
7
RA
Ctrl
Ctrl
0
1
2
3
4
5
6
7
8
Fold activation
RA
–
–
+
+
+
+
+
+
+
+
+
Hdac1
Hdac2
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
d
c
a
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
RA
Ctrl
Ctrl
–
Pano
+
+
+
+
+
+
Hdac1
Ctrl
siRNA
RA
+
Hdac1 Ctrl
+
Ctrl
Wdr5
Hdac1
Hdac2
Ctrl
siRNA
RA
+
Hdac1
Hdac2
Hdac1
Hdac2
Ctrl
+
Ctrl
Wdr5
Fold activation
0
0.5
1
1.5
2
2.5
3
RA
+
–
TSA
–
+
–
Ctrl
Fold activation
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
**
**
**
**
**
**
**
**
**
**
**
**
*
*
**
*
**
**
**
**
**
**
**
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Rere
Ctrl
Ctrl
Ctrl
RA
+
+
+
+
siRNA
+
b
**
**
**
**
Hdac1
Ctrl
+
+
+
+
Ctrl
Rere
+
+
+
+
+
+
+
+
+
+
+
–
–
Rere
Wdr5
RA
+
–
SB
–
+
–
Ctrl
+
+
+
+
+
+
+
–
–
Rere
Wdr5
Ctrl
Fold activation
0
0.5
1
1.5
2
2.5
3
RA
–
Ctrl
Ctrl
Rere
Wdr5
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Wdr5
Ctrl
RA
+
+
+
+
+
+
+
+
siRNA
**
**
**
**
Fold activation
0
1
2
3
4
5
6
7
8
Ctrl
Rere
N-Rere
Ctrl
Rere-C
RA
–
+
+
+
+
**
**
NS
Fold activation
0
1
2
3
4
5
6
7
8
RA
+
+
+
+
+
–
+
TSA
–
+
–
–
+
–
–
Ctrl
N-Rere
SB
–
–
+
–
–
–
+
**
**
**
**
Rere
Wdr5 Hdac1 Hdac2
Hdac1 Hdac2
Wdr5 Hdac1 Hdac2
Hdac1
Ctrl
RA
+
+
+
+
siRNA
Hdac2 Hdac1
Hdac2
Rere
TSA
SB
Api
LAQ
Ctrl
Wdr5
Rere
Rere
Wdr5
e
f
g
h
i
j
k
l
m
Fig. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 Student’s unpaired two-tailed t-test
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 Fold activation
0
1
2
3
4
5
6
7
RA
Ctrl
Ctrl
0
1
2
3
4
5
6
7
8
Fold activation
RA
–
–
+
+
+
+
+
+
+
+
+
Hdac1
Hdac2
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
d
c
a
**
**
**
**
**
**
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Rere
Ctrl
Ctrl
Ctrl
RA
+
+
+
+
siRNA
+
b
**
**
**
**
Rere
Wdr5 Hdac1 Hdac2
Hdac1 Hdac2
Wdr5 Hdac1 Hdac2
Hdac1
Ctrl
RA
+
+
+
+
siRNA
Hdac2 Hdac1
Hdac2 Fold activation
0
1
2
3
4
5
6
7
RA
Ctrl
Ctrl
0
1
2
3
4
5
6
7
8
Fold activation
RA
–
–
+
+
+
+
+
+
+
+
+
Hdac1
Hdac2
c
a
**
**
**
**
**
F ld
ti
ti
Ctrl
Ctrl
s
b
Rere
Wdr5 Hdac1 Hdac2
Hdac1 Hdac2 Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
d
c
**
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Rere
Ctrl
RA
+
+
+
+
siRNA
+
**
**
**
**
Wdr5 Hdac1 Hdac2
Hdac1
Ctrl
RA
+
+
+
+
siRNA
Hdac2 Hdac1
Hdac2 Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Hdac1
Ctrl
siRNA
RA
+
Hdac1 Ctrl
+
Ctrl
Wdr5
**
*
*
Hdac1
Ctrl
+
+
+
+
Rere
e Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Hdac1
Hdac2
Ctrl
siRNA
RA
+
Hdac1
Hdac2
Hdac1
Hdac2
Ctrl
+
Ctrl
Wdr5
**
*
**
Ctrl
Rere
+
+
+
+
f Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
RA
Ctrl
Ctrl
–
Pano
+
+
+
+
+
+
+
Wdr5
Hdac1
Hdac2
Ctrl
siRNA
RA
+
Hdac1
Hdac2
Hdac1
Hdac2
Ctrl
+
Ctrl
Wdr5
**
**
**
**
**
*
**
*
**
Hdac1
Ctrl
+
Ctrl
Rere
+
+
+
+
TSA
SB
Api
LAQ
f
g Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
RA
Ctrl
Ctrl
–
Pano
+
+
+
+
+
+
**
**
**
**
**
TSA
SB
Api
LAQ
g Fold activation
0
0.5
1
1.5
2
2.5
3
RA
+
–
TSA
–
+
–
Ctrl
Fold activation
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
**
**
**
**
**
**
+
+
+
+
+
+
+
–
–
Rere
Wdr5
RA
+
–
SB
–
+
–
Ctrl
+
+
+
+
+
+
+
–
–
Rere
Wdr5
Fold activation
0
1
2
3
4
5
6
7
8
Ctrl
Rere
N-Rere
Ctrl
Rere-C
RA
–
+
+
+
+
**
**
NS
h
i
j Fold activation
0
0.5
1
1.5
2
2.5
3
RA
+
–
TSA
–
+
–
Ctrl
Fold activation
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
**
**
**
**
**
**
+
+
+
+
+
+
+
–
–
Rere
Wdr5
RA
+
–
SB
–
+
–
Ctrl
+
+
+
+
+
+
+
–
–
Rere
Wdr5
Fold activation
0
1
2
3
4
5
6
7
8
RA
h
i
j i i h l Fold activation
0
1
2
3
4
5
6
7
8
RA
+
+
+
+
+
–
+
TSA
–
+
–
–
+
–
–
Ctrl
N-Rere
SB
–
–
+
–
–
–
+
**
**
**
**
k Fold activation
0
0.5
1
1.5
2
2.5
3
RA
–
Ctrl
Ctrl
Rere
Wdr5
+
+
+
+
**
**
Rere
Wdr5
l l Ctrl
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Wdr5
Ctrl
RA
+
+
+
+
siRNA
**
**
Ctrl
Wdr5
Rere
m k k m Ctrl Fig. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 2 The WHHERE complex acts as a coactivator for retinoic acid signaling. a-k RARE-Luciferase activity from NIH3T3 cells treated or not with 1 µM RA for
20 h. a Cells transfected with expression plasmids containing Rere, Wdr5, Hdac1, or Hdac2 (n = 4). b Cells transfected with expression plasmids containing
Hdac1, Hdac2, or both (n = 4). c Cells treated either with siRNA for Rere, Wdr5, Hdac1, or Hdac2 (n = 4). d Cells treated with siRNA for Hdac1, Hdac2 or both
(n = 4). e Cells overexpressing Rere or Wdr5 and treated with siRNA against Hdac1 (n = 4). f Cells overexpressing Rere or Wdr5 and treated with siRNA
against both Hdac1 and Hdac2 (n = 4). g Cells treated with the HDAC inhibitors Trichostatin A (TSA) (60 nM), sodium butyrate (SB) (3 mM), apicidin (Api)
(300 nM), LAQ824 (LAQ) (60 nM) and Panobinostat (Pano) (30 nM) (n = 4). h Cells overexpressing Rere or Wdr5 and treated with TSA (30 nM) (n = 4). i Cells overexpressing Rere or Wdr5 and treated with SB (1.5 mM) (n = 4). j Cells transfected with expression plasmids containing Rere, N-Rere (Rere
N-terminal domain) or Rere C (Rere C-terminal domain) (n = 3). k Cells overexpressing N-Rere (Rere N-terminal domain) and treated with TSA (30 nM) or SB
(1.5 mM) (n = 4). l Cells transfected with expression plasmids containing Rere, Wdr5, or both (n = 4). m Cells overexpressing Rere and treated with siRNA for
Wdr5 (n = 4). In all graphs data represent mean ± s.e.m. NS—not significant, *P < 0.05 and **P < 0.01. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 2 The WHHERE complex acts as a coactivator for retinoic acid signaling. a-k RARE-Luciferase activity from NIH3T3 cells treated or not with 1 µM RA for
20 h. a Cells transfected with expression plasmids containing Rere, Wdr5, Hdac1, or Hdac2 (n = 4). b Cells transfected with expression plasmids containing
Hdac1, Hdac2, or both (n = 4). c Cells treated either with siRNA for Rere, Wdr5, Hdac1, or Hdac2 (n = 4). d Cells treated with siRNA for Hdac1, Hdac2 or both
(n = 4). e Cells overexpressing Rere or Wdr5 and treated with siRNA against Hdac1 (n = 4). f Cells overexpressing Rere or Wdr5 and treated with siRNA
against both Hdac1 and Hdac2 (n = 4). g Cells treated with the HDAC inhibitors Trichostatin A (TSA) (60 nM), sodium butyrate (SB) (3 mM), apicidin (Api)
(300 nM), LAQ824 (LAQ) (60 nM) and Panobinostat (Pano) (30 nM) (n = 4). h Cells overexpressing Rere or Wdr5 and treated with TSA (30 nM) (n = 4). i Cells overexpressing Rere or Wdr5 and treated with SB (1.5 mM) (n = 4). j Cells transfected with expression plasmids containing Rere, N-Rere (Rere
N-terminal domain) or Rere C (Rere C-terminal domain) (n = 3). k Cells overexpressing N-Rere (Rere N-terminal domain) and treated with TSA (30 nM) or SB
(1.5 mM) (n = 4). l Cells transfected with expression plasmids containing Rere, Wdr5, or both (n = 4). m Cells overexpressing Rere and treated with siRNA for
Wdr5 (n = 4). In all graphs data represent mean ± s.e.m. NS—not significant, *P < 0.05 and **P < 0.01. Student’s unpaired two-tailed t-test We next examined the effect of null mutations of members of
the WHHERE complex on somite symmetry. In nearly half of the
Hdac1–/– embryos, we observed a right side delay of somite
formation resembling the defect observed in Rereom/om and
Raldh2–/– mutants7, 8 (Fig. 3e–g, i, j and Supplementary Fig. 4f). Similar
lateralized
somite
desynchronization
defects
were
observed in a limited number of Wdr5fl/+;T-Cre embryos which
also exhibited broad somitogenesis defects (Fig. 3h). We next
intercrossed Wdr5fl/+ and Rere;T-Cre mice. Strikingly Rereom/om;
Wdr5fl/+;T-Cre
embryos
exhibited
a
stronger
phenotype
than either Rereom/om or Wdr5fl/+;T-Cre (Fig. 3l–o) with ~ 80%
(7 out of 9) of the embryos with a right side delay in with an ubiquitous Cre suggesting that the Wdr5 mutation is
heterozygous lethal at early stages. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 Recruitment of
RNA Polymerase II (Pol II) increased following RA treatment
paralleling the WHHERE complex recruitment (Fig. 4i). Then
we investigated the requirement of Hdac1 deacetylase activity
in transcription activation and Pol II recruitment during
RA signaling. Decreased RARE-Luciferase and Rarβ expression
was observed in TSA-treated cells after RA treatment demon-
strating the importance of HDAC activity in early activation
of RA target genes (Fig. 4a, b). TSA treatment or siRNA-mediated
knockdown of Hdac1 decreased Pol II recruitment at the
Rarβ promoter (Fig. 4j, k). In contrast, no change in Pol II
occupancy
levels
could
be
detected
in
RA
target
genes
unresponsive to RA in NIH3T3 cells, such as Cyp26a1 and
Hoxa1 or in promoters of genes unresponsive to RA signaling
following
such
treatments
(Supplementary
Fig. 3k–m). In
transfected cells expressing Flag-Hdac1 alone or Flag-Hdac1 and
Rere-HA, we observed binding of Hdac1 to endogenous Rarα. This suggests that Hdac1 could bridge the WHHERE complex
to Rarα (Fig. 4l–n and Supplementary Fig. 8). Together, these
data
support
a
role
of
the
WHHERE
complex
in
the
recruitment of Pol II necessary for early activation of RA
regulated genes. This role depends on the deacetylase activity of
Hdac1 and Hdac2. somitogenesis (Fig. 3k and Supplementary Fig. 4f) and in ~ 35%
(3 out 9) of the embryos, no clear segmented structures forming
on the right side (Fig. 3o and Supplementary Fig. 4g, h). This
provides strong evidence for genetic interaction between Wdr5
and Rere in the control of somite symmetry. Consistently,
transfection of Rere and Wdr5 together in NIH3T3 cells activated
RA signaling more strongly than overexpression of either of the
proteins alone (Fig. 2l) and Rere-dependent RA activation is no
longer observed in cells depleted of Wdr5 (Fig. 2m). Overall, these
data reveal the importance of WHHERE in positive regulation of
RA signaling and in the control of somitogenesis synchronization
in mouse embryos. WHHERE is required to recruit PolII to RA targets’ promoter. Next we analyzed the binding of the WHHERE complex to
RA regulated genes by chromatin immunoprecipitation (ChIP). To that end, we first checked the expression kinetics of the RARE-
Luciferase reporter and of the endogenous Rarβ gene in NIH3T3
cells treated with RA for 1, 2, and 6 h. At 2 and 6 h, strong
transcriptional activation was observed for both RA targets
(Fig. 4a, b). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 RARE-LacZ
Wild-type
Uncx4.1
Uncx4.1
Wild-type
Number of embryos
0
1
2
3
4
5
6
7
Left-sided
Rereom/om
Number of embryos
0
1
2
3
4
5
6
7
8
9
10
Hdac1–/–
Number of embryos
0
1
2
3
4
5
6
7
8
l
h
e
c
b
a
g
f
d
m
o
n
Symmetric Right-sided
Left-sided Symmetric Right-sided
Left-sided Symmetric Right-sided
Rereom/om;Wdr5fl/+;T-Cre
Wdr5fl/+;T-Cre
Hdac1–/–
Rereom/om
Wdr5fl/+;T-Cre
Rereom/om;Wdr5+/+
Rereom/om;Wdr5fl/+
T-Cre
i
j
k
Fig. 3 The WHHERE complex controls somite bilateral symmetry. a–d RARE-LacZ expression in wild-type a, Rereom/om b, Hdac1−/−c and Wdr5fl/+; T-Cre
d embryos at E8.75-E9.0 (dorsal views). e–h In situ hybridization showing somites labeled with Uncx4.1 in wild-type e, Rereom/om f, Hdac1–/– g and Wdr5fl/+;
T-Cre h embryos at E8.75-E9.0 (dorsal views). i–k Graphs representing the number of 7- somite to 15-somite stage embryos with left-sided (orange),
symmetric (blue) or right-sided (green) delay in somite formation: Rereom/om i, Hdac1–/– j and Rereom/om;Wdr5fl/+;T-Cre k. l–o In situ hybridization for
Uncx4.1 in wild-type l, Wdr5fl/+;T-Cre m, Rereom/om;Wdr5+/+ n and Rereom/om;Wdr5fl/+;T-Cre o embryos at E8.75-E9.0 (dorsal views). For each genotype
at least 5–10 embryos were analyzed. Bar = 100 microns d Number of embryos
0
1
2
3
4
5
6
7
8
9
10
Hdac1–/–
y
g
Left-sided Symmetric Right-sided
j g o
Rereom/om;Wdr5fl/+
T-Cre Number of embryos
0
1
2
3
4
5
6
7
8
Left-sided Symmetric Right-sided
Rereom/om;Wdr5fl/+;T-Cre
k Uncx4.1 Left-sided Symmetric Right-sided Fig. 3 The WHHERE complex controls somite bilateral symmetry. a–d RARE-LacZ expression in wild-type a, Rereom/om b, Hdac1−/−c and Wdr5fl/+; T-Cre
d embryos at E8.75-E9.0 (dorsal views). e–h In situ hybridization showing somites labeled with Uncx4.1 in wild-type e, Rereom/om f, Hdac1–/– g and Wdr5fl/+;
T-Cre h embryos at E8.75-E9.0 (dorsal views). i–k Graphs representing the number of 7- somite to 15-somite stage embryos with left-sided (orange),
symmetric (blue) or right-sided (green) delay in somite formation: Rereom/om i, Hdac1–/– j and Rereom/om;Wdr5fl/+;T-Cre k. l–o In situ hybridization for
Uncx4.1 in wild-type l, Wdr5fl/+;T-Cre m, Rereom/om;Wdr5+/+ n and Rereom/om;Wdr5fl/+;T-Cre o embryos at E8.75-E9.0 (dorsal views). For each genotype
at least 5–10 embryos were analyzed. Bar = 100 microns the Rarβ promoter (Fig. 4g, h). This effect was specific, as
no significant enrichment of WHHERE complex members
could be observed in regions upstream of the Rarβ promoter
in similar conditions (Fig. 4h, bottom graph). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 To circumvent this issue, we
crossed mice homozygous for the Wdr5 conditional allele
(Wdr5fl/fl) to the mesoderm-specific T-Cre line10. Removal of a
single Wdr5 allele in Wdr5fl/+;T-Cre;RARE-LacZ embryos was
sufficient to strongly decrease β-galactosidase staining in the
mesoderm (Fig. 3d). In contrast, no significant downregulation of
Notch target genes such as Lfng or Hes7 was detected in these
mutants suggesting the Notch-dependent somite segmentation
clock appears normal (Supplementary Fig. 4b–e). Altogether,
these results support the function of the WHHERE complex in
the control of RA signaling in the embryo. with an ubiquitous Cre suggesting that the Wdr5 mutation is
heterozygous lethal at early stages. To circumvent this issue, we
crossed mice homozygous for the Wdr5 conditional allele
(Wdr5fl/fl) to the mesoderm-specific T-Cre line10. Removal of a
single Wdr5 allele in Wdr5fl/+;T-Cre;RARE-LacZ embryos was
sufficient to strongly decrease β-galactosidase staining in the
mesoderm (Fig. 3d). In contrast, no significant downregulation of
Notch target genes such as Lfng or Hes7 was detected in these
mutants suggesting the Notch-dependent somite segmentation
clock appears normal (Supplementary Fig. 4b–e). Altogether,
these results support the function of the WHHERE complex in
the control of RA signaling in the embryo. NATURE COMMUNICATIONS| 8: 728
| DOI: 10.1038/s41467-017-00593-6| www.nature.com/naturecommunications 4 4 ARTICLE ARTICLE the members of the WHHERE complex suggesting that its
binding to Rere might be transient (Supplementary Fig. 5a). In
mouse embryos deficient for Ehmt223 crossed to the RARE-LacZ
reporter22, LacZ expression is downregulated suggesting that
Ehmt2 is also implicated in positive regulation of RA signaling
(Fig. 5a, b). Furthermore, half of the Ehmt2 mutant embryos
presented a delay in somite formation on the right side Ehmt2 acts together with WHHERE to activate RA signaling. The histone methyltransferase Ehmt2 (G9a) was shown to
bind the N-terminal SANT domain of Rere, and together with
Hdac1/Hdac2 to regulate the methylation of H3K9 at specific loci
leading to the formation of compact heterochromatin and gene
silencing14. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 In mouse embryos, we observed by ChIP analysis
that the retinoic acid receptor alpha (Rarα) and all the WHHERE
complex components were present at the Rarβ promoter and
at the RARE-containing reporter RARE-LacZ (Fig. 4c–f). In
ChIP experiments performed 1 h after RA treatment of NIH3T3
cells, Rere, Wdr5, Hdac1, and Hdac2 but not Rarα increased at 5 5 NATURE COMMUNICATIONS| 8: 728
| DOI: 10.1038/s41467-017-00593-6| www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 are also required for the WHHERE-dependent Pol II recruitment
to the promoter of RA targets remains to be investigated. are also required for the WHHERE-dependent Pol II recruitment
to the promoter of RA targets remains to be investigated. resembling mutants of members of the WHHERE complex
(Fig. 5c–e; Supplementary Fig. 4f). In NIH3T3 cultures, siRNA-
mediated knockdown of Ehmt2 led to a downregulation of the
RARE-Luciferase reporter activity (Fig. 5f and Supplementary
Fig. 3h), whereas overexpressing Ehmt2 (or Ehmt1) stimulates
RA
signaling
(Fig. 5g
and
Supplementary
Fig. 5b). Co-transfection of Ehmt2 with Rere increases the RA response
more than transfection of either one alone (Fig. 5h). siRNA-
mediated knockdown of Ehmt2 inhibited Rere and Hdac1-
dependent activation of the RA pathway (Fig. 5i). In NIH3T3
cells, Ehmt2 was recruited at the RARE element of the Rarβ
promoter after 1 h of RA treatment while no such enrichment was
observed in upstream regions (Fig. 5j and Supplementary Fig. 5c). In mouse embryos Ehmt2 could also be detected at the RARE
element present in both the Rarβ promoter and the RARE-LacZ
reporter (Fig. 4o, p). Knockdown of Ehmt2 in NIH3T3 cells
reduced the occupancy levels of Rere, Wdr5, Hdac1, Hdac2, and
Pol II at the Rarβ promoter (Fig. 5k, l and Supplementary
Fig. 5d). In NIH3T3 cells, inhibition of Ehmt2 methyltransferase
activity with UNC0638 (U38) or UNC0646 (U46)24, 25 did
not alter RARE-Luciferase reporter activity or Rarβ mRNA
expression (Supplementary Fig. 5e, f). Also no difference in
H3K9me1 nor H3K9me2 levels was observed by ChIP after 1 h of
RA treatment suggesting that Ehmt2 function is independent
from its methyltransferase activity (Supplementary Fig. 5g, h). This suggests that Ehmt2 could function as a scaffold protein to
stabilize the WHHERE complex at RA regulated genes to allow
Pol
II
loading. Thus,
these
results
indicate
that
Ehmt2
acts together with the WHHERE coactivator complex in the
RA-dependent control of somite bilateral symmetry. Negative regulation of RA signaling by Ep300. The histone
acetyl transferase Ep300 (p300) has been shown to acetylate
Hdac1 leading to a decrease of its deacetylase activity29. In the
presence of RA, overexpression of Ep300 in NIH3T3 cells
decreased RA signaling and inhibited Hdac1-dependent RA
activation (Fig. 7a). In line with this, transfection of an Hdac1
mutant form resistant to Ep300 acetylation (H1-6R)29 activated
more strongly the RA pathway than wild-type Hdac1 (H1-WT)
(Fig. 7b). Fig. 4 The WHHERE complex is recruited to the promoter of retinoic acid target genes. a RARE-Luciferase activity, b qPCR analysis of Rarβ expression of
NIH3T3 treated with 1 µM RA (blue) or 1 µM RA and 100 nM TSA (green) (n = 4 each). c, d Rarβ-promoter ChIP analysis from E8.75 RARE-LacZ mouse
embryos with antibodies against Rarα c or Rere, Wdr5, Hdac1, and Hdac2 d. Top panels c, d: RARE element of the Rarβ promoter ChIP (n = 3). Bottom panels:
control ChIP from Rarβ promoter upstream region (−3 Kb). e, f ChIP analysis of the RARE sequence in the RARE-LacZ reporter with antibodies against
Rarα e or Rere, Wdr5, Hdac1, and Hdac2 f using E8.75 RARE-LacZ mouse embryos (n = 3). g–i ChIP analysis of Rarβ promoter in NIH3T3 treated or not
during 1 h with 1 µM RA using antibodies for Rarα g, Rere, Wdr5, Hdac1, and Hdac2 h, Pol II i. Top panels h, i: ChIP of the RARE element in the Rarβ-promoter
(n = 3). Bottom panels: control ChIP from Rarβ-promoter -upstream region (−3 Kb). j ChIP of the Rarβ promoter with Pol II antibody using NIH3T3 cells
treated with 1 µM RA or 1 µM RA and 100 nM TSA for 1 h (n = 3). k ChIP with Pol II antibody from NIH3T3 transfected with siRNA for Hdac1 and treated
with 1 µM RA during 1 h (n = 3). l–n Co-immunoprecipitation using NIH3T3 transfected with Flag-Hdac1 l, Rere-HA and Flag-Hdac1 m, n, with anti-Flag l, m
or anti-HA n antibody and immunoblots (IB) for Flag-Hdac1, Rere-HA, and Rarα. o Rarβ-promoter ChIP analysis from E8.75 RARE-LacZ mouse embryos
with an anti-Ehmt2. Top panel o: RARE element ChIP of the Rarβ promoter (n = 3). Bottom panel: control ChIP from Rarβ promoter upstream region (−3 Kb).
p ChIP analysis of the RARE sequence in the RARE-LacZ reporter with an anti- Ehmt2 using E8.75 RARE-LacZ mouse embryos (n = 3). Data represent
mean ± s.e.m. unless otherwise specified. NS—not significant, *P < 0.05 and **P < 0.01. Student’s unpaired two-tailed t-test ARTICLE Moreover, Rarα- and Rere-dependent RA activation was
inhibited following Ep300 overexpression whereas transfection of
the histone acetyl transferase Kat2a (Gcn5) together with Rarα or
Rere increased RA signaling more than transfection of either
construct alone (Fig. 7c, d). While treatment of fibroblast cultures
with siRNAs against Rere or Kat2a in the presence of RA
decreased RA reporter activity, knockdown of Ep300 did not
affect the RA pathway (Fig.7e and Supplementary Fig. 3i, j). Similarly in Ep300 mutant embryos (Ep300–/–)30, the RARE-LacZ
reporter22 expression appeared normal (Fig. 7f, g) and somito-
genesis progressed symmetrically (Figs. 7h, i). Altogether these
results demonstrated that Ep300 negatively regulates Hdac1
activation of RA signaling. Furthermore Kat2a can participate
together with the WHHERE complex in the activation of the RA
pathway. ARTICLE In the proteomic experiment, Ehmt2 (and the related
protein Ehmt1) were detected with low NSAF values compared to 0
5
10
15
20
25
Fold enrichment
IgG
Pol II
ChIP
RA
–
TSA
–
+
–
+
+
–
–
+
–
+
+
0
2
4
6
8
10
12
Fold enrichment
14
16
IgG
ChIP
Hdac1
Ctrl
siRNA
RA
Hdac1
Ctrl
0
0.5
1
1.5
2
2.5
3
3.5
4
Fold enrichment
4.5
IgG
ChIP
RA
–
+
–
+
Rarα
0
2
4
6
8
Fold enrichment
1
3
5
7
9
RA
IgG
ChIP
0
2
4
6
8
10
12
Fold enrichment
14
16
18
IgG
Pol II
ChIP
RA
–
–
+
–
+
–
+
–
+
–
+
+
–
+
0
2
4
6
8
Fold enrichment
1
3
5
7
9
0
2
4
6
8
10
12
Fold enrichment
14
16
18
NS
**
**
**
*
**
NS
NS
NS
NS
**
NS
**
0
0
1
2
3
4
5
6
2
4
6
8
Fold activation
10
12
RA (hours)
0
1
2
3
4
5
6
RA (hours)
RARE-luciferase
RA
TSA
0
2
4
6
5
8
Fold activation
1
3
7
9
Rarb mRNA
b
**
**
NS
NS
**
**
0
10
20
30
40
50
60
Fold enrichment
70
IgG
Wdr5
Rere
ChIP
**
**
**
**
0
5
10
15
20
25
Fold enrichment
IgG
ChIP
**
0
2
4
6
Fold enrichment
1
3
5
*
ChIP
IgG
0
10
20
30
40
50
60
Fold enrichment
70
0
5
10
15
20
25
Fold enrichment
0
2
4
6
Fold enrichment
1
3
5
0
5
10
15
20
25
Fold enrichment
30
35
**
**
*
**
0
2
4
6
Fold enrichment
1
3
5
7
8
**
c
d
e
a
f
p
g
h
i
j
k
l
m
n
0
2
4
6
8
10
12
Fold enrichment
**
Flag-Hdac1
IP: IgG
IP: Flag
Input 0.8%
IB: Flag
IB: Rarα
IB: Flag
IB: Rarα
IB: HA
Input 0.8%
IP: IgG
IP: Flag
Rere-HA + Flag-Hdac1
IB: Flag
IB: Rarα
IB: HA
Input 0.8%
IP: IgG
IP: HA
Rere-HA + Flag-Hdac1
Rarα
Hdac1Hdac2
Ehmt2
IgG
ChIP
Rarα
IgG
Rere
ChIP
Hdac2
Hdac1
Wdr5
ChIP
IgG
Ehmt2
IgG
ChIP
Rarα
IgG
Rere
ChIP
Hdac2
Hdac1
Wdr5
ChIP
IgG
Ehmt2
o
Rere
Wdr5
Hdac1
Hdac2
RA
IgG
ChIP
IgG
Pol II
ChIP
RA
–
–
+
–
+
–
+
–
+
–
+
+
–
+
Rere
Wdr5
Hdac1
Hdac2
Pol II 0
0
1
2
3
4
5
6
2
4
6
8
Fold activation
10
12
RA (hours)
RARE-luciferase
RA
TSA
**
**
NS
a 0
10
20
30
40
50
60
Fold enrichment
70
IgG
Wdr5
Rere
ChIP
**
**
**
**
0
10
20
30
40
50
60
Fold enrichment
70
d
Hdac1Hdac2
IgG
Rere
ChIP
Hdac2
Hdac1
Wdr5 0
5
10
15
20
25
Fold enrichment
IgG
ChIP
**
0
5
10
15
20
25
Fold enrichment
c
Rarα
IgG
ChIP
Rarα 0
2
4
6
Fold enrichment
1
3
5
*
ChIP
IgG
0
2
4
6
Fold enrichment
1
3
5
Ehmt2
ChIP
IgG
Ehmt2
o a d 0
1
2
3
4
5
6
RA (hours)
0
2
4
6
5
8
Fold activation
1
3
7
9
Rarb mRNA
b
NS
**
** b p
0
2
4
6
8
10
12
Fold enrichment
**
ChIP
IgG
Ehmt2 e p 0
5
10
15
20
25
Fold enrichment
IgG
Pol II
ChIP
RA
–
TSA
–
+
–
+
+
–
–
+
–
+
+
0
0.5
1
1.5
2
2.5
3
3.5
4
Fold enrichment
4.5
IgG
ChIP
RA
–
+
–
+
Rarα
0
2
4
6
8
Fold enrichment
1
3
5
7
9
RA
IgG
ChIP
0
2
4
6
8
10
12
Fold enrichment
14
16
18
ChIP
RA
–
–
+
–
+
–
+
–
+
–
+
0
2
4
6
8
Fold enrichment
1
3
5
7
9
0
2
4
6
8
10
12
Fold enrichment
14
16
18
NS
**
**
**
*
NS
NS
NS
**
RA (hours)
g
h
i
j
IgG
ChIP
Rarα
IgG
Rere
ChIP
Hdac2
Hdac1
Wdr5
Rere
Wdr5
Hdac1
Hdac2
RA
IgG
ChIP
ChIP
RA
–
–
+
–
+
–
+
–
+
–
+
Rere
Wdr5
Hdac1
Hdac2 0
0.5
1
1.5
2
2.5
3
3.5
4
Fold enrichment
4.5
IgG
ChIP
RA
–
+
–
+
Rarα
NS
NS
g 0
2
4
6
8
10
12
Fold enrichment
14
16
18
IgG
Pol II
ChIP
RA
–
+
–
+
0
2
4
6
8
10
12
Fold enrichment
14
16
18
**
NS
i
IgG
Pol II
ChIP
RA
–
+
–
+ g i j l
Flag-Hdac1
IP: IgG
IP: Flag
Input 0.8%
IB: Flag
IB: Rarα m
IB: Flag
IB: Rarα
IB: HA
Input 0.8%
IP: IgG
IP: Flag
Rere-HA + Flag-Hdac1 0
2
4
6
8
10
12
Fold enrichment
14
16
IgG
ChIP
Hdac1
Ctrl
siRNA
RA
+
+
+
+
Hdac1
Ctrl
NS
**
k
Pol II l n
IB: Flag
IB: Rarα
IB: HA
Input 0.8%
IP: IgG
IP: HA
Rere-HA + Flag-Hdac1 k m n NATURE COMMUNICATIONS| 8: 728
| DOI: 10.1038/s41467-017-00593-6| www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 generally associated to transcriptional repression34–36. However,
R
h
i
f
RA
i
li
8
d
Hdac1 and Hdac2 identified in this work might have significant
i
li
i
f
h
i l
f HDAC i hibi
f
h
RARE-LacZ
Ehmt2+/+
Uncx4.1
Ehmt2 gt/gt
Ehmt2+/+
Ehmt2 gt/gt
Number of embryos
0
1
2
3
4
5
6
7
Left-sided
Ehmt2 gt/gt
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Ehmt2
Ctrl
siRNA
RA
+
+
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Ctrl
Ctrl
RA
–
+
+
Fold activation
0
0.5
1
1.5
2
2.5
3
3.5
RA
–
+
+
+
+
Ctrl
Rere
Ctrl
Rere
Ehmt2
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Ehmt2
Ctrl
Ctrl
Ehmt2
Ctrl
RA
+
+
+
+
siRNA
+
Ehmt2
+
Ctrl
0
0.5
1
1.5
2
2.5
3
3.5
4
Fold enrichment
IgG
ChIP
RA
–
+
–
+
0
2
4
6
Fold enrichment
1
3
5
IgG
Rere
ChIP
Ctrl
siRNA
RA
Ehmt2 Ctrl Ehmt2 Ctrl Ehmt2 Ctrl Ehmt2 Ctrl Ehmt2
+
+
+
+
+
+
+
+
+
+
Hdac1
a
f
h
i
j
**
**
**
**
**
**
**
NS
*
NS
*
*
**
**
0
2
4
6
8
10
12
Fold enrichment
14
IgG
Pol II
ChIP
Ehmt2
Ctrl
siRNA
RA
+
Ehmt2
Ctrl
l
NS
**
b
c
d
e
g
k
Right-sided
Symmetric
+
+
+
Wdr5
Hdac2
Ehmt2
Rere
Hdac1
Ehmt2
Ehmt2
Fig. 5 Ehmt2 controls retinoic acid signaling and symmetric somite formation. a, b RARE-LacZ activity in control Ehmt2+/+ a and Ehmt2gt/gt b embryos at E8.75-
E9.0 (dorsal views). c, d In situ hybridization for Uncx4.1 in wild-type Ehmt2+/+ c and Ehmt2gt/gt d embryos at E8.75-E9.0 (dorsal views). e Graph representing the
number of 7- somite to 15-somite stage Ehmt2gt/gt embryos with left-sided (orange), symmetric (blue), or right-sided (green) delay in somite formation. f–i RARE-Luciferase activity from NIH3T3 cells treated with or without 1 µM RA for 20 h. f Cells treated with siRNA for Ehmt2 (n = 4). g Cells transfected with an
Ehmt2 expression plasmid (n = 4). h Cells co-transfected with Rere and Ehmt2 expression plasmids (n = 4). i Cells overexpressing Rere or Hdac1 and treated
with siRNA against Ehmt2 (n = 4). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 a, b RARE-LacZ activity in control Ehmt2+/+ a and Ehmt2gt/gt b embryos at E8.75- Fig. 5 Ehmt2 controls retinoic acid signaling and symmetric somite formation. a, b RARE-LacZ activity in control Ehmt2+/+ a and Ehmt2gt/gt b embryos at E8.75-
E9.0 (dorsal views). c, d In situ hybridization for Uncx4.1 in wild-type Ehmt2+/+ c and Ehmt2gt/gt d embryos at E8.75-E9.0 (dorsal views). e Graph representing the
number of 7- somite to 15-somite stage Ehmt2gt/gt embryos with left-sided (orange), symmetric (blue), or right-sided (green) delay in somite formation. f–i RARE-Luciferase activity from NIH3T3 cells treated with or without 1 µM RA for 20 h. f Cells treated with siRNA for Ehmt2 (n = 4). g Cells transfected with an
Ehmt2 expression plasmid (n = 4). h Cells co-transfected with Rere and Ehmt2 expression plasmids (n = 4). i Cells overexpressing Rere or Hdac1 and treated
with siRNA against Ehmt2 (n = 4). j ChIP of the Rarβ promoter with a specific antibody for Ehmt2 using NIH3T3 cells treated or not with 1 µM RA during 1 h
(n = 3). k, l ChIP analysis of the Rarβ promoter in NIH3T3 cells transfected with siRNA for Ehmt2 and treated with 1 µM RA during 1 h. ChIP was performed with
antibodies specific to Rere, Wdr5, Hdac1, and Hdac2 k or Pol II l (n = 3). In all graphs data represent mean ± s.e.m. NS—not significant, *P < 0.05 and **P < 0.01. Student’s unpaired two-tailed t-test. For each genotype at least 5–10 embryos were analyzed. Bar = 100 microns Hdac1 and Hdac2 identified in this work might have significant
implications for the potential use of HDAC inhibitors for the
treatment of RA-sensitive cancers39. generally associated to transcriptional repression34–36. However,
Rere was shown to act as a coactivator for RA signaling8 and
Hdac1/Hdac2 are required for transcriptional activation of the
MMTV promoter downstream of the glucocorticoid receptor29. Ehmt2 has also been shown to participate in the positive
regulation of a subset of glucocorticoid receptor-regulated
genes37, 38. Our data show that in the context of RA signaling
these transcriptional repressors can form a complex, which acts
as a coactivator of the pathway. We further show that the acetyl
transferase Ep300 can reduce Rere-dependent RA signaling. This raises the possibility that in this context, Ep300-dependent
acetylation
of
the
retinoic
acid
receptors
decreases
their
transcriptional activity, an effect which could be reversed by
Hdac1/2 mediated deacetylation. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 j ChIP of the Rarβ promoter with a specific antibody for Ehmt2 using NIH3T3 cells treated or not with 1 µM RA during 1 h
(n = 3). k, l ChIP analysis of the Rarβ promoter in NIH3T3 cells transfected with siRNA for Ehmt2 and treated with 1 µM RA during 1 h. ChIP was performed with
antibodies specific to Rere, Wdr5, Hdac1, and Hdac2 k or Pol II l (n = 3). In all graphs data represent mean ± s.e.m. NS—not significant, *P < 0.05 and **P < 0.01. Student’s unpaired two-tailed t-test. For each genotype at least 5–10 embryos were analyzed. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 Thus, studies like those reported
here may be necessary to fully understand the functions of
co-regulators, which may act as coactivators or corepressors
depending on the developmental context and genes involved. The
positive role of these proteins in signaling mediated by other
nuclear receptors suggests that the WHHERE complex could also
have a broader role as a coactivator downstream of nuclear
receptor signaling. The positive regulation of RA signaling by Discussion
h
l The molecular mechanisms controlling embryonic bilateral
symmetry are still poorly understood. In mouse, the only genes
shown to act in this process are Raldh2 and Rere which are
involved in the control of RA signaling7, 8. Here, we identify
a protein complex, called WHHERE which associates key
epigenetic regulators such as Wdr5, Hdac1, and Hdac2 to the
control of bilateral symmetry downstream of RA signaling. In the
background of a situs inversus mutation (a mutation that can
reverse left–right identity of the body), Raldh2 and Rere-deficient
mouse embryos can show a reversed somite defect, i.e., left-side
delay of somitogenesis8, 31. This therefore argues that WHHERE-
dependent RA signaling buffers a desynchronizing influence of
the left−right determination pathway, involved in asymmetrical
development of internal organs. RA signaling was shown to
directly
antagonize
the
left
identity
determinant
Fgf8
in
the mouse embryo through recruitment of Hdac1 and Prc2,
providing a potential explanation for this buffering effect32, 33. Thus, our work identifies a new pathway antagonizing FGF
signaling acting downstream of RA to control bilateral symmetry
in the mouse embryo. Effect of RA signaling on key histone marks. Wdr5 is part of
complexes such as ATAC, MOF, and MLL, that are involved in
histone acetylation and H3K4 methylation, which are chromatin
modifications associated with transcriptional activation26. Despite
the requirements of HDAC enzymatic activity for WHHERE
function, we observed an increase of the levels of acetylated H3
and H4 as well as H3K27ac on the Rarβ promoter upon 1 h of RA
treatment of NIH3T3 cells (Fig. 6a–c). This suggests that Hdac1
and Hdac2 might act on non-histone substrates or both could
participate to the stability of the WHHERE complex. The
H3K36me3 mark, which is associated to transcription elongation,
was also increased (Fig. 6d). We also found that in the same
conditions, H3K27me3 is absent from the Rarβ promoter
(Fig. 6e), whereas H3K4me1 increases while H3K4me2 and
H3K4me3 decrease (Fig. 6f–h). MLL3 and MLL4 are complexes
which contain Wdr5 and regulate the deposition of the H3K4me1
mark27. The MLL3 and MLL4 complexes have been shown to be
involved in RA-dependent transcription and Wdr5 might provide
a link with the WHHERE complex28. Whether these complexes Strikingly most of the members of the WHHERE complex
including Rere, Hdac1, Hdac2, and also Ehmt2 have been 7 NATURE COMMUNICATIONS| 8: 728
| DOI: 10.1038/s41467-017-00593-6| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 Bar = 100 microns RARE-LacZ
Ehmt2+/+
a Ehmt2 gt/gt
b b RARE-LacZ
Uncx4.1
Number of embryos
0
1
2
3
4
5
6
7
Left-sided
Ehmt2
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Ehmt2
Ctrl
siRNA
RA
+
+
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Ctrl
Ctrl
RA
–
+
+
Fold activation
0
0.5
1
1.5
2
2.5
3
3.5
RA
–
+
+
+
+
Ctrl
Rere
Ctrl
Rere
Ehmt2
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Ehmt2
Ctrl
Ctrl
Ehmt2
Ctrl
RA
+
+
+
+
siRNA
+
Ehmt2
+
Ctrl
f
h
i
**
**
**
**
**
**
**
g
Right-sided
Symmetric
Rere
Hdac1
Ehmt2
Ehmt2 Num
0
1
2
Left-sided
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Ehmt2
Ctrl
siRNA
RA
+
+
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Ctrl
Ctrl
RA
–
+
+
Fold activation
0
0.5
1
1.5
2
2.5
3
3.5
RA
–
+
+
+
+
Ctrl
Rere
Ctrl
Rere
Ehmt2
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Ehmt2
Ctrl
Ctrl
Ehmt2
Ctrl
RA
+
+
+
+
siRNA
+
Ehmt2
+
Ctrl
f
h
i
**
**
**
**
**
**
**
g
Right-sided
Symmetric
Rere
Hdac1
Ehmt2
Ehmt2 Ctrl
Ctrl
–
+
+
Fold activation
0
0.5
1
1.5
2
2.5
3
3.5
RA
–
+
+
+
+
Ctrl
Rere
Ctrl
Rere
Ehmt2
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Ehmt2
Ctrl
Ctrl
Ehmt2
Ctrl
RA
+
+
+
+
siRNA
+
Ehmt2
+
Ctrl
h
i
**
**
**
**
**
**
Rere
Hdac1
Ehmt2
Ehmt2 f f i h g Ctrl
0
0.5
1
1.5
2
2.5
3
3.5
4
Fold enrichment
IgG
ChIP
RA
–
+
–
+
0
2
4
6
Fold enrichment
1
3
5
IgG
Rere
ChIP
Ctrl
siRNA
RA
Ehmt2 Ctrl Ehmt2 Ctrl Ehmt2 Ctrl Ehmt2 Ctrl Ehmt2
+
+
+
+
+
+
+
+
+
+
Hdac1
j
NS
*
NS
*
*
**
**
0
2
4
6
8
10
12
Fold enrichment
14
IgG
Pol II
ChIP
Ehmt2
Ctrl
siRNA
RA
+
Ehmt2
Ctrl
l
NS
**
k
+
+
+
Wdr5
Hdac2
Ehmt2
Rere
Hdac1 j e formation. Methods
G
ti Generation of transgenic mice. The Rere-HA construct was generated by adding
two HA tags at the C-terminal extremity of Rere. Rere-HA was cloned downstream
of a loxP-STOP-loxP cassette and inserted into the Rosa26 locus by homologous
recombination in 129S6/SvEvTac-derived W4 embryonic stem (ES) cells40. Animals were maintained on a 50% C57BL/6 genetic background. fl/ Wdr5 conditional allele (Wdr5fl/+) mice were generated by the Mouse Clinical
Institute (Illkirch-Graffenstaden) by inserting two loxP sites flanking the region
encompassing exon 2 and exon 4 in C57BL/6N-derived ES cells (Supplementary
Fig. 4a). The Neo selection cassette flanked by FRT site was removed in vivo using a
FlpO deleter mice41. Animals were maintained on a C57BL/6 genetic background. Mice breeding and generation of mutant embryos. To remove the STOP
cassette from the RS-Rere-HA mouse line, we crossed this line with ZP3-Cre mice,
which express the Cre recombinase specifically in oocytes42. Mice in which the
STOP cassette was removed were subsequently intercrossed with Rereom/+ mice9. The Rere-HA construct rescued the Rereom/+ mutation indicating that the protein
Rere-HA is functional (Supplementary Fig. 1a–c). In order to obtain embryos
expressing Rere-HA specifically in the mesoderm for proteomic experiments,
we generated a homozygous line for RS-Rere-HA and crossed it with homozygous NATURE COMMUNICATIONS| 8: 728
| DOI: 10.1038/s41467-017-00593-6| www.nature.com/naturecommunications 8 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 0
50
100
150
200
250
300
Fold enrichment
350
**
IgG
H3K4me1
ChIP
RA
–
+
–
+
5
0
10
15
20
25
30
Fold enrichment
IgG
H3K4me2
ChIP
RA
–
+
–
+
NS
NS
*
5
0
10
15
20
25
30
Fold enrichment
35
IgG
H3K4me3
ChIP
RA
–
+
–
+
NS
**
h
50
0
0
100
150
200
250
300
Fold enrichment
350
IgG
H3K27ac
ChIP
RA
–
+
–
+
NS
**
0
5
10
15
20
25
30
Fold enrichment
IgG
H3ac
ChIP
RA
–
+
–
+
**
10
20
30
40
50
60
Fold enrichment
IgG
H4ac
ChIP
RA
–
+
–
+
**
NS
NS
0
0
10
20
30
40
50
60
Fold enrichment
IgG
H3K36me3
ChIP
RA
–
+
–
+
**
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2.0
Fold enrichment
IgG
H3K27me3
ChIP
RA
–
+
–
+
NS
NS
NS
c
b
e
a
d
f
g
Fig. 6 Effect of RA treatment on different histone acetylation and methylation marks. Methods
G
ti The gene-trap allele (gt) Ehmt2gt/+23
was maintained on at least 50% C57BL/6 genetic background. Embryos Ehmt2gt/gt
were obtained by mating heterozygous males and females for the Ehmt2 mutation. The mouse mutant for Ep30030 was maintained on at least 50% C57BL/6 genetic
background. Heterozygous males and females Ep300+/−were then mated to obtain
Ep300−/−embryos. To study the status of RA signaling, the RARE-LacZ reporter
mice22 in which LacZ expression is driven by the RARE of the retinoic acid receptor
beta gene (Rarβ) were crossed to: Rereom/+, Wdr5fl/+;T-Cre, Hdac1+/−, Ehmt2gt/+,
and Ep300–/– and the mutant embryos were analyzed for β-galactosidase activity
using X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). The wild-type
and mutant embryos were always stained in the same conditions and same well for
1 h 30 min. All animal procedures were performed according to a protocol
approved by IGBMC (#2012-142), in agreement with national and international
guidelines. For the generation of baculoviruses expressing Flag-Hdac1 and Flag-Hdac2, the
full-length cDNA encoding mouse Hdac1 and Hdac2 fused with a N-terminal Flag
tag, were cloned into the pVL1393 entry vector at BamHI and XbaI sites. For the
generation of baculoviruses expressing Rere-Flag, the full-length cDNA encoding
mouse Rere, was cloned into the pVL1392 entry vector at BplI and KpnI sites. The
viruses were then generated from these entry vectors following the transferring
genes protocol (Method I) described in Summers and Smith (1987) “A manual of
methods for baculo vectors and insect cell culture procedures”. The viruses
expressing HA-Wdr5 was provided by Dr. Laszlo Tora (IGBMC). Cell culture, RA reporter assay and siRNA experiments. NIH3T3 cells were
cultured in DMEM (Invitrogen) containing 4,5 g/l D-glucose, L-glutamine, pyr-
uvate, and 10% newborn calf serum8. For RA reporter assay, when cells reached
50% confluence in 24-well plates, they were co-transfected with RARE-pGL4.23
(200 ng), pGL3-Promoter (200 ng), pRL-CMV (5 ng) (Promega), TA-Luc (200 ng)
(Panomics) and the different expression constructs containing Rere, Wdr5, Hdac1,
Hdac2, Ehmt1, Ehmt2, N-Rere (Rere N-terminal region), Rere C (Rere C-terminal
region), Hdac1 wild-type (H1-WT), Hdac1 mutant (H1-6R), Ep300, Rarα and
Kat2a using Fugene 6 (Roche). For single transfection, we used 200 ng of each
construct whereas in co-transfection experiments 150 ng of each plasmid was used. One micro molar RA (Sigma) was added after 24 h of transfection, and 20 h later,
cells were collected and lysed. Methods
G
ti a–h ChIP analysis of the Rarβ promoter from NIH3T3 cells treated
with 1 µM RA during 1 h. ChIP was performed with specific antibodies to H3ac a, H4ac b, H3K27ac c, H3K36me3 d, H3K27me3 e, H3K4me1 f, H3K4me2 g,
and H3K4me3 h (n = 3). In all graphs data represent mean ± s.e.m. NS—not significant, *P < 0.05 and **P < 0.01. Student’s unpaired two-tailed t-test T-Cre animals10. The Rereom/+ mouse line was obtained in an N-ethyl-N-
9 terminal region (a.a. 1–571) (N-Rere) and Rere C-terminal region (a.a. 572–1558)
(Rere C) were subcloned into pMyc-CMV (Sigma) and pFlag-CMV (Sigma),
respectively. Human Hdac1 wild-type (H1-WT) and Hdac1 mutant (H1-6R) in
pEGFP-N1 (Clontech) was a gift from Gordon Hager. Human Ep300 in pCMVb
was a gift from Richard Eckner and mouse Kat2a (Gcn5) in pCMV-sport247
(Invitrogen) (Addgene plasmid 23098) was a gift from Sharon Dent. For the generation of baculoviruses expressing Flag-Hdac1 and Flag-Hdac2, the
full-length cDNA encoding mouse Hdac1 and Hdac2 fused with a N-terminal Flag
tag, were cloned into the pVL1393 entry vector at BamHI and XbaI sites. For the
generation of baculoviruses expressing Rere-Flag, the full-length cDNA encoding
mouse Rere, was cloned into the pVL1392 entry vector at BplI and KpnI sites. The
viruses were then generated from these entry vectors following the transferring
genes protocol (Method I) described in Summers and Smith (1987) “A manual of
methods for baculo vectors and insect cell culture procedures”. The viruses
expressing HA-Wdr5 was provided by Dr. Laszlo Tora (IGBMC). terminal region (a.a. 1–571) (N-Rere) and Rere C-terminal region (a.a. 572–1558)
(Rere C) were subcloned into pMyc-CMV (Sigma) and pFlag-CMV (Sigma),
respectively. Human Hdac1 wild-type (H1-WT) and Hdac1 mutant (H1-6R) in
pEGFP-N1 (Clontech) was a gift from Gordon Hager. Human Ep300 in pCMVb
was a gift from Richard Eckner and mouse Kat2a (Gcn5) in pCMV-sport247
(Invitrogen) (Addgene plasmid 23098) was a gift from Sharon Dent. T-Cre animals10. The Rereom/+ mouse line was obtained in an N-ethyl-N-
nitrosourea (ENU) screen9 and the line was maintained on a C57BL/6 genetic
background. To analyze conditional mutations of Wdr5, we crossed the Wdr5fl/+
mouse line with T-Cre mice. The embryos Wdr5fl/+;T-Cre used for analysis only
have one mutant allele in the mesodermal tissues. To generate the Hdac1+/– mouse
line, Hdac1fl/flanimals21 were crossed with ZP3-Cre mice42 and maintained on at
least 50% C57BL/6 genetic background. Heterozygous males and females Hdac1+/–
were then mated to obtain Hdac1–/– embryos. Methods
G
ti a–h ChIP analysis of the Rarβ promoter from NIH3T3 cells treated
with 1 µM RA during 1 h. ChIP was performed with specific antibodies to H3ac a, H4ac b, H3K27ac c, H3K36me3 d, H3K27me3 e, H3K4me1 f, H3K4me2 g,
and H3K4me3 h (n = 3). In all graphs data represent mean ± s.e.m. NS—not significant, *P < 0.05 and **P < 0.01. Student’s unpaired two-tailed t-test 50
0
0
100
150
200
250
300
Fold enrichment
350
IgG
H3K27ac
ChIP
RA
–
+
–
+
NS
**
0
5
10
15
20
25
30
Fold enrichment
IgG
H3ac
ChIP
RA
–
+
–
+
**
10
20
30
40
50
60
Fold enrichment
IgG
H4ac
ChIP
RA
–
+
–
+
**
NS
NS
0
0
10
20
30
40
50
60
Fold enrichment
IgG
H3K36me3
ChIP
RA
–
+
–
+
**
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2.0
Fold enrichment
IgG
H3K27me3
ChIP
RA
–
+
–
+
NS
NS
NS
c
b
e
a
d 50
0
0
100
150
200
250
300
Fold enrichment
350
IgG
H3K27ac
ChIP
RA
–
+
–
+
NS
**
10
20
30
40
50
60
Fold enrichment
IgG
H4ac
ChIP
RA
–
+
–
+
**
NS
c
b 0
0
10
20
30
40
50
60
Fold enrichment
IgG
H3K36me3
ChIP
RA
–
+
–
+
**
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2.0
Fold enrichment
IgG
H3K27me3
ChIP
RA
–
+
–
+
NS
NS
NS
e
d 0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2.0
Fold enrichment
IgG
H3K27me3
ChIP
RA
–
+
–
+
NS
NS
e 5
0
10
15
20
25
30
Fold enrichment
35
IgG
H3K4me3
ChIP
RA
–
+
–
+
NS
**
h 0
50
100
150
200
250
300
Fold enrichment
350
**
IgG
H3K4me1
ChIP
RA
–
+
–
+
NS
f 5
0
10
15
20
25
30
Fold enrichment
IgG
H3K4me2
ChIP
RA
–
+
–
+
NS
*
g 5
0
10
15
20
25
30
Fold enrichment
IgG
H3K4me2
ChIP
RA
–
+
–
+
NS
*
5
0
10
15
20
25
30
Fold enrichment
35
IgG
H3K4me3
ChIP
RA
–
+
–
+
NS
**
h
g g Fig. 6 Effect of RA treatment on different histone acetylation and methylation marks. Methods
G
ti Renilla luciferase and firefly luciferase activity were
measured using a dual luciferase assay kit (Promega) and a microplate lumin-
ometer (Berthold Centro XS3 LB 960). Five different histone deacetylase inhibitors
were used: Trichostatin A (30–100 nM), Sodium butyrate (1.5–3 mM), Apicidin
(300 nM) (all from Sigma) and LAQ824 (60 nM), Panobinostat (30 nM) (both from
Selleckchem). UNC0638 and UNC0646 (both from Tocris) were used to inhibit
Ehmt2 methyltransferase activity. Whole-mount in situ hybridization. Whole-mount in situ hybridizations were
performed according to the protocol described in ref. 43. Briefly, formaldehyde-fixed
embryos were treated with protease and refixed with 4% formaldehyde/0.1%
glutaraldehyde. Hybridization with DIG-labeled RNA probes was performed under
stringent conditions (1.3X SSC, 50% formamide at 65 °C, pH 5) in a buffer containing
0.2% Tween-20 and 0.5% CHAPS. Washed embryos were treated with Boehringer
blocking reagent and incubated overnight in alkaline phosphatase-coupled anti-DIG
antibody. After extensive washes, embryos were stained from 30 min to 16 h. The
probes used are described in the literature: Uncx4.144, Lfng45, and Hes746. Vector construction and baculovirus generation. To assay RA signaling in
NIH3T3 cells, we used the RA reporter RARE-Luciferase and mouse Rere-HA
subcloned into pMyc-CMV (Sigma) described in ref. 8. The mouse full-length
Wdr5, Hdac1, Hdac2, and Rarα cDNAs were subcloned into pFlag-CMV (Sigma). Human Flag-Ehmt2 in PCNF plasmid and Flag-Ehmt1 in pCDNA3 plasmid (both
from Nakatani lab) were used to assay RA signaling in NIH3T3 cells. Rere N- For siRNA experiments, NIH3T3 cells were transfected with ON-TARGETplus
Smartpool mouse siRNA (Dharmacon) or ON-TARGETplus non-targeting control
siRNA pool (Dharmacon) using DharmaFECT 1 or DharmaFECT Duo (for
luciferase experiments) reagents (Dharmacon) and following the manufacturer’s
instructions. Methods
G
ti Bar = 100 microns To immunoprecipitate the endogenous WHHERE complex from NIH3T3 cells,
whole cell protein extract prepared with Tris-HCl pH 7.5, 20% glycerol, 0.2 M KCl,
2X PIC and 1 mM DTT was mixed with Rere antibody (Santa Cruz, sc-98145)
coupled with protein G sepharose beads (protein G sepharose 4 fast flow, GE
healthcare) or protein G sepharose beads only (negative control IP) for 1 h rotating
at 4 °C. Beads were washed in a buffer containing 150 mM KCl, 25 mM Tris-HCl
pH 8.0, 10% glycerol, 5 mM MgCl2, 0.1% NP-40, 2X PIC, 1 mM DTT and eluted
after boiling beads in Laemmli buffer (4% SDS, 20% glycerol, 120 mM Tris-HCl pH
6.8, Bromophenol blue 0.02%) followed by western blot analysis against Rere
(Santa Cruz, sc-98145, dilution 1/1000), Hdac1 (Abcam, ab7028, dilution 1/1000),
Hdac2 (Abcam, ab7029, dilution 1/1000) and Wdr5 (Millipore 07-706, dilution
1/1000). All uncropped western blots can be found in Supplementary Figs. 7–9. following target genes: Rere, Wdr5, Hdac1, Hdac2, Ehmt2, Ep300, and Kat2a. For
single siRNA experiments 100 nM of each siRNA was used for the transfection. In
the case of double siRNA transfections 50 nM of each siRNA was used for
experiments. following target genes: Rere, Wdr5, Hdac1, Hdac2, Ehmt2, Ep300, and Kat2a. For
single siRNA experiments 100 nM of each siRNA was used for the transfection. In
the case of double siRNA transfections 50 nM of each siRNA was used for
experiments. Co-immunoprecipitation experiments and western blots. NIH3T3 cells were
cultured in 10 cm cell culture plates overnight to 70–80% confluence and then
transfected with pFlag-Hdac1 alone or together with pMyc-Rere-HA vectors using
Fugene 6 (Roche). NIH3T3 cells were cultured in T-25 flasks overnight to 70–80%
confluence and then transfected with the expression vectors using Fugene 6
(Roche). Cells were collected 24 h post-transfection and lysed with a hypotonic
gentle lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 10 mM EDTA, 0.5%
Triton X-100 and Complete protease inhibitors (Roche)). After the lysis, the NaCl
concentration was raised to 150 mM. Soluble extracts, together with either mouse
IgG (Upstate), M2 anti-FLAG (Sigma) or anti-HA (Sigma), were incubated with
Dynabeads M-280 sheep anti-Mouse IgG (Invitrogen) overnight at 4 °C. Methods
G
ti Bar = 100 microns Fold activation
0
0.5
1
1.5
2
2.5
3
3.5
4.5
4
5
RA
–
+
+
+
+
Ctrl
Ep300 Hdac1
Ctrl
Hdac1
Ep300
**
**
a Fold activation
0
0.5
1
1.5
2
2.5
3
3.5
4.5
4
5
RA
–
+
+
+
Ctrl
H1-6R
H1-WT
Ctrl
**
b Fold activation
0
0.5
1.5
2
2.5
3
3.5
RA
–
+
+
+
+
Ctrl
Rere
Ctrl
Rere
Ep300
Rere
Kat2a
*
**
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Rere Ep300 Kat2a
Ctrl
RA
+
+
+
+
siRNA
**
NS
**
d
e Fold activation
0
1
2
3
4
5
6
RA
–
+
+
+
+
Ctrl
Rarα
Ctrl
Rarα
Ep300
Rarα
Kat2a
*
*
c Uncx4.1
Ep300 –/–
Ep300 +/+
h
i RARE-LacZ
Ep300 –/–
Ep300 +/+
f
g f h Ep300 –/–
g g RARE-LacZ Uncx4.1 Fig. 7 Kat2a but not Ep300 acts as a coactivator for retinoic acid signaling. a–e RARE-Luciferase activity from NIH3T3 cells treated or not with 1 µM RA for
20 h. a Cells transfected with expression plasmids containing Ep300, Hdac1, or both (n = 4). b Cells transfected with expression plasmids containing human
Hdac1 (H1-WT) or Hdac1 mutant (H1-6R) (n = 4). c Cells transfected with expression plasmids containing Rarα, Rarα, and Ep300 or Rarα and Kat2a (n = 3). d Cells transfected with expression plasmids containing Rere, Rere and Ep300 or Rere and Kat2a (n = 3). e Cells treated either with siRNA for Rere, Ep300, or
Kat2a (n = 4). In all graphs data represent mean ± s.e.m. NS—not significant, *P < 0.05 and **P < 0.01. Student’s unpaired two-tailed t-test. f, g RARE-LacZ
expression in wild-type Ep300+/+ f and Ep300–/– g embryos at E8.75-E9.0 (dorsal views). h, i In situ hybridization showing somites labeled with Uncx4.1 in
wild-type Ep300+/+ h and Ep300–/– i embryos at E8.75-E9.0 (dorsal views). For each genotype at least 5–10 embryos were analyzed. Methods
G
ti The ON-TARGETplus Smartpool mouse siRNA were used against the 9 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 Fold activation
0
1
2
3
4
5
6
RA
–
+
+
+
+
Ctrl
Rarα
Ctrl
Rarα
Ep300
Rarα
Kat2a
*
*
Fold activation
0
0.5
1.5
2
2.5
3
3.5
RA
–
+
+
+
+
Ctrl
Rere
Ctrl
Rere
Ep300
Rere
Kat2a
*
**
Fold activation
0
0.2
0.4
0.6
0.8
1
1.2
Rere Ep300 Kat2a
Ctrl
RA
+
+
+
+
siRNA
**
NS
**
RARE-LacZ
Ep300 –/–
Ep300 +/+
Uncx4.1
Ep300 –/–
Ep300 +/+
Fold activation
0
0.5
1
1.5
2
2.5
3
3.5
4.5
4
5
RA
–
+
+
+
+
Ctrl
Ep300 Hdac1
Ctrl
Hdac1
Ep300
**
**
Fold activation
0
0.5
1
1.5
2
2.5
3
3.5
4.5
4
5
RA
–
+
+
+
Ctrl
H1-6R
H1-WT
Ctrl
**
a
b
c
d
e
f
h
i
g
ig. 7 Kat2a but not Ep300 acts as a coactivator for retinoic acid signaling. a–e RARE-Luciferase activity from NIH3T3 cells treated or not with 1 µM RA for
0 h. a Cells transfected with expression plasmids containing Ep300, Hdac1, or both (n = 4). b Cells transfected with expression plasmids containing human
dac1 (H1-WT) or Hdac1 mutant (H1-6R) (n = 4). c Cells transfected with expression plasmids containing Rarα, Rarα, and Ep300 or Rarα and Kat2a (n = 3). Cells transfected with expression plasmids containing Rere, Rere and Ep300 or Rere and Kat2a (n = 3). e Cells treated either with siRNA for Rere, Ep300, or
at2a (n = 4). In all graphs data represent mean ± s.e.m. NS—not significant, *P < 0.05 and **P < 0.01. Student’s unpaired two-tailed t-test. f, g RARE-LacZ
xpression in wild-type Ep300+/+ f and Ep300–/– g embryos at E8.75-E9.0 (dorsal views). h, i In situ hybridization showing somites labeled with Uncx4.1 in
wild-type Ep300+/+ h and Ep300–/– i embryos at E8.75-E9.0 (dorsal views). For each genotype at least 5–10 embryos were analyzed. ARTICLE A total of 11 and 12 MudPIT analyses were
performed for the low and high salt conditions, respectively (Fig. 1a, b and Sup-
plementary Fig. 2d–f). The same purification strategy was applied to embryos from
wild-type females to use as a negative control. Proteins were extracted in either low
or high salt conditions from 108 × 2 and 100 × 2 embryos prepared on different
days, then split in half and affinity purified using either the Sigma or Roche anti-
HA antibodies; elutions were combined into 3 digested samples as described above
and analyzed independently for a total of 12 MudPIT analyses. treated with 0.1 µM and 1 µM of RA (Sigma) 20 h before collecting. At ~80%
confluency, cells were crosslinked with 1% formaldehyde at 37°C for 15 min. Crosslinking was quenched by the addition of glycine to a final concentration of
125 mM for 5 min at room temperature. Cells were lysed in lysis buffer containing
1% SDS, 10 mM EDTA and 50 mM Tris-HCl pH 8.0 and protease inhibitors at a
concentration of 3 × 107 cells/ml. Sonication was performed to produce an average
fragment size of ~ 200–1000 bp. Chromatin was diluted fivefold in buffer con-
taining 0.005% SDS, 0.55% Triton X-100, 0.6 mM EDTA, 8.35 mM Tris pH 8.0,
8.35 mM NaCl. Antibodies (5–10 µg per immunoprecipitation) were pre-bound to
a protein A sepharose slurry (0.15 g lyophilized protein A sepharose beads (Sigma)
were resuspended in 1.3 mL 1X TE with 300 µg sonicated salmon sperm DNA
and 1.5 µg BSA). Forty microliters of the resulting slurry was used per
immunoprecipitation. Chromatin was pre-cleared using this slurry without anti-
body and pre-cleared chromatin was then added to the antibody-bead complexes
overnight. Chromatin from ~3 × 106 NIH3T3 or F9 cells was used per immuno-
precipitation. Complexes were washed the next morning for 10 min each with
Buffer A: 0.1% SDS, 1% TritonX-100, 1 mM EDTA, 20 mM Tris-HCl pH 8.0, 50
mM NaCl; Buffer B (same as buffer A except contained 500 mM NaCl; Buffer C:
0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl
pH 8.0, followed by two washes in 1X TE). Beads were then eluted at room
temperature with 1% SDS and 0.1 M NaHCO3, crosslinks were reversed, proteinase
K digestion was performed and DNA was isolated. ARTICLE During the course of a fully automated
chromatography, ten 120-minutes cycles of increasing salt concentrations followed
by organic gradients slowly released peptides directly into the mass spectrometer12. Three different elution buffers were used: 5% acetonitrile, 0.1% formic acid (Buffer
A); 80% acetonitrile, 0.1% formic acid (Buffer B); and 0.5 M ammonium acetate,
5% acetonitrile, 0.1% formic acid (Buffer C). The last two chromatography steps
consisted in a high salt wash with 100% Buffer C followed by the acetonitrile
gradient. The application of a 2.5 kV distal voltage electrosprayed the eluting
peptides directly into the mass spectrometers equipped with a nano-LC
electrospray ionization source. Each full MS scan (from 400 to 1600 m/z) was
followed by five MS/MS events using data-dependent acquisition where the first
most intense ion was isolated and fragmented by collision-induced dissociation
(at 35% collision energy), followed by the second to fifth most intense ions. g
0.1 ml/min to about 200–300 nl/min. During the course of a fully automated
chromatography, ten 120-minutes cycles of increasing salt concentrations followed
by organic gradients slowly released peptides directly into the mass spectrometer12. Three different elution buffers were used: 5% acetonitrile, 0.1% formic acid (Buffer
A); 80% acetonitrile, 0.1% formic acid (Buffer B); and 0.5 M ammonium acetate,
5% acetonitrile, 0.1% formic acid (Buffer C). The last two chromatography steps
consisted in a high salt wash with 100% Buffer C followed by the acetonitrile
gradient. The application of a 2.5 kV distal voltage electrosprayed the eluting
peptides directly into the mass spectrometers equipped with a nano-LC
electrospray ionization source. Each full MS scan (from 400 to 1600 m/z) was
followed by five MS/MS events using data-dependent acquisition where the first
most intense ion was isolated and fragmented by collision-induced dissociation
(at 35% collision energy), followed by the second to fifth most intense ions. 50 p
p
For ChIP assay with mouse embryonic tissue, we modified the protocol as
follows. E8.75-9.0 embryos were collected from transgenic RARE-LacZ mice in PBS
with Complete protease inhibitors (Roche) and crosslinked with 1% formaldehyde
at room temperature for 15 min. Crosslinking was quenched by the addition of
glycine to a final concentration of 125 mM for 5 min at room temperature. The
embryos after dounce homogenization were snap frozen in liquid nitrogen. ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 Table 1 Antibodies used for ChIP
Antibody
Catalog number (Company)
Concentration/ChIP
Rere H-113
sc-98415 (Santa Cruz)
10 μg
Wdr5
07-706 (Millipore)
~ 5 μg (1:200)
Hdac1 C-19
sc-6298 (Santa Cruz)
10 μg
Hdac2 H-54
sc-7899 (Santa Cruz)
10 μg
Rarα C-20
sc-551 (Santa Cruz)
10 μg
Rpb1 (total Pol II)
PB-7C2 (Euromedex)
~ 3 μg (1:300)
Ehmt2
07-551 (Millipore)
10 μg
H3K9me1
ab9045 (Abcam)
2 μg
H3K9me2
ab1220 (Abcam)
2 μg
H3K4me1
ab8895 (Abcam)
2 μg
H3K4me2
ab32356 (Abcam)
2 μg
H3K4me3
ab8580 (Abcam)
2 μg
H3K27ac
ab4729 (Abcam)
2 μg
H3ac
06-599 (Millipore)
2 μg
H4ac
06-598 (Millipore)
2 μg
H3K36me3
ab9050 (Abcam)
2 μg
H3K27me3
ab6002 (Abcam)
2 μg Biosystems). PCR conditions were as follows: 95 °C (20 s) (1×); 95 °C (3 s) and
60 °C (30 s) (40×). Primer pairs used for the RARE-LacZ reporter were:
5′-TGCTGCACGCGGAAGA-3′ and 5′-CCACCAATCCCCATATGGAA-3′. Biosystems). PCR conditions were as follows: 95 °C (20 s) (1×); 95 °C (3 s) and
60 °C (30 s) (40×). Primer pairs used for the RARE-LacZ reporter were:
5′-TGCTGCACGCGGAAGA-3′ and 5′-CCACCAATCCCCATATGGAA-3′. Biosystems). PCR conditions were as follows: 95 °C (20 s) (1×); 95 °C (3 s) and
60 °C (30 s) (40×). Primer pairs used for the RARE-LacZ reporter were:
5′-TGCTGCACGCGGAAGA-3′ and 5′-CCACCAATCCCCATATGGAA-3′. Table 1 Antibodies used for ChIP Antibody
Catalog number (Company)
Concentration/ChIP Catalog number (Company)
Concentration/ChIP qPCR was performed with LightCycler 480 SYBR Green I Master mix (Roche)
in a LightCycler 480 II System (Roche) using the primers described in
Supplementary Table 1. qPCR was performed with LightCycler 480 SYBR Green I Master mix (Roche)
in a LightCycler 480 II System (Roche) using the primers described in
Supplementary Table 1. Proteomic approach to identify Rere-associated proteins. To identify proteins
binding to Rere, we first generated a mouse colony homozygous for both the T-Cre
transgene and the RS-Rere-HA allele. Thus, all the embryos generated by crossing
these mice express Rere-HA only in mesodermal tissues. We collected around 600
E10.5 embryos and prepared whole cell extracts using either a low salt (350 mM
NaCl) or a high salt (420 mM NaCl) extraction buffer (Supplementary Note 1). For
the low salt condition, affinity purification was done at 150 mM NaCl; whereas for
the high salt protocol, it was performed at 300 mM NaCl. ARTICLE Each affinity purification
was performed on biological replicates of protein extracts from about 50–70
embryos prepared on different days. Two different anti-HA antibodies from Sigma
and Roche were used for the affinity purification. For each of the conditions, six
elutions were performed with a HA peptide, and the last elution with SDS 2% to
completely remove all the proteins attached to the beads. Samples were analyzed by
MudPIT as follows: elution 1 run alone, elution 2 and 3 combined and elutions 4 to
7 combined (Supplementary Note 1). A total of 11 and 12 MudPIT analyses were
performed for the low and high salt conditions, respectively (Fig. 1a, b and Sup-
plementary Fig. 2d–f). The same purification strategy was applied to embryos from
wild-type females to use as a negative control. Proteins were extracted in either low
or high salt conditions from 108 × 2 and 100 × 2 embryos prepared on different
days, then split in half and affinity purified using either the Sigma or Roche anti-
HA antibodies; elutions were combined into 3 digested samples as described above
and analyzed independently for a total of 12 MudPIT analyses. Proteomic approach to identify Rere-associated proteins. To identify proteins
binding to Rere, we first generated a mouse colony homozygous for both the T-Cre
transgene and the RS-Rere-HA allele. Thus, all the embryos generated by crossing
these mice express Rere-HA only in mesodermal tissues. We collected around 600
E10.5 embryos and prepared whole cell extracts using either a low salt (350 mM
NaCl) or a high salt (420 mM NaCl) extraction buffer (Supplementary Note 1). For
the low salt condition, affinity purification was done at 150 mM NaCl; whereas for
the high salt protocol, it was performed at 300 mM NaCl. Each affinity purification
was performed on biological replicates of protein extracts from about 50–70 p
g
p
p
embryos prepared on different days. Two different anti-HA antibodies from Sigma
and Roche were used for the affinity purification. For each of the conditions, six
elutions were performed with a HA peptide, and the last elution with SDS 2% to
completely remove all the proteins attached to the beads. Samples were analyzed by
MudPIT as follows: elution 1 run alone, elution 2 and 3 combined and elutions 4 to
7 combined (Supplementary Note 1). ARTICLE Homogenized tissue from 24 embryos was suspended in 960 µl of lysis buffer and
40 µl of tissue lysate was subjected to five cycles of sonication in 0.5 ml Protein
LoBind Tube (Eppendorf) by Bioruptor (Diagenode) to an average DNA fragment
size of 500 bp, and sonicated chromatin was pooled and homogenized, and then
divided into 24 specimens to a concentration of one embryo per specimen. Twenty
microgram of chromatin were used per immunoprecipitation. A volume of 50 µl of
Dynabeads M-280 Sheep anti-Rabbit or Mouse IgG (Invitrogen) was coupled with
5 µg of antibodies in Blocking Solution (0.1% SDS, 1% TritonX-100, 2 mM EDTA,
20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1 mg/ml sonicated salmon sperm DNA
and 0.2 mg/ml BSA) at room temperature for 1 h and was used per
immunoprecipitation. Antibody-Protein complex was generated in blocking
solution at 4 °C overnight and then washed for 5 min each with Buffer D: 0.1%SDS,
1% TritonX-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mg/ml;
Buffer E (same as buffer D except contained 500 mM NaCl); 1X TE containing
0.1% SDS, and all solutions contain 10 mg/ml Linear Acrylamide. Beads were
eluted at 55 °C for 1 h with Elution Buffer (1% SDS, 10 mM EDTA, 50 mM
Tris-HCl pH 8.0, 1 mg/ml Proteinase K and 10 mg/ml Linear Acrylamide) and then
heated at 95 °C for 15 min. Eluted DNA was purified with QIAGEN PCR
purification kit. PCR was performed with the Fast SYBR Green Master Mix
(Applied Biosystems) using a 7500 Fast Real-Time PCR System (Applied (
gy)
y
RAW files were extracted into ms2 file format50 using RAW_Xtract (J.R. Yates,
Scripps Research Institute). MS/MS spectra were queried for peptide sequence
information on a 157-node dual processor Beowulf Linux cluster dedicated to
SEQUEST analyses51. MS/MS spectra were searched without specifying differential
modifications against a protein database consisting of 73626 non-redundant mouse
proteins (NCBI, 2006-09-05 release), 177 sequences for usual contaminants
(such as keratins, proteolytic enzymes…), as well as the sequence for mouse Rere
with two HA tags at the C-terminus. In addition, to estimate false discovery rates,
each non-redundant protein entry was randomized. The resulting “shuffled”
sequences were added to the database and searched at the same time as the
“forward” sequences. To account for carboxamidomethylation by IAM, +57 Da
were added statically to cysteine residues for all the searches. ARTICLE PCR was performed with the
Fast SYBR Green Master Mix (Applied Biosystems) using a 7500 Fast Real-Time
PCR System (Applied Biosystems). PCR conditions were as follows: 95 °C (20 s)
(1×); 95 °C (3 s) and 60 °C (30 s) (40×). Primer pairs used for the Rar-beta
promoter were: 5′-CAGACTGGTTGGGTCATTTGAA-3′ and 5′-GCGAGT-
GAACTTTCGGTGAAC-3′. Immunoprecipitated DNA amounts were calculated
from a standard curve of input DNA and normalized to rabbit or mouse IgG
immunoprecipitations. Ch
h
b
d fid h
l Multidimensional protein identification technology. TCA-precipitated protein
samples from mouse HA immunoprecipitations were solubilized in either 30 µl
or 60 µl (for pooled elutions only) of freshly made 0.1 M Tris-HCl pH 8.5, 8 M
urea, 5 mM TCEP (Tris(2-Carboxylethyl)-Phosphine Hydrochloride, Pierce). After
30 min at room temperature, freshly made 0.5 M IAM (Iodoacetamide, Sigma) was
added to a final concentration of 10 mM, and the samples were left at room
temperature for another 30 min in the dark. Endoproteinase Lys-C (Roche) was
first added at an estimated 1:100 (wt/wt) enzyme to protein ratio, for at least 6 h at
37 °C. Urea was then diluted to 2 M with 0.1 M Tris-HCl pH 8.5, CaCl2 was added
to 0.5 mM and modified trypsin (Promega), 1:100 (wt/wt), was added for over 12 h
at 37 °C. All enzymatic digestions were quenched by adding formic acid to 5%. Each trypsin digested sample was analyzed independently by Multidimensional at 37 C. All enzymatic digestions were quenched by adding formic acid to 5%. Each trypsin-digested sample was analyzed independently by Multidimensional
Protein Identification Technology (MudPIT)11, 48. Peptide mixtures were pressure-
loaded onto a 100 µm fused-silica column pulled to a 5 μm tip using a P 2000 CO2
laser puller (Sutter Instruments). The microcapillary columns were packed first
with 8 cm of 5 μm C18 reverse phase (RP) particles (Aqua, Phenomenex), followed
by 3 cm of 5 μm strong cation exchange material (Partisphere SCX, Whatman), and
by 2 cm of RP particles49. Loaded microcapillaries were placed in line with LTQ
linear ion trap mass spectrometers (Thermo Scientific, San Jose, CA, USA)
interfaced with quaternary Agilent 1100 quaternary pumps (Agilent Technologies,
Palo Alto, CA, USA). Overflow tubing was used to decrease the flow rate from
0.1 ml/min to about 200–300 nl/min. Methods
G
ti Beads
were washed 10 times with 50 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.05%
Triton-X100, and then bound protein was eluted using Laemmli sample buffer
(Sigma) and heated at 100 °C for 5 min. Elutions were resolved on 7.5%
polyacrylamide Tris-HCl gels (BioRad) and transferred to polyvinylidene difluoride
membrane (Millipore). Blots were treated according to a standard western blot
protocol. Real-time PCR assays. Total mRNA was isolated from NIH3T3 cells using
FastLane Cell One-Step Buffer Set (Qiagen). qPCR experiments were performed
using Quantifast SYBR Green RT-PCR Kit (Qiagen) in a LightCycler 480 II System
(Roche). The following primers from Qiagen (Qiagen Quantitect Primer Assay)
were used—with catalog numbers in brackets: Rere (QT01075774); Wdr5
(QT00111685), Hdac1 (QT02273418), Hdac2 (QT01161657), Ehmt2
(QT00115262), Rarβ (QT00151956), and Rplp0 (QT00249375). Chromatin immunoprecipitation assay. NIH3T3 cells were grown in 15 cm
dishes and treated with 1 µM of RA (Sigma) for 1 h before collecting. At 70–80%
confluency, cells were crosslinked with 1% formaldehyde at room temperature for
10 min. For ChIP experiments with NIH3T3 cells, 50 µg of chromatin were used
per immunoprecipitation. F9 or NIH3T3 cells were grown in 15 cm dishes and The following reagents and antibodies were used: mouse IgG (Upstate), M2
anti-Flag (Sigma), anti-HA (Sigma), Dynabeads M-280 sheep anti-mouse IgG
(Invitrogen), Flag-coupled HRP (Sigma), HA-coupled HRP (Roche) and Rarα C-20
(Santa Cruz). 10 NATURE COMMUNICATIONS| 8: 728
| DOI: 10.1038/s41467-017-00593-6| www.nature.com/naturecommunications NATURE COMMUNICATIONS| 8: 728
| DOI: 10.1038/s41467-017-00593-6| www.nature.com/naturecommunications FDR ¼ 2 ´ SHUFFLED Spectral Counts
Total Spectral Counts 5. Xu, L., Glass, C. K. & Rosenfeld, M. G. Coactivator and corepressor complexes
in nuclear receptor function. Curr. Opin. Genet. Dev. 9, 140–147 (1999). Protein level FDR is calculated as: 6. Rosenfeld, M. G., Lunyak, V. V. & Glass, C. K. Sensors and signals: a
coactivator/corepressor/epigenetic code for integrating signal-dependent
programs of transcriptional response. Genes Dev. 20, 1405–1428 (2006). Under these criteria the final FDRs at the protein and spectral levels were 1.9%
and 0.14% ± 0.085, respectively. p
g
p
p
7. Vermot, J. et al. Retinoic acid controls the bilateral symmetry of somite
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ð
Þi ¼
Spectral Count=Length
ð
Þi
P
N
k¼1
Spectral Count=Length
ð
Þk
: NSAF
ð
Þi ¼
Spectral Count=Length
ð
Þi
P
N
k¼1
Spectral Count=Length
ð
Þk
: NSAF
ð
Þi ¼
Spectral Count=Length
ð
Þi
P
N
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Spectral Count=Length
ð
Þk
: NSAF
ð
Þi ¼
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ð
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h l
f
ll
h
l
(
) 24. Vedadi, M. et al. A chemical probe selectively inhibits G9a and GLP
methyltransferase activity in cells. Nat. Chem. Biol. 7, 566–574 (2011) p
y
methyltransferase activity in cells. Nat. Chem. Biol. 7, 566–574 (2011). Statistics. Statistics were performed using Excel software (Microsoft). Significance
of difference was analyzed with a Student’s unpaired two-tailed t-test. All data are
presented as mean ± s.e.m. FDR ¼ 2 ´ SHUFFLED Spectral Counts
Total Spectral Counts For immunoprecipitation of the WHHERE complex from
SF21 cells, cells co-expressing Rere-Flag, Flag-Hdac1, Flag-Hdac2, and HA-Wdr5
were lyzed with a buffer containing 0.4 M KCl, 25% glycerol, 20 mM Tris-HCl pH
8.0, 5 mM MgCl2, 0.5% NP-40, 2X complete protease inhibitor cocktail EDTA-free
(Roche) and 1 mM DTT. After centrifugation of the lysate for 10 min at 8000 r.p.m. at 4 °C, the supernatant containing the whole cell protein extract was collected and
incubated with anti-Flag antibody coupled with agarose beads (anti-Flag M2 affi-
nity gel, Sigma) for 1 h at 4 °C, to perform the Flag-IP. After washing beads in a
buffer containing 150 mM KCl or 500 mM KCl, 25 mM Tris-HCl pH 8.0, 10%
glycerol, 5 mM MgCl2, 0.1% NP-40, proteins were eluted by peptide binding
competition using the Flag-tag peptide DYKDDDDK (2 mg/ml final) dissolved into
a buffer containing 100 mM KCl, 25 mM Tris-HCl pH 8.0, 10% glycerol, 5 mM
MgCl2, 0.1% NP-40. Proteins eluate was then concentrated on amicon-ultra
centrifugal filter units (Millipore, membrane cutoff of 30 kDa) before gel filtration
analysis. To study the direct protein–protein interaction between Rere and Wdr5,
Rere-Flag, and HA-Wdr5 were co-expressed and Flag-tagged purified as described
above. The gel filtration analysis of the purified complexes was performed on a
Superose 6 PC 3.2/30 (GE Healthcare) mounted on an AKTA micro chromato-
graphy system (GE Healthcare) using a buffer containing 25 mM Tris-HCl pH 8.0,
100 mM KCl, 5 mM MgCl2, and 2% glycerol. Calibration of the column was
performed using a standard calibration kit (BioRad), containing Thyroglobulin
(670 kDa), ɣ-globulin (158 kDa), ovalbumin (44 kDa), myoglobulin (17 kDa),
and Vitamin B12 (1355 Da). The exclusion volume V0 was determined using
calf thymus DNA (> 2000 bp), while the total volume of the column was
measured using L-Tyrosine. For the experiment, 80–100 µl of purified sample was
injected through an injection loop to the column. Flow rate was set to 40 µl/min
and SEC fractions of 75 µl each were collected throughout the gel filtration run and
analyzed by western blotting to check for the presence of Rere, Hdac1, Hdac2, and
Wdr5. The following antibodies were used for western blot: Rere (Santa Cruz, sc-
98415), Hdac1 (Abcam, ab7028), Hdac2 (Abcam, ab7029), and Wdr5 (Abcam,
ab22512). 12. Florens, L. & Washburn, M. P. Proteomic analysis by multidimensional protein
identification technology. Methods Mol. Biol. 328, 159–175 (2006). 13. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00593-6 minimum XCorrs of 1.8 for singly- charged, 2.0 for doubly- charged, and 3.0 for
triply-charged spectra, and a maximum Sp rank of 10. Finally, combining all runs,
proteins had to be detected by at least 2 such peptides, or 1 peptide with two
independent spectra. Proteins that were subset of others were removed. minimum XCorrs of 1.8 for singly- charged, 2.0 for doubly- charged, and 3.0 for
triply-charged spectra, and a maximum Sp rank of 10. Finally, combining all runs,
proteins had to be detected by at least 2 such peptides, or 1 peptide with two
independent spectra. Proteins that were subset of others were removed. ARTICLE g
p
p
immunoprecipitation. Antibody-Protein complex was generated in blocking
solution at 4 °C overnight and then washed for 5 min each with Buffer D: 0.1%SDS,
1% TritonX-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mg/ml;
Buffer E (same as buffer D except contained 500 mM NaCl); 1X TE containing
0.1% SDS, and all solutions contain 10 mg/ml Linear Acrylamide. Beads were
eluted at 55 °C for 1 h with Elution Buffer (1% SDS, 10 mM EDTA, 50 mM
Tris-HCl pH 8.0, 1 mg/ml Proteinase K and 10 mg/ml Linear Acrylamide) and then
heated at 95 °C for 15 min. Eluted DNA was purified with QIAGEN PCR
purification kit. PCR was performed with the Fast SYBR Green Master Mix
(Applied Biosystems) using a 7500 Fast Real-Time PCR System (Applied Results from different runs were compared and merged using CONTRAST52. Spectrum/peptide matches were only retained if peptides had to be at least seven
amino acids long and be fully tryptic. The DeltCn had to be at least 0.08, with 11 NATURE COMMUNICATIONS| 8: 728
| DOI: 10.1038/s41467-017-00593-6| www.nature.com/naturecommunications NATURE COMMUNICATIONS| 8: 728
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| DOI: 10.1038/s41467-017-00593-6| www.nature.com/naturecommunications NATURE COMMUNICATIONS| 8: 728
| DOI: 10.1038/s41467-017-00593-6| www.nature.com/naturecommunicatio 13
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Banana leaf in the diet of laying hens in cage free system
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Acta Scientiarum
http://periodicos.uem.br/ojs/acta
ISSN on-line: 1807-8672
Doi: 10.4025/actascianimsci.v41i1.46908
NONRUMINANTS NUTRITION
Banana leaf in the diet of laying hens in cage free system
Nayara Emanoelle Matos e Silva1, Heder José D'Ávila Lima1, Jean Kaique Valentim2* , Fernandes
Jesuino Muquissai Tossué1, Tatiana Marques Bittencourt1 and Jonatan Mikhail Del Solar Velarde3
Departamento de Zootecnia e Extensão Rural, Faculdade de Agronomia e Zootecnia, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brasil.
Departamento de Zootecnia, Faculdade de Agronomia e Zootecnia, Universidade Federal da Grande Dourados, Rodovia Dourados-Itahum, Km 12, Unidade
II, 79804-970, Dourados, Mato Grosso do Sul, Brasil. ³Departamento de Zootecnia, Faculdade de Zootecnia, Campus São Cristóvão, Universidade Federal
de Sergipe, São Cristóvão, Sergipe, Brasil. *Author for correspondence. E-mail: kaique.tim@hotmail.com
1
2
ABSTRACT. The aim of this research was evaluate the productive performance, egg quality and the
endoparasites presence on Carijó laying hens in Cage Free system, fed with two experimental diets, one of
them supplemented with fresh banana leaf in natura. It were used 196 laying hens with twenty-four weeks
of age, distributed in a completely randomized design with 14 boxes, two experimental diets and seven
replicates, by treatment with fourteen birds per experimental unit. The variables analyzed were: feed
intake; egg production; egg mass: feed conversion per egg mass; feed conversion per dozen eggs; egg
weight; albumen weight; yolk weight; shell weight; yolk percentage; albumen percentage shell
percentage; yolk pigmentation and the presence of endoparasites. No influence (p > 0.05) were found, for
performance and egg production, in Carijó laying hens fed supplemented with banana leaf in natura. For
the treatment with inclusion of banana leaf, yolk pigmentation were superior (p < 0.05) when compared to
control diet, producing eggs with more yolks orange color. In respect to endoparasite analysis, there was
significant effect (p < 0.05) in the percentage of infections present in excretas. Among the treatments
evaluated, the one with banana leaf supplementation reduced in 26.54% the endoparasites presence. The
inclusion of banana leaf in the diet of Carijo laying hens does not affect performance and eggs quality and
decreases the amount of endoparasites of the genus Strogyloides spp.
Keywords: banana production; Cage-Free; endoparasites; performance.
Received on March 9, 2019.
Accepted on March 27, 2019.
Introduction
Brazilian poultry is one of the leading animal production agriculture industries, becoming an example of
activity and productive chain of success. Production costs related to food in this segment has generated
relevant concerns for industry and poultry producers, since production cost affects directly in the final
profitability (Rufino, Cruz, Tanaka, Melo, & Feijó, 2017). The increase of corn and soybean prices or their
low availability has encouraged the research, looking for alternative foods reducing poultry production
costs, without harming animal performance (Lopes et al., 2014).
According to Diniz, Granja-Salcedo, Oliveira and Viegas (2014), banana (Musa spp) cultivation has grown
considerably in the last three decades, together with soil improvement and the country climatic characteristics
favoring its production, this crop shows rapid economic return on invested capital since the production begin can
occur the first year, and for being a perennial crop with continuous flow, for that, becoming attractive to farmers.
Bezerra, Sousa and Oliveira (2002) reported that banana tree nutritional composition varies according to
the variety, age and plant component, and the leaf can have 12 to 13% protein in the dry matter. The banana
is still rich in organic pigment beta-carotene, which can confer more orange color to yolk, also can be used
as a prophylactic agent and precursor of vitamin A.
Carotenoid pigments are normally found in vegetables, and are responsible for feathers, skin and egg
yolk pigmentation. Yellow coloration is typical effected by carotenes (Mendonça, Correa, Benevides, Mota &
Franca, 2018). Another benefit of plant extracts regards the natural anthelmintic capacity (Soares, Welter,
Gonzaga, Alessandro, Mancini-Filho & Roseane, 2008). Vegetable extracts are available in nature and they
are easy to reproduce and plant, although they are less expensive compared to other nutritional fonts.
Researchers found positive results of using plants in the control of helminthes, enabling to decrease in
the parasite load in extensive poultry, obtaining high quality products with lower costs and increasing the
poultry producer profitability (Fernandes, Rodrigues, Borba, Fernandes & Amorim, 2004).
Acta Scientiarum. Animal Sciences, v. 41, e46908, 2019
Page 2 of 6
Silva et al.
Thus, this research aimed to evaluate the use of banana leaf provided in natura as supplementation in
the diet and its influence on productive performance, egg quality and the endoparasites presence on Carijó
laying hens raised in Cage Free system.
Material and methods
The experiment was conducted in the Federal University of Mato Grosso (UFMT), in the Poultry Sector of the
Experimental Farm located in Santo Antônio municipality – Leverger. The project was submitted, analyzed and
approved by the ethics committee on the use of animals (CEUA) under number 23108.092960/2015-80.
In this context, 196 Carijó laying hens with twenty four weeks of age, 2.235 kg of live weight (LW) and
69% of posture rate, were distributed in a completely randomized design with seven experimental units (EU)
as replicates and fourteen birds per EU. The birds were fed with experimental diet (Table 1), which was
based on corn and soybean meal, formulated as recommended by Rostagno et al. (2011) for semi-heavy birds
adding, as supplementation, banana leaf in natura.
The experiment lasted 63 days, divided in three periods of 21 days each. All birds were weighed at the
beginning and end of the experiment to determine body weight (BW) variation. The birds were housed in
experimental masonry shed, with ceramic tile roof, concrete floor, side walls screened with external
protective curtain, equipped with sprinklers and fans.
The 14 experimental boxes used were 2.22 m wide and 3.27 m long, totaling 7.25 m 2, with 14 birds in
each box, obtaining a housing density of 1.93 bird’s m-2. The birds of both treatments were fed with a basal
diet provided twice a day in the morning and afternoon, and a treatment was supplemented with fresh
banana leaf during whole experiment.
The banana leaf were collected every morning in the fruit sector of the UFMT experimental farm and
taken to the shed where the birds were housed, after which they were processed by a leaf shredder to make
the particles smaller after this was weighed 570 g per experimental boxing corresponding to 40g per bird
housed and supplied in feeders for that purpose. Next day in the morning the leftovers were removed and
weighed, thus being able to estimate the actual consumption of the banana leaf in natura. The lighting
program adopted during the entire experimental phase was 16 hours of light per day.
Performance
The feed intake (FI) was obtained through the amount of feed offered during whole experimental period
and discarded leftovers at the end of the experimental period divided by the number of birds of each
treatment and then divided by sixty-three days expressed in grams of consumed feed bird day-1.
Table 1. Percentage and calculated composition of the diet.
Ingredients
Ground corn %
Soybean meal %
Nucleus¹ %
Dicalcium phosphate %
Calcic limestone %
Common Salt %
Oil %
Nutrition composition calculated
Metabolizable energy (kcal kg-1)
Crude protein%
Calcium %
Available phosphorus %
Sodium %
Digestible Lysine %
Methionine + cystine Digestible %
Digestible Threonine %
Digestible Tryptophan %
Value
62.5
25.0
1.5
1.1
7.6
0.5
1.8
2900
16.02
3.90
0.291
0.218
0.777
0.707
0.591
0.179
¹Guarantee level per kg of core, Calcium (max) 210g.; Calcium (min) 170g.; Phosphorus (min) 45g.;
Methionine (min) 10g.; Vitamin A (min) 140000 IU.; Vitamin D3 (min) 35000 IU.; Vitamin E (min) 140 IU.;
Thiamine (B1) (min) 10 mg.; Riboflavin (B2) 20 mg.; Vitamin B12 (min) 120 mcg.; Vitamin K3 (min) 30 mg.;
Folic Acid (min) 6 Mg.; Niacin (min) 300 mg.; Calcium Pantothenate (min) 120 mg.; (Min) 6 mg.; Iron (min)
630 mg.; Iodine (min) 20 mg.; Selenium (min) 6 mg.; Sodium (min) 30 g.; Manganese (min) 1600 mg.; Zinc
(min) 1300 mg.; Copper (min) 10000 FTU and Zinc Bacitracin 500 mg.
Acta Scientiarum. Animal Sciences, v. 41, e46908, 2019
Banana farming co-product in poultry production
Page 3 of 6
The feed conversion per egg mass (FCEM) was obtained by dividing FI in kilograms by the egg mass (EM)
produced in kilograms (kg kg-1), during the entire experimental period.
Feed conversion per dozen eggs (FC12E) was expressed as the total feed consumption in kilograms
divided by the dozen eggs produced (kg dz-1).
To analyze egg production (EP), they were collected and recorded daily. After that, the values were
transformed in percentage, adding the yields per repetition of each period and divided by the total amount
of days of the experiment, that is 63 days, and then divided by 14, number of experimental repetitions,
obtaining the average production (%) in each period.
Eggs quality
All intact eggs produced at the end of each experimental period consisting of 21 days were collected and
afterwards, three eggs were randomly selected from each replicate to perform egg quality analyzes daily for
three consecutive days and weighed in a precision scale of 0.01 g. The mean egg weight (EW) in grams was
obtained by weighing the eggs. After weighed individually, the eggs were broken and then the yolk weight
(YW) in grams. The shells were washed and air dried for two days to obtain shell weight (BW) in grams.
Finally, the albumen weight (AW) was obtained by subtracting the EW and YW in grams. The percentage (%)
in composition of the egg, was composed by EW considered as 100% and the other egg measures were
divided by it to obtain its respective percentage as yolk (Y%), albumen (A%) and shell (B%) percentage.
In order to analyze the color of the yolk, a colorimetric tool DSM 14 tons was used to obtain yolk
pigmentation (YP) which varies from opaque yellow to intense orange, the higher the score value obtained
in the fan, the greater the degree of pigmentation of the yolk. The three samples of each repetition were
placed on a flat surface, the colorimetric tool was placed above the buds, and it was possible to observe the
staining according to the colorimetric tool scale, these analyzes were always performed by the same person
from the beginning to the end of the experiment, always in the same place and daytime, in order to avoid
variations, mainly for being a subjective analysis, and thus calculated the average per period.
The specific gravity of the eggs was evaluated from collected intact eggs, which were properly identified
according to the treatment and repetition, were immersed and evaluated in NaCl solutions with density
varying from 1.070 to 1.095 g cm-3. The density was measured and confirmed with the aid of a floating glass
densitometer specific for salinity, according to the method suggested by Hamilton (1982), after immersing
the eggs that corresponded to the specified density fluctuated, then the value was recorded manually in
worksheets.
Presence of endoparasites
Excreta samples were collected on the last day of the experimental period in the morning. The excreta
were collected with the aid of wooden spatulas without touching the floor and stored in specific plastic pots
for analysis and packed in iceboxes. Subsequently they were transported to the Laboratory. Three samples of
each replicate were used for analyzes. The analysis for endoparasite presence was performed using Willis
(1921) method, at the UFMT - Medicine Veterinary Hospital, Animal Parasitology Laboratory, with the
principle of fluctuation through a high density NaCl solution, to which the eggs adhere to the lower surface
of the slide followed by direct qualitative microscopic analysis after excreta concentration. Parasites
nematodes and cestodes eggs and larvae, and oocysts of Eimeriidae and Strongyloidea families were
observed.
Statistical analysis
The variables statistical analysis was performed by the Assistat Statistical Program (Silva & Azevedo,
2002). Performance bird’s means were compared using the F Test, while the means of endoparasite presence
(%) were compare whitin with SNK test. Both performance and endoparasite presence were analyzed with
5% probability level.
Results and discussion
There was no significant influence (p > 0.05) on productive performance and egg quality of Carijo laying
hens supplemented with banana leaf in natura in the diet (Table 2). The daily addition of banana leaf did not
influence (p < 0.05) FI, EP, EM, EM, FC12E, YW, BW, AW, Y%, B% and A%.
Acta Scientiarum. Animal Sciences, v. 41, e46908, 2019
Page 4 of 6
Silva et al.
Table 2. Performance of Carijó laying hens due to dietary supplementation with banana leaf. Control diet (CD) (Diet based on corn and
soybean meal); Supplementation diet (SD) (Diet based on corn and soybean meal + supply of banana leaf in natura).
Variables
FI (g bird day-1)
Banana leaf consumption (g bird day-1)*
EP (%)
EW (g)
EM (g bird day-1)
FCEM (kg kg-1)
FC12E (kg dz-1)
YW (g)
BW (g)
AW (g)
YP **
Y%
B%
A%
Live weight variation (g)*
CD
SD
p-value
0.173
0.0
57.74
64.25
32.72
4.64
3.57
19.26
5.83
39.27
4.78b
29.98
9.08
61.12
388.0
0.171
40.7
52.52
64.99
30.68
4.89
3.82
19.65
5.86
39.54
6.99a
30.24
9.02
60.83
410.0
0.087
---0.054
0.089
0.006
0.0078
0.34
0.21
0.075
0.26
0.0002
0.056
0.067
0.074
---
C.V.
(%)
1.55
----9.31
1.34
9.43
8.92
8.87
2.08
1.77
1.55
3.95
1.55
1.39
0.71
---
FI: feed intake; EP: egg production; EM: egg mass; FCEM: feed conversion per egg mass; FC12E: Feed conversion per dozen
eggs; EW: The mean egg weight; YW: yolk weight; BW: shell weight in grams; AW: albumen weight in grams; YP: yolk
pigmentation; Y%: yolk percentage; A%: albumen percentage and B%: shell percentage. ns - No statistical significance by F test
(p > 0.05). Means followed by the same letter on the line do not differ at the 5% level of significance. *Descriptive analysis.
**Statistical significance with 1% probability level.
The addition of banana leaf in natura did not alter birds performance (p > 0.05), which may be a
nutritional complement that benefits other variables, such as the color of the yolk. Significant results were
found at the 1% probability level (p < 0.01) in the yolk color (Table 2). The treatment based on corn and
soybean meal, with the supplementation of fresh leaves provided greater egg yolk pigmentation.
The change in the YP to more orange may be justified by the presence of carotenes in the
composition of the banana leaf, such as carotene, which is an organic substance that gives pigment to
the plants and when used in poultry, feeding birds in adequate quantities, might help to intensify both
bird skin color and egg yolk.
According to Silva, Albino and Souza (2000), YP is an important trait for egg valorization and acceptance
by pasta consumers and manufacturers, deserts and other products that have their value maximized by
offering more vivid and attractive colors to the consumer. Therefore, foods that may be favored by YP may
result in better price to producers.
Foliar residues from banana farming can also be added as fiber source in feed for monogastric animals.
Musmanni, Campabadal and Vargas (1979) evaluated biologically and economically the use of different
protein levels in pigs fed supplementing banana waste, and concluded that production cost reduced using it.
Marín, Carías, Cioccia and Hevia (2003), in their research using banana leaf in broilers feeding, showed
that the treatment with banana leaf provided to the broiler chickens higher growth performance when
compared to the other diets containing ingredients derived from legumes.
The data obtained after endoparasite excreta tests (Table 3), showed that Carijo laying hens
supplemented with banana leaf dah low load (p < 0.05) in percentage of Strongyloides spp. and Eimeira spp.
present in the excreta, compared with the control diet.
In this experiment, parasites of the genus Strongyloides spp. and Eimeria spp. Were observed, also those
were found in Siqueira (2016) research. Although the author has detected genres Capillaria, Heterakis,
Choanotaenia, Ascaridia, Stronyloidea, Strongyloides and Eimeria protozoan, the birds showed no clinical
signs of parasitic infection.
Table 3. Endoparasites presence (%) in excreta of Carijó laying hens due to dietary supplementation with banana leaf. Control diet
(CD) (Diet based on corn and soybean meal); Supplementation diet (SD) (Diet based on corn and soybean meal + supply of banana leaf
in natura).
Variables
Oocytes of Strogyloides spp (%)
Oocytes of Eimeira spp*
CD
63.26a
1
SD
36.73b
0
p-value
0.0034
----
CV
10.92
---
Means followed by different letters in the row differ among themselves at the 5% level of significance by SNK test. *Descriptive analysis.
Acta Scientiarum. Animal Sciences, v. 41, e46908, 2019
Banana farming co-product in poultry production
Page 5 of 6
These data corroborate those found by Silva, Amorim, Santos-Serejo, Ferreira and Rodriguez (2013),
which found that banana can be used for feeding cattle in order to deworm the animals, being supplied in
natura mixed in meal. It is possible to find the presence of seven species of endoparasites in birds raised in
intensive systems: the protozoa Eimeria sp. and Entamoeba sp.; Nematoda helminthes Heterakis gallinarum,
Strongyllus avium, Tetrameres sp, Ascaridia galii; although found a diversity of cestodes.
Gomes, Simões Machado, Lemos, Almeida and Daher (2009) verified the presence of species Eimeria, Hetrakis
gallinarium, Strongyloides, and Ascaridia galii in bird excreta samples raised in Cage free systems. According to
Olivo et al. (2007) banana species present tannins in their constitution. These compounds could be responsible
for anthelmintic activity, since plants containing tannins are often used in the fight against verminosis.
Acorda, Mangubat and Divina (2019) working with the addition of pumpkin seeds as an alternative
natural extracts for laying hens suggested that pumpkin seed has the potential to be used as an alternative
anthelmintic for chickens.
From this, banana farming co-products can be used, such as the stem and its leaves. Proving that, this
research showed the use of fresh banana leaf as supplementation for feeding Carijo laying hens, leading to
the control intestinal endoparasites of the Nematoda class.
Conclusion
Feed supplementation with banana leaf in the diet of Carijó laying hens in Cage free system increases
yolk pigmentation, does not interfere on performance and reduces the endoparasites presence.
References
Acorda, J. A., Mangubat, I. Y. E. C., & Divina, B. P. (2019). Evaluation of the in vivo efficacy of pumpkin
(Cucurbita pepo) seeds against gastrointestinal helminths of chickens. Turkish Journal of Veterinary and
Animal Sciences, 43(2), 206-211. doi: 10.3906/vet-1807-39
Bezerra, L. J. D., Sousa, E. B. C., & Oliveira, M. (2002). Estudo bromatológico da bananeira (Musa spp.) e sua
utilização na alimentação de bovinos. Agronline, 18(12), 1-7. Retrieved from
http://www.agronline.com.br/agrociencia/pdf/public_37.pdf
Diniz, T. T., Granja-Salcedo, Y., Oliveira, E. M. Z., & Viegsas, C. R. (2014). Uso de subprodutos da
bananicultura na alimentação animal. Revista Colombiana de Ciencias Animal, 6(1), 194-212. doi:
10.24188/recia.v6.n1.2014.260
Fernandes, R. M., Rodrigues, M. L. A., Borba, H. R., Fernandes, M. Z. C. M., & Amorim, A. (2004). Ausência
da atividade anti-helmíntica de plantas em frangos de corte naturalmente infectados com Heterakis
gallinarum (Schranck, 1788) Madsen, 1949. Ciência Rural, 34, 1629-1682. Retrieved from
http://www.scielo.br/pdf/%0D/cr/v34n5/a50v34n5.pdf
Gomes, F. F., Simões Machado, H. H., Lemos, L. S., Almeida, L. G., & Daher, R. F. (2009). Principais parasitos
intestinais diagnosticados em galinhas domésticas criadas em regime extensivo na municipalidade de
Campos dos Goytacazes, RJ. Ciência Animal Brasileira, 10(3), 818-822. Retrieved from
https://www.revistas.ufg.br/vet/article/view/1095
Hamilton, R. M. G. (1982). Methods and factors that affect the measurement of egg shell quality. Poultry
Science, 61(10), 2022-2039. Retrieved from https://academic.oup.com/ps/articleabstract/61/10/2022/1489695
Lopes, I. R. V., Freitas, E. R., Nascimento, G. A. J., Viana Neto, J. L., Cruz, C. E. B., & Braz, N. M. (2014).
Inclusão de fenos de folha de leucena e de cunhã na ração de poedeiras. Archivos de zootecnia, 63(241),
183-190. doi: 10.4321/S0004-05922014000100018
Marín, A., Carías, D., Cioccia, A. M., & Hevia, P. (2003). Valor nutricional de los follajes de musa paradisiaca
y clitoria ternatea como diluyentes de raciones para pollos de engorde. Interciencia, 28(1), 50-56.
Retrieved from http://www.scielo.org.ve/scielo.php?script=sci_arttext&pid=S0378-18442003000100008
Mendonça, A. S. A., Correa, R. E. A., Benevides, P. R., Mota, A. V., & Franca, E. B. A. (2018). Pigmentante alternativo
para gema em ovos de galinha caipira no município de Garrafão-do-Norte/PA. Cadernos de Agroecologia, 13(2),
1-10. Retrieved from http://cadernos.aba-agroecologia.org.br/index.php/cadernos/article/view/2127
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Silva et al.
Musmanni, M., Campabadal, C., & Vargas, E. (1979). Suplementación proteíca del banano en la
alimentación de cerdos en desarrollo y engorde. Agronomía Costarricense, 3(2), 129-135. Retrieved from
http://www.mag.go.cr/rev_agr/v03n02_129.pdf
Olivo, C. J., Pereira, L. E. T., Carvalho, N. M., Vogel, F. F., Heinzmann, B. M., & Neves, A. P. (2007). Uso da
bananeira (Musa spp.) no controle de parasitas de animais domésticos: do empirismo à ciência. Livestock
Research for Rural Development, 19(11), 1-5. Retrieved from
https://lrrd.cipav.org.co/lrrd19/11/oliv19158.htm
Rostagno, H. S., Albino, L. F. T., Donzele, J. L., Gomes, P. C., Oliveira, R., Lopes, D. C., ... Euclides, R. F.
(2011). Composição de alimentos e exigências nutricionais (2ed. Vol. 2). Viçosa, MG: Universidade Federal
de Viçosa.
Rufino, J. P. F., Cruz, F. G. G., Tanaka, E. S., Melo, R. D., & Feijó, J. C. (2017). Análise econômica da inclusão
de farinha do resíduo de buriti na alimentação de poedeiras comerciais. Revista Ciência Agronômica,
48(4), 732-738. Retrieved from http://www.ccarevista.ufc.br/seer/index.php/ccarevista/article/view/4308
Silva, F., & Azevedo, C. (2002). Versão do programa computacional Assistat para o sistema operacional
Windows. Revista Brasileira de Produtos Agroindustriais, 4(1), 71-78.
Silva, J. H. V., Albino, L. F. T., & Souza, G., Mauro José. (2000). Efeito do extrato de urucum na pigmentação
da gema dos ovos. Revista Brasileira de Zootecnia, 29(5), 1435-1439. Retrieved from
http://www.scielo.br/pdf/%0D/rbz/v29n5/5665.pdf
Silva, S. O., Amorim, E. P., Santos-Serejo, J. A. s., Ferreira, C. F., & Rodriguez, M. A. D. (2013).
Melhoramento genético da bananeira: estratégias e tecnologias disponíveis. Revista Brasileira de
Fruticultura, 35(3), 919-931. Retrieved from http://www.musalit.org/seeMore.php?id=15013
Siqueira, G. B. D. (2016). Parasitos intestinais em galinhas caipiras da região metropolitana de Porto Alegre,
RS. PUBVET, 10(9), 690-695. Retrieved from http://hdl.handle.net/10183/148224
Soares, M., Welter, L., Gonzaga, L., Alessandro, L., Mancini-Filho, J., & Roseane, F. E. T. T. (2008). Avaliação
da atividade antioxidante e identificação dos ácidos fenólicos presentes no bagaço de maçã cv. Gala.
Ciência e Tecnologia de Alimentos, 28(3), 727-732. Retrieved from
https://www.redalyc.org/html/3959/395940088032
Willis, H. H. (1921). A simple levitation method for the detection of hookworm ova. Medical Journal of
Australia, 2(18), 375-376. Retrieved from https://www.cabdirect.org/cabdirect/abstract/19222900461
Acta Scientiarum. Animal Sciences, v. 41, e46908, 2019
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Student Handbook1, 3 (TTEL, Ghana: Component 2, Teaching Practice - materials for lecturers (tutors), school-based mentors and students, Student,Handbook1-3)
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Transforming Teacher Education and Learning i Year 1 - 3: Teaching Practice Schools Partnership Programme Year 1-3: Teaching Practice
Student Teacher Handbook The Government of Ghana The Government of Ghana The Government of Ghana T-TEL Schools Partnership Programme. Year 1-3: Teaching Practice (Student Teacher Handbook). Published by the Ministry of Education (Ghana), under Creative Commons Attribution-ShareAlike 4.0
International. Available online at http://www.t-tel.org. Version 1.0, January 2016. Student Teacher Handbook for Teaching Pratice 2 INTRODUCTION TABLE OF CONTENTS Abbreviations ............................................................................................................................................4
Introduction
Year 1, Year 2 and Year 3 Teaching Practice arrangements .........................................................8
Student Teacher interaction with pupils in their classrooms .................................................................8
Student Teacher interaction with other Student Teachers - TP partner or TP buddy ......................9
Use of the Teaching Practice Journal (TP Journal) .........................................................................9
Roles and responsibilities outlined .....................................................................................................10
Assessment of Student Teacher performance ...............................................................................13
Guidelines for protection strategies for Student Teachers on teaching practice .............13
Year 1 Observation Teaching Practice
Purpose: In Year 1 Observation Teaching Practice: .............................................................................15
Topics and activities for Year 1 Observation Teaching Practice ..........................................................16
Teaching Practice Framework: Year 1 Observation Teaching Practice
Year 2 On-Campus Teaching Practice
Purpose: In Year 2 On-Campus Teaching Practice: .................................................................................43
Topics and activities for Year 2 On-Campus Teaching Practice ...........................................................44
Teaching Practice Framework: Year 2 On-Campus Teaching Practice
Year 3 Off-Campus Teaching Practice
Purpose: In Year 3 Off-Campus Teaching Practice: ................................................................................63
Topics and activities for Year 3 Off-Campus Teaching Practice ...........................................................64
Teaching Practice Framework: Year 3 Off-Campus Teaching Practice
Annex 1. Teacher Competencies ............................................................................................................. 109
Annex 2. Lesson Plan Format ................................................................................................................... 113
Annex 3. Lesson Observation Format .................................................................................................... 114
Annex 4. Assessment Procedures ........................................................................................................... 116
Annex 5. Guidelines for Protection Strategies for Student Teachers on Teaching Practice ...... 124
Annex 6. Example Page from a TP Journal ........................................................................................... 127 Abbreviations ............................................................................................................................................4
Introduction
Year 1, Year 2 and Year 3 Teaching Practice arrangements .........................................................8
Student Teacher interaction with pupils in their classrooms .................................................................8
Student Teacher interaction with other Student Teachers - TP partner or TP buddy ......................9
Use of the Teaching Practice Journal (TP Journal) .........................................................................9
Roles and responsibilities outlined .....................................................................................................10
Assessment of Student Teacher performance ...............................................................................13
Guidelines for protection strategies for Student Teachers on teaching practice .............13
Year 1 Observation Teaching Practice
Purpose: In Year 1 Observation Teaching Practice: .............................................................................15
Topics and activities for Year 1 Observation Teaching Practice ..........................................................16
Teaching Practice Framework: Year 1 Observation Teaching Practice
Year 2 On-Campus Teaching Practice
Purpose: In Year 2 On-Campus Teaching Practice: .................................................................................43
Topics and activities for Year 2 On-Campus Teaching Practice ...........................................................44
Teaching Practice Framework: Year 2 On-Campus Teaching Practice
Year 3 Off-Campus Teaching Practice
Purpose: In Year 3 Off-Campus Teaching Practice: ................................................................................63
Topics and activities for Year 3 Off-Campus Teaching Practice ...........................................................64
Teaching Practice Framework: Year 3 Off-Campus Teaching Practice
Annex 1. Teacher Competencies ............................................................................................................. 109
Annex 2. Lesson Plan Format ................................................................................................................... 113
Annex 3. Lesson Observation Format .................................................................................................... 114
Annex 4. Assessment Procedures ........................................................................................................... 116
Annex 5. TABLE OF CONTENTS Guidelines for Protection Strategies for Student Teachers on Teaching Practice ...... 124
Annex 6. Example Page from a TP Journal ........................................................................................... 127 .4 Introduction Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 3 i Year 1 - 3: Teaching Practice Student Teacher Handbook for Teaching Pratice FOREWORD The quality of education in every society is largely determined by the quality of its teachers. Conversely, the quality of teachers is determined by the level of their subject matter mastery
and how they pass on that subject matter to their learners, among many other factors. Teachers’
ability to do this depends on how the Initial Teacher Education (ITE) prepares them for teaching. The ITE should provide teachers with intellectual and professional background through the study
of academic content, professional and pedagogical studies, and create the opportunity for student
teachers to practise teaching. In many contexts, it is this foundation that informs teachers’ practice
the most. The initial teacher education in Africa, and sub-Saharan Africa particularly, has been criticised for
failing to prepare teachers adequately for the conditions they face in the field (Lewin and Stuart,
2003; Bakari, 2009). In some cases, weaknesses in teachers’ capabilities in promoting effective
learning at the classroom level have been cited. There is also the argument that the ITE curricula
in many African countries, including Ghana, are too theoretical, with little emphasis on practical
knowledge and practice (Akyeampong, Lussier, Pryor & Westbrook, 2013). To address these issues,
the ITE programme must be reformed to make it practice-based, giving student teachers a number
of opportunities to observe and practise a variety of different forms of professional knowledge and
skills through direct practical experience in schools. Teaching practice is an essential component of all ITE programmes. During teaching practice,
Student Teachers are placed under the guidance of qualified professionals (Mentors, College Tutors,
District Education Officers, etc.) who introduce them to teaching and its routines in order to
develop in them the required professional skills and competencies, and a positive attitude towards
the teaching profession. Teaching practice also provides the right environment for Student Teachers
to familiarise themselves with all the processes of the school setting, and to observe examples of
good practice. The Transforming Teacher Education and Learning (T-TEL) programme is supporting the Ministry of
Education, the National Council for Tertiary Education and the Ghana Education Service, including all
their related agencies to transform the ITE programme in all public colleges of education in Ghana. ABBREVIATIONS CoE
College of Education
CS
Circuit Supervisor
DBE
Diploma in Basic Education
GEO
Girls Education Officer
GES
Ghana Education Service
ICT
Information Communication Technologies
ITE
Initial Teacher Education
JHS
Junior High School
LP
Lower Primary
PCK
Pedagogical Content Knowledge
P-D-R
Plan Together-Do- Reflect Together
RPK
Relevant Previous Knowledge
SEM
Semester
SPA
School Partnership Adviser (T-TEL)
STPD
Student Teacher Professional Development
T
Theme
TLA
Teaching Learning Adviser (T-TEL)
TLM
Teaching Learning Materials
TP
Teaching Practice
TPC
Teaching Practice Coordinator
T-TEL
Transforming Teacher Education and Learning
UP
Upper Primary
Y1, Y2, Y3
Year One, Year Two, Year Three Student Teacher Handbook for Teaching Pratice 4 INTRODUCTION FOREWORD The structuring of three teaching practice periods for Student Teachers while in college, the
provision of handbooks to guide the three teaching practice periods and the training of teacher
educators and related stakeholder groups are some of the ways in which T-TEL is partnering with
these institutions to transform the initial teacher education programme. In this initiative, there are
three handbooks for College Tutors, Mentors and Student Teachers respectively, each containing
information on the three structured and complementary teaching practice activities. The handbooks
provide a vitally important resource for Student Teachers as well as their Mentors and College
Tutors who are directly involved in supporting and directing Student Teachers throughout the
teaching practice periods. It is expected that by the end of the three-year Diploma in Basic
Education programme at the Colleges of Education, having gone through the three structured
teaching practice sessions, Student Teachers will be competent and capable of providing quality
education for children in basic schools across Ghana. Transforming Teacher Education and Learning
www.t-tel.org 5 Transforming Teacher Education and Learning i Year 1 - 3: Teaching Practice Student Teacher Handbook for Teaching Pratice ACKNOWLEDGEMENTS This handbook has been developed as a result of the financial support of the Government of the
United Kingdom through its aid agency, the Department for International Development (DFID), with
technical assistance from Cambridge Education and Mott MacDonald Limited, all in the United
Kingdom. We wish to acknowledge and thank the many institutions in Ghana that contributed in
various ways to making the production of this handbook possible: The Ministry of Education
The National Council for Tertiary Education
The National Teaching Council
The Ghana Education Service
Teacher Education Division
University of Cape Coast
University of Education, Winneba
Colleges of Education
Principals of Colleges of Education All other governmental and non-governmental institutions and agencies that were consulted during
the writing of this handbook are also duly acknowledged. We also acknowledge the contributions of the following persons who made available written
materials, ideas and time to write this handbook: Emmanuel T. Aboagye, James Adefrah, Christine
Adu-Yeboah, Samuel Agyemang, Daniel Amofa, Harriet Amui, Augustus Asah Awuku, Eric Ofosu
Dwamena, Jonathan Fletcher, Emma Fynn, Issa Musah, Robert Owusu-Ansah, Cynthia Yalley and
Marion Young. Special thanks are in order for our publishers, who were extremely patient and
worked tirelessly to produce the handbooks in record time. The views expressed in this handbook are those of the writing team and do not represent the views
of DFID, Cambridge Education or Mott MacDonald Limited. Student Teacher Handbook for Teaching Pratice 6 INTRODUCTION Introduction This Teaching Practice (TP) Handbook has been prepared to provide you with a set of topics and
activities that you will undertake before, during and after your three TP periods in college. There
is also a Teaching Practice Handbook for College of Education (CoE) Tutors and a Teaching Practice
Handbook for Mentors. Note that Student Teacher is the terminology used consistently throughout the T-TEL Teaching
Practice Handbooks, and is also used when referring to the DBE trainee and the Year 3 Student
Teacher. The topics and activities are designed to focus on key skills and competencies you will
develop step-by-step throughout the teaching practice cycle. Each topic and activity will be planned
to fit into the daily routines of the school week. You will undertake all the activities described in this
Handbook at the designated time. In addition your Tutor or the head teacher of your teaching
practice school may assign other activities as part of the normal teaching practice timetable to
further expand your skills and enrich your teaching practice experience. Some activities will be
undertaken during your non-contact time, for example lesson planning and preparing teaching and
learning materials (TLM). Other activities are planned for you to undertake during the classroom
contact time, either observing the teacher and pupils or for you to practise your own teaching skills. Each activity has the following format: summary of the task; Plan Together; Do; Reflect Together
(P-D-R). The P-D-R activities provide a step-by-step description of the tasks and whom you should
work with during each activity – either your TP partner, Tutor or Mentor/class teacher. In some activities there is a self-assessment or peer assessment task. At the end of each Year 2 and Year 3 topic you will evaluate your work with your Tutor or Mentor
and complete a simple check box to reflect on where you need to improve in your teaching skills. As a self-evaluation question, each day you should answer the question: ‘What new thing have
I learnt today?’ I learnt today?’
In some activities there is additional information that will help you to undertake the task. Introduction In the Annexes at the end of the Student Teacher Handbook, there is additional information that
you will need to refer to during your teaching practice including Teacher Competencies (Annex 1),
Lesson Plan (Annex 2), Lesson Observation format (Annex 3), Assessment Procedures (Annex 4),
Protection Strategies for Student Teachers on Teaching Practice (Annex 5), and an example page
from a TP Journal (Annex 6). Evaluation of Performance
This week my teaching practice activities were VERY SUCCESSFUL/SATISFACTORY/COULD BE BETTER
These are the main points for me to improve:
Student Teacher: _________________________________________________________________________________
Mentor: _______________________________________________________
Date: ___________________________ Evaluation of Performance Evaluation of Performance Evaluation of Performance
This week my teaching practice activities were VERY SUCCESSFUL/SATISFACTORY/COULD BE BETTER
These are the main points for me to improve:
Student Teacher: _________________________________________________________________________________
Mentor: _______________________________________________________
Date: ___________________________ Evaluation of Performance
This week my teaching practice activities were VERY SUCCESSFUL/SATISFACTORY/COULD BE BETTER
These are the main points for me to improve: In some activities there is additional information that will help you to undertake the task. In the Annexes at the end of the Student Teacher Handbook, there is additional information that
you will need to refer to during your teaching practice including Teacher Competencies (Annex 1),
Lesson Plan (Annex 2), Lesson Observation format (Annex 3), Assessment Procedures (Annex 4),
Protection Strategies for Student Teachers on Teaching Practice (Annex 5), and an example page
from a TP Journal (Annex 6). Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning
www.t-tel.org 7 i Year 1 - 3: Teaching Practice YEAR 1, YEAR 2 AND YEAR 3 TEACHING PRACTICE ARRANGEMENTS During the three-year Diploma in Basic Education (DBE) programme you, as a Student Teacher, will
be provided with three structured and complementary teaching practice experiences through which
you will observe and practise new skills. You will be assessed in each of the three TPs and the scores
will be included as part of your final TP grade. Y1 Observation TP: In DBE Year 1, you will join a host school for two weeks during the vacation
between Semesters 1 and 2 to undertake school and classroom observation. Y2 On-Campus TP: In DBE Year 2, you will actively participate in On-Campus demonstration and
teaching practice lessons for three weeks in Semester 1 and three weeks in Semester 2. Whenever
possible your Tutor will arrange for the peer teaching and individual teaching to take place with a
class of pupils in one of the schools close to your CoE. Y3 Off-Campus TP: In DBE Year 3, you will undertake a 12-week Off-Campus Teaching Practice
(school-based) during Semester 1. During the Y3 teaching practice you will be assigned to a Mentor
in the TP school. You will observe the Mentor teaching some lessons and you will co-teach some
lessons with your TP Partner and the Mentor, sharing parts of the lesson among you. You will plan
the lesson together and then reflect on the lesson together. For each of the three TPs, there will also be one week of pre-TP activities and one week of post-TP
activities in your CoE organised by your Tutor. In the pre-TP activities, the TPC and Tutors will help
you to prepare and plan; the post-TP activities will provide you with an opportunity to reflect and
evaluate your TP experience with your TPC, Tutors and other Student Teachers. HOW TO KEEP THE TP JOURNAL HOW TO KEEP THE TP JOURNAL
•
Keep your TP Journal up-to-date and available at all times throughout the teaching practice
periods. •
Use your TP Journal with your Tutor, Mentor and TP partner following the three P-D-R steps
for each activity. •
Clearly write the date, the activity number and the title of the activity at the start of the
section. Your Tutor and Mentor will have the same activities in their respective Handbooks. •
Plan and work together with your TP partner or TP buddy, but both of you should maintain
your own TP Journal, recording and reflecting your own individual ideas. •
Keep your TP Journal in a tidy condition with anything you record entered neatly. You may
want to draft your entries on paper first and then write in the TP Journal when ready. Y1 Observation TP: In the pre-TP week of orientation Your Tutor will instruct you on how to use the
TP Journal during your 10-day classroom observation. Complete the TP Journal entries each day of
the Observation TP following the instructions for each activity in the Student Teacher Handbook. You should also show the TP Journal to the head teacher and class teacher of your host school as
they may want to check your entries. Your TP Journal will be used by you and your Tutor in the first
week of Y1 Semester 2 during the post-TP activities. Y2 On-Campus TP: During the Pre-On-Campus TP week your Tutor will give you instructions to
ensure that you know how to use the TP Journal for the peer-teaching and individual teaching
practice activities. Complete the TP Journal entries following the instructions for each activity in
the Student Teacher Handbook. If you conduct your peer or individual TP in a school show the TP
Journal to the head teacher and class teacher of your host school as they may want to check your
entries. Your TP Journal will be used by you and your Tutor in the post-TP activities week. Y3 Off-Campus TP: Your Mentor will ensure that you maintain your TP Journal in accordance with
the activities described in this Student Teacher Handbook. Your TP Journal entries will be used as
the basis for weekly Reflective Practice meetings and may also be used in the assessment of your
TP performance. YEAR 1, YEAR 2 AND YEAR 3 TEACHING PRACTICE ARRANGEMENTS TP Timetable
Pre-TP Activities
TP Activities
Post-TP Activities
Y1 Observation
TP
Before end of Y1 SEM1
- on campus
2 weeks (10 days) during
the end of Y1 SEM1
vacation
- in schools
First week of Y1
SEM2
- on campus
Y2 On-Campus
TP
1 week in Y2 SEM1
- on campus
3 weeks in Y2 SEM1
3 weeks in Y2 SEM2
- on campus/in schools
1 week in Y2 SEM2
- on campus
Y3 Off-Campus
TP
First week of Y3 SEM1
- on campus
12 weeks in Y3 SEM1
- in schools
Last week of Y3
SEM1
- on campus Student Teacher interaction with pupils in their classrooms Student Teacher interaction with pupils in their classrooms
It is important for you to be given opportunities to interact with pupils in their classrooms
throughout each stage of the DBE programme. This classroom-based interaction with pupils will be
a core element of your professional development. In addition to the Y1, Y2 and Y3 TP where you
should work with pupils at every opportunity, your Tutor will also arrange demonstration lessons
either in the demonstration school or neighbouring schools close to your CoE. On some occasions,
your Tutor will arrange for some pupils to be brought into the CoE for a demonstration lesson or for
peer and individual teaching practice. Student Teacher Handbook for Teaching Pratice 8 INTRODUCTION Student Teacher interaction with other Student Teachers – TP partner or TP buddy
It is important to have a TP partner to work with so that you can plan together and reflect together
on the activities. In Y1 TP, you will be posted to a school close to your home. You may be the only
Student Teacher in the school. If this is the situation ask the head teacher of the observation school
to assign a teacher on the staff to be your Observation Teaching Practice buddy. The assigned buddy
can take the place of the TP partner in any of the activities where a partner is required for planning
and reflection. USE OF THE TEACHING PRACTICE JOURNAL (TP JOURNAL) You will be required to keep a regular and systematic record of teaching practice tasks, activities
and assignments for the full three years of the DBE programme, as described in each section of this
Student Teacher Handbook. The TP Journal will be your personal record of your own professional
development and learning experiences and will be used by your Tutor and/or Mentor for the
purpose of assessing your progress and performance as a Student Teacher (see Annex 6, Example
Page from a TP Journal). CoE Tutor and Link Tutor: CoE Tutors concentrate on content acquisition and project work as well as Student Teach CoE Tutors concentrate on content acquisition and project work as well as Student Teachers’
practical teaching. CoE Tutors must: practical teaching. CoE Tutors must: • guide Student Teachers through the Y1, Y2 and Y3 pre- and post-TP activities; • guide Student Teachers through the Y1, Y2 and Y3 pre- and post-TP activities;
• guide Student Teachers through the Y2 On-Campus TP activities;
• visit Student Teachers during Y3 TP to guide their progress and monitor their performance;
• assist Student Teachers with their Y3 TP project work;
• encourage Student Teachers to complete their Y3 project work as scheduled;
• provide subject matter content knowledge support for Student Teachers;
• help Student Teachers with appropriate skills and competencies for teaching each subject. • guide Student Teachers through the Y2 On-Campus TP activities; • visit Student Teachers during Y3 TP to guide their progress and monitor their performance;
• assist Student Teachers with their Y3 TP project work;
• encourage Student Teachers to complete their Y3 project work as scheduled;
• provide subject matter content knowledge support for Student Teachers;
• help Student Teachers with appropriate skills and competencies for teaching each subject • visit Student Teachers during Y3 TP to guide their progress and monitor their performance; • assist Student Teachers with their Y3 TP project work; • encourage Student Teachers to complete their Y3 project work as scheduled; • provide subject matter content knowledge support for Student Teachers; • help Student Teachers with appropriate skills and competencies for teaching each subjec CoE Link Tutors work closely with the Mentors when they visit Student Teachers to resolve any
academic, professional and social problems that affect Student Teachers, and to discuss and share
ideas on the progress of Student Teachers. ROLES AND RESPONSIBILITIES OUTLINED FOR TEACHING PRACTICE Student Teachers: In Y1 TP you will work closely with the class teacher/TP buddy in your
observation school. In Y2 your Tutor will guide you through the TP activities. During the Y3 TP,
co-operate with the Mentor and your TP partner/buddy and also interact actively with other staff
and pupils in the host school. During the three TP periods your main areas of responsibility include the following (those specific to
Y3 TP are indicated in parenthesis): • plan, prepare and present a scheme of work and expanded scheme of work (lesson notes) to the
Mentor for guidance regularly and prior to the Link Tutor’s supervision visit (Y3 TP); • teach using the prepared expanded scheme of work (Y3 TP); • participate actively in extra-curricular activities, e.g. sports; • participate actively in extra-curricular activities, e.g. sports;
• ensure that discipline is maintained in the class and the school as a whole (Y3 TP); • pay individual attention to pupils’ learning needs; • give and mark pupils’ class exercises, assignments/homework and provide feedback through
discussion and corrections accordingly (Y3 TP);
• ensure that instructional periods are used to improve teaching and learning of all pupils (Y3 TP); • give and mark pupils’ class exercises, assignments/homework and provide feedback through • ensure that instructional periods are used to improve teaching and learning of all pupils (Y3 • ensure that instructional periods are used to improve teaching and learning of all pupils (Y3 TP);
• ensure good relationships with Student Teachers, Mentors, community members and others;
• create a learning environment conducive to the active learning of all pupils (Y3 TP); • acquire additional skills and knowledge from the Mentor/class teacher as you observe them
teach various subjects and lessons in class and ask questions for clarification where necessary;
i
l id
if
hi
kill
d
i
h
d
i
d
k • acquire additional skills and knowledge from the Mentor/class teacher as you observe them
teach various subjects and lessons in class and ask questions for clarification where necessary;
• actively identify teaching skills and competencies that you need to improve on and work on
these following the advice given by the Mentor and Tutor. • actively identify teaching skills and competencies that you need to improve on and work on
these following the advice given by the Mentor and Tutor. HOW TO KEEP THE TP JOURNAL When your Tutor visits you at the TP school they will ask to see your TP Journal. Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 9 i Year 1 - 3: Teaching Practice CoE Tutor and Link Tutor: Link Tutors must: • liaise between CoEs and TP schools;
• assess Student Teachers’ teaching to achieve the basic goals of teaching practice;
• assist Student Teachers to carry out Action Research for their project work;
• give feedback to Tutors on challenges Student Teachers may face which may require assistance;
• provide Student Teachers with material resources, such as reference books from the library;
• oversee the Mentorship exercise in a cluster of schools;
• collaborate with Lead Mentors/Mentors on Student Teacher professional development; • collaborate with Lead Mentors/Mentors on Student Teacher professional development;
• resolve conflict that may arise among Student Teachers and others at the school level;
• ensure all reports are received from Tutors who have supervised Student Teachers on TP. Student Teacher Handbook for Teaching Pratice 10 INTRODUCTION Teaching Practice Coordinator (TPC): The TPC is a member of the Teaching Practice Implementing
Committee and is responsible for: Teaching Practice Coordinator (TPC): The TPC is a member of the Teaching Practice Implementing
Committee and is responsible for: • selection of schools for your TP placement; • administration arrangements for posting you to the TP schools, including providing letters of
introduction; • pastoral arrangements, including ensuring provision of suitable accommodation through liaiso
with district and community officials; • monitoring and evaluation of your teaching practice placements; • assessment of Student Teacher performance during TP. See also the TPC Guidelines (T-TEL) for additional details on the roles and responsibilities of the TPC. Head Teacher/Lead Mentor: the Lead Mentor will provide you with an introduction and
orientation to the school and will introduce you to classes and to teachers who are trained as
Mentors. The Lead Mentor will be familiar with the TP guidelines including the need to ensure
your professional and pastoral well-being during the TP period. CoE Tutor and Link Tutor: Your Mentor should co-operate and work closely with the Link Tutor and Tutor, and should: • endeavour to be regular and punctual at school to support Student Teachers;
• supervise and observe Student Teachers’ work in the classroom;
• assist Student Teachers to plan and organise their lessons;
• help Student Teachers to choose their Action Research topic and carry out project work;
• guide Student Teachers to practise classroom organisation, control and management;
• help Student Teachers to prepare appropriate TLMs for lesson delivery;
• arrange for regular meetings with Student Teachers to discuss issues regarding teaching
practice;
• write a comprehensive report on Student Teachers’ performance during their teaching practice; • endeavour to be regular and punctual at school to support Student Teachers;
• supervise and observe Student Teachers’ work in the classroom;
• assist Student Teachers to plan and organise their lessons;
• help Student Teachers to choose their Action Research topic and carry out project work;
• guide Student Teachers to practise classroom organisation, control and management;
• help Student Teachers to prepare appropriate TLMs for lesson delivery;
• arrange for regular meetings with Student Teachers to discuss issues regarding teaching
practice;
h
d
h
’
f
d
h
h • write a comprehensive report on Student Teachers’ performance during their teaching practice;
• hold regular meetings with the Lead Mentor to address issues relating to Student Teachers. See also the Y1 TP Class Teacher Guidelines (T-TEL) for additional details on their TP roles and
responsibilities in supporting you during the Y1 School Observation TP. Girls Education Officer (GEO): The GEO has responsibility for ensuring that girls are given equita-
ble access to good quality education opportunity by visiting schools on a regular basis to advise and
strengthen gender-sensitive approaches. They will also be responsible for implementing and moni-
toring the Gender Policy and Sexual Harassment Framework for CoEs, and the related Guidelines for
Protection Strategies for Student Teachers on Teaching Practice (see Annex 5). During your teaching practice, you will learn how to adopt gender-sensitive approaches to teaching
and learning. The GEO may visit your school during the teaching practice and should discuss with
you how to improve your skills in gender-sensitive education. See also the GEO Guidelines (T-TEL) for additional details on their TP roles and responsibilities. See also the GEO Guidelines (T-TEL) for additional details on their TP roles and responsibilities. Student Teacher Handbook for Teaching Pratice CoE Tutor and Link Tutor: The following are the main
responsibilities of the Lead Mentor: • organise orientation for Student Teachers; • arrange and hold regular Reflective Practice meetings with Mentors and Student Teachers;
h h
f
k
d
d
h • co-operate with the activities of Mentors, Link Tutors, Tutors and Student Teachers at
school level; • monitor and evaluate the roles and responsibilities of Mentors;
• ensure that Mentors are punctual and remain at post during school hours;
• hold regular meetings with Mentors to monitor the progress of Student Teachers;
• provide guidance, counselling and professional development of Mentors and Student Teachers
• assume overall responsibility for the welfare of Student Teachers;
• ensure that Student Teachers are punctual and make good use of class contact hours;
• provide Student Teachers with appropriate TLMs to assist them in lesson preparation;
• assess the performance of Student Teachers in Y3 TP and report back to the CoE. See also the Lead Mentor Guidelines (T-TEL) for additional details on their TP roles and
responsibilities. Mentor: The Mentor will work closely alongside you throughout the full period of your Y3
Off-Campus TP, providing you with professional guidance and encouragement, acting as a role
model for you to follow and coordinating classroom activities to gradually give you more and
more responsibility as your skills and confidence grow. The Mentor will be guided by the Mentor
Handbook which parallels the activities detailed in your Student Teacher Handbook. The Mentor will
conduct assessment as part of the Reflective Practice process in Y3 TP. Your final assessment will be
conducted by the Tutor. 11 Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning i Year 1 - 3: Teaching Practice Mentors focus on planning of lessons and practical teaching in the school providing psychological
and professional support in real classroom teaching situations. ASSESSMENT OF STUDENT TEACHER PERFORMANCE In Teaching Practice, assessment is the process by which the quality of your work or performance
during the three TPs will be judged. The Assessment Procedures are summarised in Annex 4. The Y1, Y2 and Y3 teaching practices will be assessed based on the Teacher Competencies set out in
the GES Policy with which you will be familiar (see Annex 1). Competencies develop from one year to
the next and the TP tasks and activities are designed to gradually develop the range of competen-
cies that are needed to become a qualified teacher. You are required to monitor your own progress
against the Teacher Competencies and review them regularly with your Tutor and Mentor to identify
the areas of Teacher Competencies that you need to work on to improve. CoE Tutor and Link Tutor: Circuit Supervisor (CS): The CS has responsibility for monitoring and quality assurance in schools. The CS must therefore: Circuit Supervisor (CS): The CS has responsibility for monitoring and quality assurance in schools. The CS must therefore: • provide support in respect of the availability of curriculum materials, e.g. syllabus for Student
Teachers; • enquire of Student Teachers whether they are facing any challenges in their teaching practic
and provide support accordingly; • ensure that Student Teachers have cordial relationships with staff and community members
• motivate Student Teachers to be committed and dedicated to their tasks. See also the CS Guidelines (T-TEL) for additional details on their TP roles and responsibilities. 12 Student Teacher Handbook for Teaching Pratice INTRODUCTION Establishing and maintaining good relationships between mentor and student teacher is really important in teaching practice Establishing and maintaining good relationships between mentor and student teacher is really important in teaching practice Student Teacher Handbook for Teaching Pratice GUIDELINES FOR PROTECTION STRATEGIES FOR STUDENT TEACHERS ON
TEACHING PRACTICE The Ghana Education Service (GES) recognises the importance of ensuring the well-being and safety
of Student Teachers, particularly female Student Teachers, during their teaching practice. The GES
Gender Policy and Sexual Harassment Framework for CoEs will be implemented in all TP schools,
ensuring that the necessary arrangements and safeguards are in place. As a Monitoring and Evaluation component of the T-TEL Project, a Matrix of Gender-Responsive
Competencies has been developed, and is included in this Handbook as Guidelines for Protection
Strategies for Student Teachers on Teaching Practice (see Annex 5). Your Tutor will familiarise you with the Gender-Responsive Competencies and how you can use
these as strategies to protect yourself and other Student Teachers while on teaching practice. It will
be your responsibility to ensure you are familiar with these strategies and to safeguard your own
welfare and that of your fellow-Student Teachers. Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 13 1 Year 1 - 3: Teaching Practice Student Teacher Handbook for Teaching Pratice 14 YEAR 1 • SCHOOL OBSERVATION TP Y1 Observation Teaching Practice Activities •
pre-TP observation activities to be undertaken by the Tutors in pre-observation orientation
before the end of Semester 1, On-campus for all Year 1 Student Teachers; •
pre-TP observation activities to be undertaken by the Tutors in pre-observation orientation
before the end of Semester 1, On-campus for all Year 1 Student Teachers; •
one topic for each of the ten TP observation days during the Y1 end of Semester 1 vacation,
with two to three activities to be undertaken by the Student Teacher and recorded in their TP
Journal •
post-TP observation activities to be undertaken by the Tutors during the first week of
Semester 2, On-campus for all Year 1 Student Teachers. TOPICS AND ACTIVITIES FOR YEAR 1 OBSERVATION TEACHING PRACTICE TOPICS AND ACTIVITIES FOR YEAR 1 OBSERVATION TEACHING PRACTIC Y1 Pre-TP Topic: Pre-Observation Activities (college-based)
Purpose: This activity will prepare you for the two-week Observation Teaching Practice
Pre-TP Activity 1: Orientation for Student Teachers – School Observation Checklist
Pre-TP Activity 2: Using the TP Journal Pre-TP Activity 3: Establishing good working relationships DO In your TP Journal, make a checklist of what you want to observe during the two-week
Observation TP. Purpose: In Year 1 Observation Teaching Practice Student Teachers will be able to demonstrate communication and interpersonal skills, technical/
pedagogical skills, subject mastery skills and classroom management and leadership skills. Host schools for Y1 Observation TP will provide professional and pastoral support for Student
Teachers in alignment with the Y1 Observation TP Guidelines and instructions from the Student
Teacher’s CoE. The CoE Tutors will conduct pre- and post-Y1 Observation TP activities and, where possible,
supervision visits to Observation TP schools in accordance with the Tutor Handbook. The TPC will undertake all administration and communication with the host schools for Y1
Observation TP in accordance with the TPC Guidelines. Teaching Practice Framework: Year 1 Observation Teaching Practice
Pre-Observation Activities (college-based)
Topic 1. Familiarisation with School Organisation
Topic 2. Observing a Lesson – What to look for
Topic 3. Lesson Planning
Topic 4. Stimulating and Sustaining Pupils’ Interest
Topic 5. Developing the Lesson
Topic 6. Classroom Organisation, Management and Control
Topic 7. Teacher Talk: Assessing Pupils’ Learning Achievement – Communication and
Questioning Skills
Topic 8. Co-Curriculum Activities
Topic 9. Professional Commitment of the Teacher
Topic 10. School Leadership
Post-Observation Teaching Practice Activities (college-based) Teaching Practice Framework: Year 1 Observation Teaching Practice
Pre-Observation Activities (college-based)
Topic 1. Familiarisation with School Organisation
Topic 2. Observing a Lesson – What to look for
Topic 3. Lesson Planning
Topic 4. Stimulating and Sustaining Pupils’ Interest
Topic 5. Developing the Lesson
Topic 6. Classroom Organisation, Management and Control
Topic 7. Teacher Talk: Assessing Pupils’ Learning Achievement – Communication and
Questioning Skills
Topic 8. Co-Curriculum Activities
Topic 9. Professional Commitment of the Teacher
Topic 10. School Leadership
Post-Observation Teaching Practice Activities (college-based) Transforming Teacher Education and Learning
www.t-tel.org 15 Transforming Teacher Education and Learning 1 Year 1 - 3: Teaching Practice How to Prepare for the School Observation TP When you have completed these three Y1 Pre-TP activities with your Tutor you will have: • prepared a school observation checklist; • understood how to use the TP Journal and have your TP Journal ready to take with you to yo
Observation TP; • discussed how to establish good working relationships with the head teacher, classroom
teachers and pupils in your host school. You will also have a good understanding of how to
conduct yourself in a professional manner in the community where the school is located. 1 Pre-TP Activity 1: Orientation for Student Teachers – School Observation Check PLAN TOGETHER
Before the end of Y1 Semester 1 your Tutor will help you to prepare for your two-week
Observation Teaching Practice. First, with your TP partner think about the school information you will need, and the things you
want to observe in the school. For example you will need information about the school rules,
the classroom timetable, the teachers in the school, and the pupils in your class and the local
community. What do you need to know before you arrive at the school and what will you need to
find out about as soon as you arrive at the school? For the classroom observation you will need a
checklist of what to observe in the lessons (see Annex 3). DO
Make a checklist of the most important information you will need (a) before you arrive at
the school, and (b) when you first arrive at the school. Write down where you think you can
find out this information. REFLECT TOGETHER
Compare your checklist with the checklist your TP partner has made. • Are there any ideas you thought of that your TP partner did not think of? • Are there any ideas your TP partner thought of that you did not? Listen to ideas from other members of your class and add any new ideas to your list. In your TP Journal, make a checklist of what you want to observe during the two-week
Observation TP. Student Teacher Handbook for Teaching Pratice Y1 Pre-TP Activity 2: Using the TP Journal Y1 Pre-TP Activity 2: Using the TP Journal
PLAN TOGETHER
You will keep a TP Journal covering all three TPs (Y1, Y2 and Y3) as a record of all the important
information and activities. To start your TP Journal, discuss with your TP partner and choose the
top ten points on your school information checklist and the top ten points on your classroom
observation checklist. DO
REFLECT TOGETHER
Share the TP Journal entries with others in your class and exchange ideas. Y1 Pre-TP Activity 3: Establishing good working relationships
PLAN TOGETHER
Review the GES requirements for Teaching Practice, covering professional and personal behaviour
of Student Teachers. Discuss each one with your TP partner. It is important to realise that in
Ghanaian society the status of a teacher, including a Student Teacher, is highly respected and as such
you will be a role model to the pupils in the school. Think about who you will be interacting with dur-
ing the Observation TP. DO
With your class, brainstorm all the positive ways of making and keeping good relationships with
adults and children in the Observation TP school. REFLECT TOGETHER
With your TP partner think of 2-3 difficult situations you might experience and how you can ensure
that you behave in a positive way in order to maintain good relationships. Consider the three case
study examples below and discuss with your TP partner how you should respond in these situations. Case Studies for discussion - what would you do in each of these situations? Case Study 1: When you arrive at the school the classroom is locked and you do not have a key. Case Study 2: Three boys arrive late for the class and have forgotten to do their homework. Case Study 3: During the lessons you notice that the teacher rarely asks any questions to the girls
in the class and sometimes the teacher uses corporal punishment to discipline pupils who are not
paying attention to the lesson. Self-Assessment Questions – to guide your learning and to help prepare for the observation TP
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ti
Write your school information checklist and classroom observation checklist in your
TP Journal. In your TP Journal, make a list of all the negative behaviours that must be avoided. PLAN TOGETHER REFLECT TOGETHER
With your TP partner think of 2-3 difficult situations you might experience and how you can ensure
that you behave in a positive way in order to maintain good relationships. Consider the three case
study examples below and discuss with your TP partner how you should respond in these situations. Case Studies for discussion - what would you do in each of these situations? Case Study 1: When you arrive at the school the classroom is locked and you do not have a key. Case Study 2: Three boys arrive late for the class and have forgotten to do their homework. Case Study 3: During the lessons you notice that the teacher rarely asks any questions to the girls
in the class and sometimes the teacher uses corporal punishment to discipline pupils who are not
paying attention to the lesson. Self-Assessment Questions – to guide your learning and to help prepare for the observation TP
ask yourself these questions:
1. How well prepared do you feel for the School Observation TP? 2. What are the two most important ideas you have learned from doing these pre-TP tasks? NOTE for the attention of Student Teachers: the activities included in the following Y1 TP Topics 1-10 will be
conducted by you, as Student Teachers, in the absence of your Tutors during the 10 days of School Observation TP. Therefore your Tutor should familiarise you with the Topic 1-10 activities during Y1 Pre-TP. Your Tutor will explain
how your Y1 TP observation will be assessed. You should remind yourself of the assessment procedures that are
included in Annex 4 of this Student Teacher Handbook. REFLECT TOGETHER REFLECT TOGETHER
Compare your checklist with the checklist your TP partner has made. • Are there any ideas you thought of that your TP partner did not think of? • Are there any ideas your TP partner thought of that you did not? Listen to ideas from other members of your class and add any new ideas to your list. Student Teacher Handbook for Teaching Pratice 16 Y1 Pre-TP Activity 2: Using the TP Journal REFLECT TOGETHER NOTE for the attention of Student Teachers: the activities included in the following Y1 TP Topics 1-10 will be
conducted by you, as Student Teachers, in the absence of your Tutors during the 10 days of School Observation TP. Therefore your Tutor should familiarise you with the Topic 1-10 activities during Y1 Pre-TP. Your Tutor will explain
how your Y1 TP observation will be assessed. You should remind yourself of the assessment procedures that are
included in Annex 4 of this Student Teacher Handbook. Transforming Teacher Education and Learning
www.t-tel.org 17 Transforming Teacher Education and Learning 1 Year 1 - 3: Teaching Practice Y1 TP Topic 1: Familiarisation with School Organisation
Purpose: In this Topic you will become familiar with the way the school is organised and managed
Activity 1.1: School Information Checklist
Activity 1.2: Observe 2-3 daily routines and how they are managed
Activity 1.3: Classroom Rules
Competencies: Student Teachers observe the teacher demonstrating:
(i) Effective classroom management: Competency 13(a-d) and 11(b); (ii) Gender sensitivity:
Competency 11(b); (iii) Gender stereotype: Competency 12(a-c) (see Annex 1) How is the TP School Organised? When you have completed these Y1 Day 1 TP activities in your observation school you will have: When you have completed these Y1 Day 1 TP activities in your observation school you will ha • prepared a check list of basic information about the school; • prepared a check list of basic information about the school; • written notes on 2-3 daily routines and how they are managed; • found out the classroom rules. Y1 TP Activity 1.1: Make a checklist of basic information about the TP School PLAN TOGETHER
Brainstorm with your TP partner on the kind of information you want to know about the school
and who you need to contact e.g. number of pupils (boys and girls), number of teachers (male and
female), starting time and closing time, daily timetable, school rules for teachers and pupils, map of
the school, etc. Look at the checklist you made with your Tutor during the pre-TP activities and compare with what
you brainstormed. Have a checklist ready to ask the teacher and head teacher for information when
you arrive at the school. PLAN TOGETHER
Brainstorm with your TP partner on the kind of information you want to know about the school
and who you need to contact e.g. number of pupils (boys and girls), number of teachers (male and
female), starting time and closing time, daily timetable, school rules for teachers and pupils, map of
the school, etc. PLAN TOGETHER
Brainstorm with your TP partner on the kind of information you want to know about the school
and who you need to contact e.g. number of pupils (boys and girls), number of teachers (male and
female), starting time and closing time, daily timetable, school rules for teachers and pupils, map of
the school, etc. Look at the checklist you made with your Tutor during the pre-TP activities and compare with what
you brainstormed. Have a checklist ready to ask the teacher and head teacher for information when
you arrive at the school. Student Teacher Handbook for Teaching Pratice DO DO
Select the 2-3 routines that you want to observe. Observe these routines and make notes about how
they are organised and how the girls and boys participate in them. Compare your notes with your
TP partner’s notes. REFLECT TOGETHER REFLECT TOGETHER
Did you get all the information you required from the class teacher/head teacher? What information
was missing? Discuss with your TP partner/TP group the criteria you used for selecting the three
most important things you found out about the school that will help you during the two-week
observation TP. Was there anything that was more important than those you first recorded? REFLECT TOGETHER
Did you get all the information you required from the class teacher/head teacher? What information
was missing? Discuss with your TP partner/TP group the criteria you used for selecting the three
most important things you found out about the school that will help you during the two-week
observation TP. Was there anything that was more important than those you first recorded? Student Teacher Handbook for Teaching Pratice 18 Y1 TP Activity 1.2: Observation of 2-3 daily routines and how they are manage PLAN TOGETHER PLAN TOGETHER
Identify routines in the school and the duration of each routine, e.g. cleaning the compound,
conducting morning assembly, calling the register, checking teacher and pupil attendance and
punctuality, dress code, etc. Make a list of the main school routines you are interested in finding
out about. Identify the people in charge of daily routines in the school and the people who are
involved in the routines. What leadership roles are the pupils given? Brainstorm with your TP partner on the reasons for the routines. Identify the rules governing the
routines and how they are enforced. Discuss the strengths and limitations of daily routines. REFLECT TOGETHER What criteria did you use to select the routines you observed? Discuss with your TP partner any
differences and similarities in the information you recorded on the routines you observed. What
challenges do pupils face when undertaking the routines? Are the challenges different for girls and
boys – describe any such differences? How can pupils overcome these challenges? Y1 TP Activity 1.3: Classroom rules and observations on how they are implemented PLAN TOGETHER
Request information on classroom rules from the head teacher and/or TP classroom teacher. Brainstorm with your TP partner on the rules you think are most important and state why they are
important. Brainstorm with your TP partner on how the rules should support teaching and learning. Find out how the rules are communicated to the pupils. DO
Observe a class of pupils throughout the day and record in your TP Journal any of the rules you
see used in the classroom or in the playground. Observe how the rules are enforced and the
consequences for pupils if they break the rules. Classroom
Rule
Why this rule is important
How this rule is enforced
Consequences if the
rule is broken
1
2
3
4
In your TP Journal make a table (below) for the four most important rules you observe. REFLECT TOGETHER
Reflect together with your TP partner on the effectiveness of classroom rules and why classroom
rules are broken. Reflect together on whether the classroom rules supported teaching and learning. Reflect on the strengths and limitations of the classroom rules. REFLECT TOGETHER
Reflect together with your TP partner on the effectiveness of classroom rules and why classroom
rules are broken. Reflect together on whether the classroom rules supported teaching and learning. Reflect on the strengths and limitations of the classroom rules. T TOGETHERl
together with your TP partner on the effectiveness of classroom rules and why classroom
e broken. Reflect together on whether the classroom rules supported teaching and learning. l
on the strengths and limitations of the classroom rules. Transforming Teacher Education and Learning
www.t-tel.org 19 1 Year 1 - 3: Teaching Practice Competencies: Student Teachers observe the teacher demonstrating:
(i) Strategies to start and close the lesson: Competencies 1 and 15; (ii) Effective classroom
management: Competency 13(a-d); (iii) Pair/Group work: Competency 7(a-c); (iv) Mixed Ability:
Competency 10(b); (see Annex 1) How to Observe a Lesson
When you have completed th How to Observe a Lesson
When you have completed these Y1 Day 2 TP activities in your observation school you will have: • observed a lesson using the lesson observation checklist you developed in college; • observed how the lesson started, how it was delivered, how the lesson ended and what the
teacher did after the lesson; • observed how the teacher supported and involved all pupils in the lesson. You will have
interacted with at least two pupils during the lesson. Y1 TP Activity 2.1: The Start of the Lesson: How to introduce the lesson Y1 TP Activity 2.1: The Start of the Lesson: How to introduce the lesson PLAN TOGETHER
In the pre-observation orientation, the Tutor guided you to make a list of the things to look for at
the start of a lesson (see Lesson Observation Checklist, Annex 3). These include how the pupils
come into the classroom, what the teacher does at the start of the lesson such as marking the
register, getting the pupils seated, getting the pupils’ attention and so on. The list of things to be
observed corresponds to the Teacher Competencies (see Annex 1). Think about how you will keep a record of your TP observations in your TP Journal, describing what
happens at the start of the lesson. Think about the 5-6 most important details you should observe at
the start of the lesson. Check Teacher Competency 1 (see Annex 1) for some key ideas. DO
Observe the start of the lesson using the checklist you and your TP partner have developed. Put a
tick (√) against any item on the checklist that the teacher covers at the start of the lesson. Record
any activity that the teacher engages in which is not on the checklist you are using. REFLECT TOGETHER
Compare your ticked checklist with your TP partner’s ticked checklist. Are there any differences in
the specific checklists? Did you and/or your TP partner observe and record any critical incident at the
start of the lesson? What new things have you learned about what the teacher should or shouldn’t
do at the start of a lesson? Discuss anything new you had not captured in college with your
TP partner and add to the checklist you have developed for observing the start of a lesson. NOTE: if you do not have a TP partner in the Observation School ask the class teacher to discuss
your checklist with you at the end of the lesson. Make a note of any critical incident at the start of the lesson which you did not
expect to happen. Record in your TP Journal two key points that you have learned about the start of a lesson. PLAN TOGETHER In the pre-observation orientation, the Tutor guided you to make a list of the things to look for at
the start of a lesson (see Lesson Observation Checklist, Annex 3). These include how the pupils
come into the classroom, what the teacher does at the start of the lesson such as marking the
register, getting the pupils seated, getting the pupils’ attention and so on. The list of things to be
observed corresponds to the Teacher Competencies (see Annex 1). In the pre-observation orientation, the Tutor guided you to make a list of the things to look for at
the start of a lesson (see Lesson Observation Checklist, Annex 3). These include how the pupils
come into the classroom, what the teacher does at the start of the lesson such as marking the
register, getting the pupils seated, getting the pupils’ attention and so on. The list of things to be
observed corresponds to the Teacher Competencies (see Annex 1). Think about how you will keep a record of your TP observations in your TP Journal, describing what
happens at the start of the lesson. Think about the 5-6 most important details you should observe at
the start of the lesson. Check Teacher Competency 1 (see Annex 1) for some key ideas. Observe the start of the lesson using the checklist you and your TP partner have developed. Put a
tick (√) against any item on the checklist that the teacher covers at the start of the lesson. Record
any activity that the teacher engages in which is not on the checklist you are using. DO DO
Observe the lesson delivery using the checklist you and your TP partner have developed. Put a tick
(√) against any item on the checklist that the teacher covers during the lesson. Record any activity
that the teacher engages in which is not on the checklist you are using. Interact with at least two
pupils (a girl and a boy) during the lesson carefully observing how they participate in the lesson. DO
Observe the lesson delivery using the checklist you and your TP partner have developed. Put a tick
(√) against any item on the checklist that the teacher covers during the lesson. Record any activity
that the teacher engages in which is not on the checklist you are using. Interact with at least two
pupils (a girl and a boy) during the lesson carefully observing how they participate in the lesson. Make a note of any critical incident in the lesson which you did not expect to happen. REFLECT TOGETHER REFLECT TOGETHER
Compare your ticked checklist with your TP partner’s ticked checklist. Are there any differences in
the specific checklists? Did you and/or your TP partner observe and record any critical incident at the
start of the lesson? What new things have you learned about what the teacher should or shouldn’t
do at the start of a lesson? Discuss anything new you had not captured in college with your
TP partner and add to the checklist you have developed for observing the start of a lesson. NOTE: if you do not have a TP partner in the Observation School ask the class teacher to discuss
your checklist with you at the end of the lesson. Student Teacher Handbook for Teaching Pratice 20 YEAR 1 • SCHOOL OBSERVATION TP Y1 TP Activity 2.2: During the Lesson: How does the teacher develop the lesson? Y1 TP Activity 2.3: The End of the Lesson: How does the teacher end the lesson? PLAN TOGETHER
Your Tutor might have helped you to make a list of the things to look for at the end of a lesson. These include how the teacher ends the lesson, how he/she collects/marks pupils’ work, how he/she
summarises the lesson, tidies the classroom, and dismisses the pupils (see Annex 1, Teacher
Competency 15). You will need to think about how you will record into your TP Journal two key
points about what happens at the end of the lesson in such a way that you will not miss any
important details. DO
Observe the end of the lesson using the checklist you and your TP partner have developed. Put a
tick (√) against any item on the checklist that the teacher covers at the end of the lesson. Record any
activity that the teacher engages in which is not on the checklist you are using. Make a note of any
critical incident at the end of the lesson which you did not expect to happen. Observe the end of the lesson using the checklist you and your TP partner have developed. Put a
tick (√) against any item on the checklist that the teacher covers at the end of the lesson. Record any
activity that the teacher engages in which is not on the checklist you are using. Make a note of any
critical incident at the end of the lesson which you did not expect to happen. REFLECT TOGETHER REFLECT TOGETHER
Compare your ticked checklist with your TP partner’s ticked checklist. Are there any differences in
the specific checklists ticked as covered by the teacher? Did you and/or your TP partner record any
critical incident during the lesson? What new things have you learned about the delivery of a lesson? Discuss any new things you had not captured in your checklist with your TP partner and add to the
checklist you have developed for observing a lesson. Discuss with your TP partner, and later with
your TPC, what you learned by interacting with at least two pupils (a girl and a boy) in the lesson. Compare your ticked checklist with your TP partner’s ticked checklist. Are there any differences in
the specific checklists ticked as covered by the teacher? Did you and/or your TP partner record any
critical incident during the lesson? What new things have you learned about the delivery of a lesson? Discuss any new things you had not captured in your checklist with your TP partner and add to the
checklist you have developed for observing a lesson. Discuss with your TP partner, and later with
your TPC, what you learned by interacting with at least two pupils (a girl and a boy) in the lesson. .Record in your TP Journal two key points that you have learned about the delivery of a lesson PLAN TOGETHER OG
Your Tutor will help you to make a list of the things to look for in a lesson delivery. These include
how the teacher organises the class and supports the pupils, teacher movement and position, how
the teacher involves the pupils and what the pupils do during the lesson. You will also look out for
any differences in participation of the boys and the girls in the class. Your Tutor guided you in the
pre-TP sessions on how to interact with at least two pupils during the lesson and how to access and
review pupils’ exercise books and their teacher’s note book (see Teacher Competencies 2-14 in
Annex 1). Think about how you will record into your TP Journal what happens in the lesson in such a
way that you record at least two key points. Think about the most important thing you learned from
your interaction with at least two pupils during the lesson. How to Plan a Lesson n you have completed these two Y2 Day 3 TP activities in your observation school you will have When you have completed these two Y2 Day 3 TP activities in your observation school you w • studied the class teacher’s lesson plan; • studied the class teacher’s lesson plan;
• observed the beginning, main activities and end of a lesson and compared the lesson delivery
and lesson plan; • observed the beginning, main activities and end of a lesson and compared the lesson delivery
and lesson plan; • observed how the teacher assesses pupils’ learning needs. • observed how the teacher assesses pupils’ learning needs. REFLECT TOGETHER REFLECT TOGETHER
Compare your ticked checklist with your TP partner’s ticked checklist. Are there any differences
in the specific checklists ticked by either of you as covered by the teacher? Did you and/or your
TP partner record any critical incident at the end of the lesson? What new things have you learned
regarding the end of a lesson? Discuss any new things you had not captured in college with your
TP partner and add to the checklist you have developed for observing the end of a lesson. Compare your ticked checklist with your TP partner’s ticked checklist. Are there any differences
in the specific checklists ticked by either of you as covered by the teacher? Did you and/or your
TP partner record any critical incident at the end of the lesson? What new things have you learned
regarding the end of a lesson? Discuss any new things you had not captured in college with your
TP partner and add to the checklist you have developed for observing the end of a lesson. Record in your TP Journal two important points that you have learned on Day 2. Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 21 1 Year 1 - 3: Teaching Practice Y1 TP Topic 3: Lesson Planning
Purpose: In this Topic you will study the teacher’s lesson plan and observe the delivery of lesson
activities
Activity 3.1: The Lesson Plan
Activity 3.2: Observe a Lesson
Competencies: Student Teachers observe the teacher demonstrating:
(i) Components of the Lesson Plan: Competency 16; (ii) Assessment of pupil’s understanding:
Competency 8 (see Annex 1) Y1 TP Topic 3: Lesson Planning
Purpose: In this Topic you will study the teacher’s lesson plan and observe the delivery of lesson
activities
Activity 3.1: The Lesson Plan
Activity 3.2: Observe a Lesson
Competencies: Student Teachers observe the teacher demonstrating:
(i) Components of the Lesson Plan: Competency 16; (ii) Assessment of pupil’s understanding:
Competency 8 (see Annex 1) PLAN TOGETHER DO
The class teacher will take you through the objectives for the lesson, the sequence and the phases
of the lesson plan. DO
The class teacher will take you through the objectives for the lesson, the sequence and the phases
of the lesson plan. DO
The class teacher will take you through the objectives for the lesson, the sequence and the phases
of the lesson plan. O
he class teacher will take you through the objectives for the lesson, the sequence and the phas
the lesson plan. The class teacher will take you through the objectives for the lesson, the sequence and the phases
of the lesson plan. REFLECT TOGETHER
Ask the class teacher what they use when they prepare the lesson, for example the syllabus, the
TLMs they need for the activities, the number of pupils in the class, ratio of girls and boys in the
class, Relevant Previous Knowledge (RPK), reference books, etc. Write down two key points you want to observe about the lesson and compare with your Y1 TP Activity 3.1: Study the TP class teacher’s Lesson Plan PLAN TOGETHER
In this activity you and your TP Partner or class teacher will look at the class teacher’s lesson plan. The lesson plan should include these sections (see Annex 2 Lesson Plan):
• Day/Duration
• Topic/Sub-Topic/Aspect
• Objectives/Rpk
• Teacher/Learner Activity
• Teaching/Learning Materials
• Core Points
• Evaluation/Remarks REFLECT TOGETHER REFLECT TOGETHER
Ask the class teacher what they use when they prepare the lesson, for example the syllabus, the
TLMs they need for the activities, the number of pupils in the class, ratio of girls and boys in the
class, Relevant Previous Knowledge (RPK), reference books, etc. REFLECT TOGETHER
Ask the class teacher what they use when they prepare the lesson, for example the syllabus, the
TLMs they need for the activities, the number of pupils in the class, ratio of girls and boys in the
class, Relevant Previous Knowledge (RPK), reference books, etc. Ask the class teacher what they use when they prepare the lesson, for example the syllabus, the
TLMs they need for the activities, the number of pupils in the class, ratio of girls and boys in the
class, Relevant Previous Knowledge (RPK), reference books, etc. Write down two key points you want to observe about the lesson and compare with your
TP partner. Write down two key points you want to observe about the lesson and compare with your
TP partner. 22 Student Teacher Handbook for Teaching Pratice YEAR 1 • SCHOOL OBSERVATION TP Y1 TP Activity 3.2: Observe the lesson - the main activities at the beginning, during
and end of the lesson. PLAN TOGETHER In this activity you will observe the class teacher introducing the lesson, the activities the pupils and
teacher are involved in, and the conclusion of the lesson.You will also observe how the teacher asks
questions and how the pupils answer questions during the lesson. REFLECT TOGETHER Y1 TP Topic 4: Stimulating and Sustaining Pupils’ Interest
Purpose: This Topic will give you practice in observing activities that stimulate and sustain
pupils’ interest
Activity 4.1: Review of pupils’ Relevant Previous Knowledge (RPK)
Activity 4.2: Activities to stimulate pupils’ interest and participation
Activity 4.3: Use of appropriate ICT and TLM in the classroom
Competencies: Student Teachers observe the teacher demonstrating:
(i) Pair/Group work: Competency 7(d); (ii) Gender Equality: Competency 11(b and d); (iii) Teacher
Feedback: Competency 9(a-d); (iv) TLMs: Competency 3(a-h) (see Annex 1)
Stimulating and Sustaining Pupils’ Interest
When you have completed these Y1 Day 4 TP activities in your observation school you will have:
• learned how teachers review pupils’ relevant previous knowledge (RPK);
• become familiar with activities teachers use to stimulate pupils’ interest and participation;
• identified ways in which teachers use appropriate ICT and TLMs in their lessons. Y1 TP Activity 4.1: How does the class teacher review pupils’ RPK? PLAN TOGETHER
Your Tutor has shown you examples of how teachers can review pupils’ RPK
DO
Make your own table, similar to the example above, to record how the teacher you are working with
reviews pupils’ RPK. REFLECT TOGETHER
Answer the following questions with your TP partner or class teacher:
• How did the teacher state the RPK? • Did they state the RPK in one of the ways your Tutor described? H
th RPK h
b
t t d diff
tl ? Y1 TP Day 4 - Summary of the Task
Subject
Type of RPK Activity
Examples
English
Uses Q & A to review a previous lesson
How is the last lesson topic related to
the new topic? Mathematics
Links pupils’ RPK with lesson topic
What do you know about this topic? Science
Shares lesson objectives and link them
to pupils’ RPK
How can we achieve the objectives
using what we know already? Y1 TP Topic 4: Stimulating and Sustaining Pupils’ Interest
Purpose: This Topic will give you practice in observing activities that stimulate and sustain
pupils’ interest
Activity 4.1: Review of pupils’ Relevant Previous Knowledge (RPK)
Activity 4.2: Activities to stimulate pupils’ interest and participation
Activity 4.3: Use of appropriate ICT and TLM in the classroom
Competencies: Student Teachers observe the teacher demonstrating:
(i) Pair/Group work: Competency 7(d); (ii) Gender Equality: Competency 11(b and d); (iii) Teacher
Feedback: Competency 9(a-d); (iv) TLMs: Competency 3(a-h) (see Annex 1) Stimulating and Sustaining Pupils’ Interest Y1 TP Activity 4.1: How does the class teacher review pupils’ RPK? PLAN TOGETHER
Your Tutor has shown you examples of how teachers can review pupils’ RPK
Subject
Type of RPK Activity
Examples
English
Uses Q & A to review a previous lesson
How is the last lesson topic related to
the new topic? Mathematics
Links pupils’ RPK with lesson topic
What do you know about this topic? Science
Shares lesson objectives and link them
to pupils’ RPK
How can we achieve the objectives
using what we know already? PLAN TOGETHER
Your Tutor has shown you examples of how teachers can review pupils’ RPK
Subject
Type of RPK Activity
Examples
English
Uses Q & A to review a previous lesson
How is the last lesson topic related to
the new topic? Mathematics
Links pupils’ RPK with lesson topic
What do you know about this topic? Science
Shares lesson objectives and link them
to pupils’ RPK
How can we achieve the objectives
using what we know already? DO
Make your own table, similar to the example above, to record how the teacher you are working with
reviews pupils’ RPK. REFLECT TOGETHER
Answer the following questions with your TP partner or class teacher:
• How did the teacher state the RPK? • Did they state the RPK in one of the ways your Tutor described? • How can the RPK have been stated differently? • How did the pupils respond to the teacher’s RPK prompts? DO Student Teacher Handbook for Teaching Pratice REFLECT TOGETHER Discuss with your TP partner the steps that are to be observed in lesson delivery following some of
the ideas given in the Teacher Competencies Checklist (Annex 1). What strategies can you use to ensure equal participation of all pupils in the class? Transforming Teacher Education and Learning 23 Transforming Teacher Education and Learning
www.t-tel.org 1 Year 1 - 3: Teaching Practice Y1 TP Topic 4: Stimulating and Sustaining Pupils’ Interest
Purpose: This Topic will give you practice in observing activities that stimulate and sustain
pupils’ interest
Activity 4.1: Review of pupils’ Relevant Previous Knowledge (RPK)
Activity 4.2: Activities to stimulate pupils’ interest and participation
Activity 4.3: Use of appropriate ICT and TLM in the classroom
Competencies: Student Teachers observe the teacher demonstrating:
(i) Pair/Group work: Competency 7(d); (ii) Gender Equality: Competency 11(b and d); (iii) Teacher
Feedback: Competency 9(a-d); (iv) TLMs: Competency 3(a-h) (see Annex 1)
Stimulating and Sustaining Pupils’ Interest
When you have completed these Y1 Day 4 TP activities in your observation school you will have:
• learned how teachers review pupils’ relevant previous knowledge (RPK);
• become familiar with activities teachers use to stimulate pupils’ interest and participation;
• identified ways in which teachers use appropriate ICT and TLMs in their lessons. Y1 TP Activity 4.1: How does the class teacher review pupils’ RPK? PLAN TOGETHER
Your Tutor has shown you examples of how teachers can review pupils’ RPK
DO
Make your own table, similar to the example above, to record how the teacher you are working with
reviews pupils’ RPK. REFLECT TOGETHER
Answer the following questions with your TP partner or class teacher:
• How did the teacher state the RPK? • Did they state the RPK in one of the ways your Tutor described? • How can the RPK have been stated differently? • How did the pupils respond to the teacher’s RPK prompts? Y1 TP Day 4 - Summary of the Task
Subject
Type of RPK Activity
Examples
English
Uses Q & A to review a previous lesson
How is the last lesson topic related to
the new topic? Mathematics
Links pupils’ RPK with lesson topic
What do you know about this topic? Science
Shares lesson objectives and link them
to pupils’ RPK
How can we achieve the objectives
using what we know already? REFLECT TOGETHER Student Teacher Handbook for Teaching Pratice 24 Y1 TP Activity 4.2: Activities used to stimulate pupils interest and participation? DO Observe a lesson and identify at least one activity which the pupils most enjoyed and participated in
fully. Identify at least one activity which the pupils least enjoyed and barely participated in. Identify the activity which the pupils you have been interacting with enjoyed most (girls and boys). Was there any difference in the enjoyment of girls compared to that of boys? Record two key findings in your TP Journal. REFLECT TOGETHER REFLECT TOGETHER
Share your findings with your TP partner or class teacher. Did you have the same ideas for activities
that pupils find interesting or not interesting? • What made an activity enjoyable? • What made an activity boring? • How could the teacher make the lessons more interesting and participatory? Were the activities you saw in the lesson included in those you listed in the Plan Together discussion? Suggest ways in which the lesson could have been more stimulating, interesting and participatory. Y1 TP Activity 4.3: How does the teacher use appropriate ICT and TLM? Use a blank version of the above table to record in your TP Journal how the teacher you are
working with used appropriate ICTs and TLMs in a lesson you observed. Y1 TP Activity 4.3: How does the teacher use appropriate ICT and TLM? PLAN TOGETHER
Discuss with your TP partner the resources the class teacher used in English, mathematics and
science lessons in basic schools. Identify TLMs that are ICT-based, such as CDs/DVDs, mobile phones,
projectors, television, radio, videos, etc. Identify TLMs that do not use ICT, such as books, Cuisenaire
rods, sets of mathematical instruments, word games (e.g. Scrabble), science equipment, etc. Make a
table showing how ICTs and TLMs can be used in lessons, as in the example below:
Subject
Type of TLM
Examples
How TLMs were used
English
Cards showing key words
Flashcards for teaching
Mathematics
Cuisenaire rods
Used in the teaching of fractions
Science
Pictures downloaded from
the Internet
Matching pictures and names PLAN TOGETHER
Discuss with your TP partner the resources the class teacher used in English, mathematics and
science lessons in basic schools. Identify TLMs that are ICT-based, such as CDs/DVDs, mobile phones,
projectors, television, radio, videos, etc. Identify TLMs that do not use ICT, such as books, Cuisenaire
rods, sets of mathematical instruments, word games (e.g. Scrabble), science equipment, etc. Make a
table showing how ICTs and TLMs can be used in lessons, as in the example below: Subject
Type of TLM
Examples
How TLMs were used
English
Cards showing key words
Flashcards for teaching
Mathematics
Cuisenaire rods
Used in the teaching of fractions
Science
Pictures downloaded from
the Internet
Matching pictures and names DO
REFLECT TOGETHER
You and your TP partner should compare notes on ICT-based resources and non-ICT-based TLMs
used in the lesson. • How did the TLMs contribute to the lesson? • Were the TLMs adequate for the class? How did the pupils use the TLMs? • Could different TLMs have been used in the same lesson? Use a blank version of the above table to record in your TP Journal how the teacher you are
working with used appropriate ICTs and TLMs in a lesson you observed. DO REFLECT TOGETHER
You and your TP partner should compare notes on ICT-based resources and non-ICT-based TLMs
used in the lesson. • How did the TLMs contribute to the lesson? • Were the TLMs adequate for the class? How did the pupils use the TLMs? • Could different TLMs have been used in the same lesson? PLAN TOGETHER Your class teacher will discuss with you the various ways a teacher can make lessons interesting
and participatory. These will include activities involving stories, puzzles, songs, role play, etc. • Think of lessons you have enjoyed. What activities made the lessons interesting? i • Think of lessons you have enjoyed least. Why did you find the lesson less interesting? Make a list of activities to include in an interesting lesson. Stimulating and Sustaining Pupils’ Interest hen you have completed these Y1 Day 5 TP activities in your observation school you will have: • observed the pupils. Record in your TP Journal the answers to the following questions as you observe the les • How does the teacher use the chalk board during the lesson? Can all the pupils see the chalk
board easily? • Can the furniture (chairs, benches, tables, desks, etc) be moved around or is it fixed? Is the
arrangement of the furniture suitable for the lesson? If not how could it be rearranged? • Which TLMs does the teacher use in the lesson? Are there enough TLMs for all the pupils • Can the furniture (chairs, benches, tables, desks, etc) be moved around or is it fixed? Is the
arrangement of the furniture suitable for the lesson? If not how could it be rearranged? • Which TLMs does the teacher use in the lesson? Are there enough TLMs for all the pupils
to use? DO DO
Study the seating arrangement pattern in the classroom and the activities that the pupils are
involved in. DO
Study the seating arrangement pattern in the classroom and the activities that the pupils are
involved in. REFLECT TOGETHER Transforming Teacher Education and Learning
www.t-tel.org 25 1 Year 1 - 3: Teaching Practice Y1 TP Topic 5: Developing the Lesson
Purpose: In this Topic you will become familiar with the arrangement of the classroom, and the
activities of teacher and pupils
Activity 5.1: Observe the classroom environment
Activity 5.2: Observe the teacher
Activity 5.3: Observe the pupils
Competencies: Student Teachers observe the teacher demonstrating:
(i) T/L Activities: Competency 4(a-i); (ii) Gender Equality: Competency 11(a-g); (iii) Teacher
Feedback: Competency 9(a and d); (iv) Assessment(F&S): Competency 8(a, b and e); (see Annex 1) PLAN TOGETHER PLAN TOGETHER
In this activity you will consider how the teacher organises the classroom for effective teaching and
learning to take place. For example you will observe the teacher’s use of chalk board, sitting ar-
rangements, use of TLMs, re-arranging tables and chairs for group work and so on. PLAN TOGETHER
In this activity you will consider how the teacher organises the classroom for effective teaching and
learning to take place. For example you will observe the teacher’s use of chalk board, sitting ar-
rangements, use of TLMs, re-arranging tables and chairs for group work and so on. Y1 TP Activity 5.3: Observe the Pupils PLAN TOGETHER
Your Tutor has discussed with you how to observe the pupils in the class and has guided you to make
a checklist of activities to look for when observing pupils in the classroom during a lesson. These
activities may include pupil attentiveness, participation, response to questions, physical appearance,
seating arrangements, different ways of grouping pupils (mixed, by ability, friendship) and so on. PLAN TOGETHER PLAN TOGETHER
In the TP preparation activities your Tutor helped you to identify the core Teacher Competencies
during a lesson observation (see Annex 1). ! HAVE THESE QUESTIONS READY TO GUIDE YOU WHEN YOU OBSERVE THE TEACHER:
• How does the teacher begin the lesson? • How does the teacher use voice modulation in the lesson? • What do you observe about teacher positioning during the lesson? • How does the teacher use questioning including pupils’ responses to questions? • Does the teacher listen carefully to the pupils’ answers and give them feedback? • Does the teacher involve all pupils, girls and boys, in the lesson, addressing their needs and
treating them equally? In the TP preparation activities your Tutor helped you to identify the core Teacher Competencies
during a lesson observation (see Annex 1). ! HAVE THESE QUESTIONS READY TO GUIDE YOU WHEN YOU OBSERVE THE TEACHER:
• How does the teacher begin the lesson? • How does the teacher use voice modulation in the lesson? • What do you observe about teacher positioning during the lesson? • How does the teacher use questioning including pupils’ responses to questions? • Does the teacher listen carefully to the pupils’ answers and give them feedback? • Does the teacher involve all pupils, girls and boys, in the lesson, addressing their needs and
treating them equally? DO
Observe the class teacher for 5 minutes at the beginning of the lesson, five minutes in the middle of
the lesson and 5 minutes at the end of the lesson. DO • the teacher’s position in the classroom;
• the teacher’s use of questioning;
• the teacher’s attention to pupil responses; and
• the teacher’s encouragement of girls’ participation in the lesson. REFLECT TOGETHER
Compare what you and your TP partner have recorded in your TP Journal on your observation of
the teacher. Discuss the key competencies that you need to practise from your observation of the
teacher during this lesson. REFLECT TOGETHER • How does the teacher use voice modulation in the lesson? • What do you observe about teacher positioning during the lesson? • How does the teacher use questioning including pupils’ responses to questions? • Does the teacher listen carefully to the pupils’ answers and give them feedback? • Does the teacher involve all pupils, girls and boys, in the lesson, addressing their needs and
treating them equally? Record in your TP Journal two points that you observe about each of these teacher activities: REFLECT TOGETHER REFLECT TOGETHER
Compare with your TP partner what you have recorded from your observation of the classroom
environment. Are there any differences in what you observed? Discuss the different points you
recorded. Compare with your TP partner what you have recorded from your observation of the classroom
environment. Are there any differences in what you observed? Discuss the different points you
recorded. Student Teacher Handbook for Teaching Pratice 26 YEAR 1 • SCHOOL OBSERVATION TP Y1 TP Activity 5.2: Observe the Teacher Y1 TP Activity 5.2: Observe the Teacher Y1 TP Activity 5.2: Observe the Teacher PLAN TOGETHER
In the TP preparation activities your Tutor helped you to identify the core Teacher Competencies
during a lesson observation (see Annex 1). DO
Observe the class teacher for 5 minutes at the beginning of the lesson, five minutes in the middle of
the lesson and 5 minutes at the end of the lesson. • the teacher’s position in the classroom;
• the teacher’s use of questioning;
• the teacher’s attention to pupil responses; and
• the teacher’s encouragement of girls’ participation in the lesson. REFLECT TOGETHER
! HAVE THESE QUESTIONS READY TO GUIDE YOU WHEN YOU OBSERVE THE TEACHER:
• How does the teacher begin the lesson? • How does the teacher use voice modulation in the lesson? • What do you observe about teacher positioning during the lesson? • How does the teacher use questioning including pupils’ responses to questions? • Does the teacher listen carefully to the pupils’ answers and give them feedback? • Does the teacher involve all pupils, girls and boys, in the lesson, addressing their needs and
treating them equally? Record in your TP Journal two points that you observe about each of these teacher activities: PLAN TOGETHER
In the TP preparation activities your Tutor helped you to identify the core Teacher Competencies
during a lesson observation (see Annex 1). DO
Observe the class teacher for 5 minutes at the beginning of the lesson, five minutes in the middle of
the lesson and 5 minutes at the end of the lesson. • the teacher’s position in the classroom;
• the teacher’s use of questioning;
• the teacher’s attention to pupil responses; and
• the teacher’s encouragement of girls’ participation in the lesson. ! HAVE THESE QUESTIONS READY TO GUIDE YOU WHEN YOU OBSERVE THE TEACHER:
• How does the teacher begin the lesson? PLAN TOGETHER DO
Observe what the pupils are doing during the lesson using the list you and your Tutor have
developed. Observe 2 girls and 2 boys: for 5 minutes in the beginning stage of the lesson, for
5 minutes in the middle of the lesson and for 5 minutes at the end of the lesson. REFLECT TOGETHER
After you have observed both the teacher and the pupils in the class, discuss with your TP partner
any new observations that you have not been introduced to in your college studies so far. Record, in your TP Journal, two key points you noticed from the pupil observations at
the beginning, middle and end of the lesson. Note down any critical incident during the
lesson which you did not expect to happen. Record it in your TP Journal. Transforming Teacher Education and Learning
www.t-tel.org 27 Transforming Teacher Education and Learning 1 Year 1 - 3: Teaching Practice Y1 TP Topic 6: Classroom Organisation, Management and Control
Purpose: In this Topic you will become familiar with ways in which the teacher manages the
classroom and the pupils
Activity 6.1: Management of the learning environment
Activity 6.2: Management of pupils’ behaviour
Competencies: Student Teachers observe the teacher demonstrating:
(i) Class management: Competency 13(a-c); (ii) Class organisation: Competency 14(c) (see Annex 1) Ways of organising, managing and controlling the class When you have completed these Y1 Day 6 TP activities in your observation school you will have: hen you have completed these Y1 Day 6 TP activities in your observation school you will have: • observed class management strategies including management of large classes/multi-grade
classes; • observed the teacher’s management of classroom behaviour. • observed the teacher’s management of classroom behaviour. Y1 TP Activity 6.1: Observation of the management of the learning environment Y1 TP Day 6 - Summary of the Task Ways of organising, managing and controlling the class PLAN TOGETHER PLAN TOGETHER
In this activity you will observe how the teacher manages the learning environment including large
classes/ multigrade classes. These include observation of the ways in which the teacher prepares
for and delivers classroom activities, for example organising the TLMs, pupils’ exercise books and
textbooks/reading materials, giving clear instructions to pupils, using a variety of teaching methods
that will enable all children to participate in the learning process, setting up good seating
arrangements that favour all pupils in the class, management of classroom resources during the
lesson and at the end of the lesson, and so on. DO
REFLECT TOGETHER
Why do you think class management strategies are important? Discuss with your TP partner or class teacher ways of achieving good classroom management. Record in your TP Journal 5-6 examples of class management you observed during the
lesson linked to any of the ideas listed in the Plan Together section above. DO Record in your TP Journal 5-6 examples of class management you observed during
lesson linked to any of the ideas listed in the Plan Together section above. REFLECT TOGETHER
Why do you think class management strategies are important? Discuss with your TP partner or class teacher ways of achieving good classroom management. Student Teacher Handbook for Teaching Pratice REFLECT TOGETHER REFLECT TOGETHER
Why do you think class management strategies are important? Discuss with your TP partner or class teacher ways of achieving good classroom management. REFLECT TOGETHER
Why do you think class management strategies are important? Discuss with your TP partner or class teacher ways of achieving good classroom manageme Why do you think class management strategies are important? Discuss with your TP partner or class teacher ways of achieving good classroom management 28 Student Teacher Handbook for Teaching Pratice PLAN TOGETHER PLAN TOGETHER
In this activity you will be observing how the teacher manages pupils’ behaviour in the class from
the beginning to the end of the lesson. This may include observing how the teacher encourages
shy pupils to participate in class and rewards hard work, whether the teacher treats boys and girls
equally, fairly and with respect, how the teacher establishes guidelines for pupil behaviour, how the
teacher deals with bullying, and what alternative ways are used by the teacher to discipline children
instead of using corporal punishment. PLAN TOGETHER
In this activity you will be observing how the teacher manages pupils’ behaviour in the class from
the beginning to the end of the lesson. This may include observing how the teacher encourages
shy pupils to participate in class and rewards hard work, whether the teacher treats boys and girls
equally, fairly and with respect, how the teacher establishes guidelines for pupil behaviour, how the
teacher deals with bullying, and what alternative ways are used by the teacher to discipline children
instead of using corporal punishment. DO
Ask your class teacher for ideas on how bullying and corporal punishment can be avoided in the
classroom. Observe how the teacher disciplines pupils in the class and record three examples in
your TP Journal Observe how the teacher disciplines pupils in the class and record three examples in
your TP Journal. REFLECT TOGETHER REFLECT TOGETHER
With your TP partner reflect on one new strategy you have learned today in relation to pupil
behaviour in the classroom. Discuss with your TP partner why do you think bullying and corporal
punishment should be discouraged in the classroom environment. Discuss with your TP partner alternative ways to discipline children in the class instead of bullying
d
l
i h
t C
OG
With your TP partner reflect on one new strategy you have learned today in relation to pupil
behaviour in the classroom. Discuss with your TP partner why do you think bullying and corporal
punishment should be discouraged in the classroom environment. Discuss with your TP partner alternative ways to discipline children in the class instead of bullying p
g
Discuss with your TP partner alternative ways to discipline children in the class instead of bullying
and corporal punishment. Record in your TP Journal three ideas for alternatives to corporal punishment. Record in your TP Journal three ideas for alternatives to corporal punishmen Record in your TP Journal three ideas for alternatives to corporal punishment. Lesson observation is an important activity for student teachers and mentors to undertake during teaching practice. Lesson observation is an important activity for student teachers and mentors to undertake during teaching practice. Transforming Teacher Education and Learning
www.t-tel.org 29 Transforming Teacher Education and Learning www.t-tel.org 1 Year 1 - 3: Teaching Practice Y1 TP Topic 7: Teacher Talk: Assessing Pupils’ Learning Achievement - Communication and
Questioning Skills
Purpose: In this Topic you will observe and analyse the ways in which the teacher and pupils com-
municate during lessons
Activity 7.1: Teacher’s use of questioning skills to elicit pupils’ views
Activity 7.2: Teacher’s use of oral and written exercises to evaluate pupils’ understanding
Activity 7.3: Teacher’s feedback to pupils (positive and negative)
Competencies: Student Teachers observe the teacher demonstrating: (i) Use of questions:
Competency 5(b and d); (ii) Assessment: Competency 8(a, b and e); (iii) Teacher feedback:
Competency 9(a-d) (see Annex 1) Y1 TP Topic 7: Teacher Talk: Assessing Pupils’ Learning Achievement - Communication and
Questioning Skills
Purpose: In this Topic you will observe and analyse the ways in which the teacher and pupils com-
municate during lessons
Activity 7.1: Teacher’s use of questioning skills to elicit pupils’ views
Activity 7.2: Teacher’s use of oral and written exercises to evaluate pupils’ understanding
Activity 7.3: Teacher’s feedback to pupils (positive and negative)
Competencies: Student Teachers observe the teacher demonstrating: (i) Use of questions:
Competency 5(b and d); (ii) Assessment: Competency 8(a, b and e); (iii) Teacher feedback:
Competency 9(a-d) (see Annex 1) DO During the lesson observation, record in your TP Journal at least four of the questions the
teacher asks in class and four questions the pupils ask. eacher Talk: questioning skills, student assessment and teacher feedback When you have completed these Y1 Day 7 TP activities in your observation school you will have: When you have completed these Y1 Day 7 TP activities in your observation school you will have: • learned how teachers use questioning skills to elicit pupils’ views; • learned how teachers use questioning skills to elicit pupils’ views; • developed a good understanding of how teachers evaluate pupils’ level of understanding;
• been able to differentiate between positive feedback and negative feedback and comment on
the impact of feedback on learning. Y1 TP Activity 7.1: How does the teacher use questioning skills to elicit pupil PLAN TOGETHER In the pre-observation orientation before
the Y1 TP your Tutor discussed with you the
reasons for questioning pupils in class during
a lesson. Your Tutor also guided you to
brainstorm and come up with questions that
can be asked in class by both teachers and
pupils. You and your TP partner should now
think of how teachers’ questions can be
used to elicit pupils’ views in class. Sort the
questions in the text box into teacher
questions and pupil questions and justify
your classification. In the pre-observation orientation before
the Y1 TP your Tutor discussed with you the
reasons for questioning pupils in class during
a lesson. Your Tutor also guided you to
brainstorm and come up with questions that
can be asked in class by both teachers and
pupils. You and your TP partner should now
think of how teachers’ questions can be
used to elicit pupils’ views in class. Sort the
questions in the text box into teacher
questions and pupil questions and justify
your classification. Y1 TP Day 7 - Summary of the Task Teacher Talk: questioning skills, student assessment and teacher feedback REFLECT TOGETHER Review the ‘teachers’ questions’ you identified in the Plan Together activity and those you observed
the teacher ask in the lesson observation. Which of the teacher’s questions elicited pupils’ views? If any question did not elicit pupils’ views discuss with your TP partner how the question can be
reframed to elicit pupils’ views. Record in your TP Journal an example of a question that did not elicit pupils’ views and the
reframed question that would elicit pupils’ views. Student Teacher Handbook for Teaching Pratice 30 YEAR 1 • SCHOOL OBSERVATION TP Y1 TP Activity 7.2: How does the teacher use oral/written exercises to evaluate pupils’
level of understanding of the lesson? DO Observe the lesson and list in your TP Journal two oral or written activities the teacher uses to
assess pupils’ understanding of relevant concepts. Observe two girls and two boys in the class. List one response from each pupil to these activities that shows their level of understanding of
the concept. Observe the lesson and list in your TP Journal two oral or written activities the teacher uses to
assess pupils’ understanding of relevant concepts. Observe two girls and two boys in the class. List one response from each pupil to these activities that shows their level of understanding of
the concept. PLAN TOGETHER Your Tutor might have discussed with you the
various methods of assessment. These include
both oral and written assessment. Discuss
with your TP partner or class teacher these
formative assessment methods and think
about how you will observe pupil assessment
during the lesson observation. You and your
TP partner or class teacher should plan
together which parts of the lesson you will
observe and how you can record what the
teacher does to evaluate individual pupils’
level of understanding. Your Tutor might have discussed with you the
various methods of assessment. These include
both oral and written assessment. Discuss
with your TP partner or class teacher these
formative assessment methods and think
about how you will observe pupil assessment
during the lesson observation. You and your
TP partner or class teacher should plan
together which parts of the lesson you will
observe and how you can record what the
teacher does to evaluate individual pupils’
level of understanding. Oral and Written examples for Formative
Assessment
• observing pupils while they work
• asking questions and listening to pupils’
answers
• pupil participation in classroom
discussion
• Review / marking pupils’ written work REFLECT TOGETHER REFLECT TOGETHER
With your TP partner compare the oral and written activities you observed that provided the
teacher with some indication of the pupils’ understanding of the concept. From your observation
were there any differences in the responses from the girls and from the boys? With your TP partner compare the oral and written activities you observed that provided the
teacher with some indication of the pupils’ understanding of the concept. From your observation
were there any differences in the responses from the girls and from the boys? PLAN TOGETHER
With your TP partner, brainstorm two examples of positive feedback and two examples of negative
feedback you received from your school teachers in the past. Record your examples in your
TP Journal, grouping them into ‘positive feedback’ and ‘negative feedback’. Discuss with your
TP partner how pupils respond to teachers’ questions and activities when they receive positive
feedback compared to when they receive negative feedback. DO
Observe the lesson and record in your TP Journal, two responses from pupils to which the teacher
gave positive feedback and two responses from pupils to which the teacher gave negative feedback. Observe and record how the pupils reacted to negative and positive feedback. REFLECT TOGETHER Explain how this can happen
Y1 TP Day 8 - Summary of the Task
Record the activities observed into the TP Journal so that you do not miss any
important details. Y1 TP Topic 8: Co-Curriculum Activities
Purpose: In this Topic you will familiarise yourself with the arrangements for school activities
beyond the classroom
Activity 8.1: Club meetings; Sports and culture activities
Activity 8.2: School clean-up exercise; Community clean-up
Activity 8.3: Parents and community involvement in the life of the school – PTA activities
Competencies: Student Teachers observe the teacher demonstrating:
(i) Gender stereotype: Competency 12(a-d) (see Annex 1) Y1 TP Topic 8: Co-Curriculum Activities
Purpose: In this Topic you will familiarise yourself with the arrangements for school activities
beyond the classroom
Activity 8.1: Club meetings; Sports and culture activities
Activity 8.2: School clean-up exercise; Community clean-up
Activity 8.3: Parents and community involvement in the life of the school – PTA activities
Competencies: Student Teachers observe the teacher demonstrating:
(i) Gender stereotype: Competency 12(a-d) (see Annex 1) Finding out about co-curriculum and community activities , p
;
• found out what routines and activities are organised by the school to get pupils involved in
cleaning up the school and the community; • observed how parents and community are involved in school activities. PLAN TOGETHER PLAN TOGETHER
In this activity you will find out from the class teacher and other teachers what clubs, sports and
cultural activities are conducted in your observation schools and how those activities are organised. PLAN TOGETHER
In this activity you will find out from the class teacher and other teachers what clubs, sports and
cultural activities are conducted in your observation schools and how those activities are organised. DO
Choose two of the clubs, sports and cultural activities to participate in while you are in the TP
School. Ask permission from the teacher in charge before joining the activities. You can ask the
teacher in charge how you can assist with the running of the activities. You should also find out in
which activities the girls participate and in which activities the boys participate and whether there
are some activities that are only for girls or only for boys and some that are mixed (girls and boys). REFLECT TOGETHER REFLECT TOGETHER
With your TP partner or class teacher review the two pupil responses that attracted positive
feedback and the two pupil responses that attracted negative feedback. Why did some responses
attract positive feedback and others attract negative feedback. How can negative feedback be given
in such a way that it also includes constructive feedback to help the pupil? With your TP partner or class teacher review the two pupil responses that attracted positive
feedback and the two pupil responses that attracted negative feedback. Why did some responses
attract positive feedback and others attract negative feedback. How can negative feedback be given
in such a way that it also includes constructive feedback to help the pupil? Transforming Teacher Education and Learning
www.t-tel.org 31 1 Year 1 - 3: Teaching Practice Y1 TP Topic 8: Co-Curriculum Activities
Purpose: In this Topic you will familiarise yourself with the arrangements for school activities
beyond the classroom
Activity 8.1: Club meetings; Sports and culture activities
Activity 8.2: School clean-up exercise; Community clean-up
Activity 8.3: Parents and community involvement in the life of the school – PTA activities
Competencies: Student Teachers observe the teacher demonstrating:
(i) Gender stereotype: Competency 12(a-d) (see Annex 1)
Finding out about co-curriculum and community activities
When you have completed these Y1 Day 8 TP activities in your observation school you will have:
• observed how clubs, sports and cultural activities are conducted;
• found out what routines and activities are organised by the school to get pupils involved in
cleaning up the school and the community;
• observed how parents and community are involved in school activities. Y1 TP Activity 8.1: Club meetings; Sports and Culture activities
PLAN TOGETHER
In this activity you will find out from the class teacher and other teachers what clubs, sports and
cultural activities are conducted in your observation schools and how those activities are organised. DO
Choose two of the clubs, sports and cultural activities to participate in while you are in the TP
School. Ask permission from the teacher in charge before joining the activities. You can ask the
teacher in charge how you can assist with the running of the activities. You should also find out in
which activities the girls participate and in which activities the boys participate and whether there
are some activities that are only for girls or only for boys and some that are mixed (girls and boys). Y1 TP Day 8 - Summary of the Task Finding out about co-curriculum and community activities Student Teacher Handbook for Teaching Pratice REFLECT TOGETHER REFLECT TOGETHER
What new activities have you learnt about or participated in today? What actions did you engage in, besides observation that enabled you to learn about these new
activities? Do you think these activities can promote learning? Explain how this can happen
Y1 TP Day 8 - Summary of the Task
Record the activities observed into the TP Journal so that you do not miss any
important details. Y1 TP Topic 8: Co-Curriculum Activities
Purpose: In this Topic you will familiarise yourself with the arrangements for school activities
beyond the classroom
Activity 8.1: Club meetings; Sports and culture activities
Activity 8.2: School clean-up exercise; Community clean-up
Activity 8.3: Parents and community involvement in the life of the school – PTA activities
Competencies: Student Teachers observe the teacher demonstrating:
(i) Gender stereotype: Competency 12(a-d) (see Annex 1)
Finding out about co-curriculum and community activities
When you have completed these Y1 Day 8 TP activities in your observation school you will have:
• observed how clubs, sports and cultural activities are conducted;
• found out what routines and activities are organised by the school to get pupils involved in
cleaning up the school and the community;
• observed how parents and community are involved in school activities. Y1 TP Activity 8.1: Club meetings; Sports and Culture activities
PLAN TOGETHER
In this activity you will find out from the class teacher and other teachers what clubs, sports and
cultural activities are conducted in your observation schools and how those activities are organised. DO
Choose two of the clubs, sports and cultural activities to participate in while you are in the TP
School. Ask permission from the teacher in charge before joining the activities. You can ask the
teacher in charge how you can assist with the running of the activities. You should also find out in
which activities the girls participate and in which activities the boys participate and whether there
are some activities that are only for girls or only for boys and some that are mixed (girls and boys). REFLECT TOGETHER
What new activities have you learnt about or participated in today? What actions did you engage in, besides observation that enabled you to learn about these new
activities? Do you think these activities can promote learning? REFLECT TOGETHER REFLECT TOGETHER
With your TP group, discuss the following questions:
Why do you think it is important to keep the school compound clean? What can happen if the school or community environment is not kept clean? Which other ways do you think the school and the community environment can be kept clean? Y1 TP ACTIVITY 8.3: Parents and community involvement in the life of the school – REFLECT TOGETHER
With your TP group, discuss the following questions:
Why do you think it is important to keep the school compound clean? What can happen if the school or community environment is not kept clean? Which other ways do you think the school and the community environment can be kept clean? Y1 TP ACTIVITY 8.3: Parents and community involvement in the life of the school –
PTA activities PLAN TOGETHER
Find out how the parents and community are involved in the school activities. Do community
members come to the school? How often and for what purpose? On the other hand, do teachers visit
pupils’ homes and for what purpose? Is there a PTA in the school? How is it organised and who is
involved? How many PTA members are female and how many are male? How often does the PTA
meet and for what purpose? How does the PTA help to solve problems in the school? Draw a simple map with the school in the centre of a piece of paper and the villages around the
school to show where the pupils live. Choose three girls to talk to about their journey to school. Find out the distance they have to travel and by what means they come to school, either on foot,
by bicycle or taxi. REFLECT TOGETHER What new activities have you learnt about or participated in today? What actions did you engage in, besides observation that enabled you to learn about these new
activities? Do you think these activities can promote learning? Explain how this can happen. 32 Student Teacher Handbook for Teaching Pratice Y1 TP Activity 8.2: School clean-up exercise; Community clean-up PLAN TOGETHER
In this activity you should find out what routines and activities are organised by the TP school to
encourage pupils to get involved in cleaning the school and around the community. Find out how
this is organised and who is involved. Does the clean-up activity work well or how could it be
improved? In preparation think about the importance of keeping the environment clean and tidy
both at home and at school. DO
Brainstorm with a small group of six pupils (three girls and three boys) to identify the activities they
do keep the school and home clean. Ask the pupils which are the most important clean-up activities
and why. REFLECT TOGETHER
With your TP group, discuss the following questions:
Why do you think it is important to keep the school compound clean? What can happen if the school or community environment is not kept clean? Which other ways do you think the school and the community environment can be kept clean? Y1 TP ACTIVITY 8.3: Parents and community involvement in the life of the school –
PTA activities
PLAN TOGETHER
Find out how the parents and community are involved in the school activities. Do community
members come to the school? How often and for what purpose? On the other hand, do teachers visit
pupils’ homes and for what purpose? Is there a PTA in the school? How is it organised and who is
involved? How many PTA members are female and how many are male? How often does the PTA
meet and for what purpose? How does the PTA help to solve problems in the school? DO
Draw a simple map with the school in the centre of a piece of paper and the villages around the
school to show where the pupils live. Choose three girls to talk to about their journey to school. REFLECT TOGETHER Find out the distance they have to travel and by what means they come to school either on foot
Record in your TP Journal the two most important clean-up activities identified by the
pupils and the reasons given by the pupils. DO
Brainstorm with a small group of six pupils (three girls and three boys) to identify the activities they
do keep the school and home clean. Ask the pupils which are the most important clean-up activities
and why. Record in your TP Journal the two most important clean-up activities identified by the
pupils and the reasons given by the pupils. REFLECT TOGETHER Discuss these questions with your TP partner or class teacher/buddy: • Is it safe for the girls to come to school if they have to come a long way or alone via busy
places, foot bridges or by crossing rivers? • What new knowledge have I learned today about the ways the school and community are
working together? • What club, sports or cultural activities could I run when I become a qualified teacher and what
have I learned about how to organise those activities? 33 Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 1 Year 1 - 3: Teaching Practice Y1 TP Topic 9: Professional Commitment of the Teacher
Purpose: In this Topic you will observe the ways in which the teacher undertakes specific
professional responsibilities
Activity 9.1: Teacher behaviour and attitude to work
Activity 9.2: Teacher relationship with pupils, other teachers, school authorities and parents
Activity 9.3: Record keeping by the teacher
Competencies: Student Teachers observe the teacher demonstrating good professional practice Competencies: Student Teachers observe the teacher demonstrating good professional practice,
values and attitudes Observing the teacher as a professional role-model When you have completed these Y1 Day 9 TP activities on the professional commitment of the
teacher, you will have: • learned how teachers behave in the classroom and how this can affect the learning of pupils • learned how teachers behave in the classroom and how this can affect the learning of pupils;
• noted the differences between teachers’ relationships with pupils, other teachers and different
stakeholders in education; • noted the differences between teachers’ relationships with pupils, other teachers and different
stakeholders in education; • learned how teachers keep records on both their own work and pupils’ work. PLAN TOGETHER Teacher behaviour has a strong impact on pupil performance. A good teacher provides positive
encouragement and gives pupils the confidence to achieve to their full potential. In this activity you
will be trying to find out ‘What makes a good teacher?’ in terms of their professional behaviour. You
will be looking for a role-model of good professional practice. In your college, your Tutor might have
guided you to plan what to look for when observing teacher behaviour in the Observation School
such as the teacher’s approachability, punctuality, dress code, and tolerance. You may also observe
teachers’ attitude to work, their enthusiasm for teaching and how they reflect on their own practice. You can also look for ways in which teachers demonstrate positive attitudes towards pupils with
special educational needs, and ways in which they encourage girls and ensure their protection
(see Annex 5). NOTE: take care not to criticise the teacher you are observing. This activity is NOT intended to be
an ‘inspection’ of the teacher you observe but rather the observation will trigger some ideas in your
mind about how you will in future try to be a good role model of teacher behaviour. DO Make a checklist in your TP Journal, as shown in the example below, and add some more
ideas to it during a lesson observation. Complete all sections of the checklist: 34 Student Teacher Handbook for Teaching Pratice YEAR 1 • SCHOOL OBSERVATION TP Behaviour
A teacher who is a good role
model
A teacher who is a poor role model
Approachability
…. is very friendly and makes
pupils feel confident
…. shows no interest in the pupils as
individuals
…. doesn’t observe when pupils are
unhappy
Punctuality
…. always arrives in class
before the pupils
…. arrives late and is not well-organised
Dress code
…. dresses tidily
…. wears clothes that are always full of
creases
Tolerance
…. listens to pupils ideas and
gives them time to talk about
their worries
…. shouts and makes the pupils nervous REFLECT TOGETHER
Compare your completed checklist with that of your TP partner. What ideas do you and your TP
partner have regarding the role model of positive teacher behaviour? How can the good teacher be
friendly with pupils and maintain discipline without being overly strict? PLAN TOGETHER Y1 TP Activity 9.2: Teacher relationships with pupils, other teachers, school authorities
and parents
PLAN TOGETHER
The teacher has to build good relationships with pupils and their parents, other teachers and the
school authorities. In this activity you will think about the different relationships between the
teacher and each of these stakeholders. REFLECT TOGETHER
Compare your completed checklist with that of your TP partner. What ideas do you and your TP
partner have regarding the role model of positive teacher behaviour? How can the good teacher be
friendly with pupils and maintain discipline without being overly strict? Y1 TP Activity 9.2: Teacher relationships with pupils, other teachers, school authorities
and parents
PLAN TOGETHER
The teacher has to build good relationships with pupils and their parents, other teachers and the
school authorities. In this activity you will think about the different relationships between the
teacher and each of these stakeholders. PLAN TOGETHER The teacher has to build good relationships with pupils and their parents, other teachers and the
school authorities. In this activity you will think about the different relationships between the
teacher and each of these stakeholders. Transforming Teacher Education and Learning 35 Transforming Teacher Education and Learning
www.t-tel.org 1 Year 1 - 3: Teaching Practice DO
As in Activity 9.1 this is not an inspection of the school or the class teacher but a way to help you
think about how to establish good relationships:
Relationship with
…
A teacher who has established good
relationships …. A teacher who has poor
relationships …. Pupils
- knows the name of all the pupils in
the class and some information about
their family background
- gives pupils support and encourage-
ment according to their individual
needs
- encourages girls in the classroom
- shows little interest in the
pupils’ and only knows a few by
name
- is unkind to pupils and uses
sarcasm
Other teachers
- plans together with other teachers
- does not collaborate much with
other teachers
Head teacher
- does not respect the authority
of the head teacher
Parents
Girls Education
Officer
Make a checklist in your TP Journal, as shown in the example below and use it during the
school observation. Complete all sections of the checklist. - does not collaborate much with
other teachers REFLECT TOGETHER
Compare your completed checklist with that of your TP partner. Discuss with your TP partner and
agree the importance of relationships you will need to develop when you become teachers and how
you can practise these during each of your Y2 and Y3 teaching practices. PLAN TOGETHER PLAN TOGETHER
In this activity you will need to ask the observation classroom teacher for permission to look at the
class records including the teacher’s lesson plans and record of pupils’ achievement. In the pre-TP
sessions at your CoE, your Tutor might have told you about which documents you should request
from the class teacher and how to study the teachers’ records. You will need to discuss with your TP
partner how you will record in your TP Journal what they will observe about the teachers’ records. In this activity you will need to ask the observation classroom teacher for permission to look at the
class records including the teacher’s lesson plans and record of pupils’ achievement. In the pre-TP
sessions at your CoE, your Tutor might have told you about which documents you should request
from the class teacher and how to study the teachers’ records. You will need to discuss with your TP
partner how you will record in your TP Journal what they will observe about the teachers’ records. Make a list of the documents that your class teacher shares with you and, for each one,
write in your TP Journal the main purpose of that document. Review the class teacher’s records of information about their pupils, e.g. record of assessment and
test scores, and record of background information about the pupils. Identify from the records of
information about the pupils: the headings of what information is recorded e.g. name, date of birth,
home town, parents’ occupation, test scores for English, maths, science, etc. Student Teacher Handbook for Teaching Pratice 36 Choose four pupils (two girls and two boys) and look at the teacher’s records of their test scores
and other information the teacher has recorded about their learning achievement. Record in your
TP Journal what you can find out about the level of achievement of each child in English, maths and
science from the teacher’s record. Make note of any record that the teacher keeps which you had not thought about with your Tutor
and Student Teachers during the pre-TP discussion. REFLECT TOGETHER REFLECT TOGETHER
With your TP partner reflect on these questions:
• What is the main purpose of the morning and afternoon assemblies? id
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d b
i i
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h
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Y1 TP Day 10 - Summary of the Task
In your TP Journal, write down how the class teacher manages the children coming into and
leaving the assembly. Y1 TP Topic 10: School Leadership
Purpose: In this Topic you will observe the ways in which the head teacher and teachers
demonstrate leadership skills
Activity 10.1: School leadership during assembly
Activity 10.2: School leadership for monitoring/supervision of teaching and learning
Activity 10.3: School leadership in vetting of teachers’ notes
Competencies: Student Teachers observe the teachers demonstrating:
(i) Gender stereotype: Competency 12(b and d); (ii) Leadership skills generalised to school level:
Competencies 6-13 (see Annex 1) Y1 TP Topic 10: School Leadership
Purpose: In this Topic you will observe the ways in which the head teacher and teachers
demonstrate leadership skills
Activity 10.1: School leadership during assembly
Activity 10.2: School leadership for monitoring/supervision of teaching and learning
Activity 10.3: School leadership in vetting of teachers’ notes
Competencies: Student Teachers observe the teachers demonstrating:
(i) Gender stereotype: Competency 12(b and d); (ii) Leadership skills generalised to school level:
Competencies 6-13 (see Annex 1) Y1 TP Activity 10.1: School leadership during assembly PLAN TOGETHER
In this activity you will observe how the two main school assemblies are organised every day in the
life of the school. These are the morning and afternoon assemblies. Discuss with your TP partner the
essence of assemblies, who are involved, what they do, and how it is organised DO
In your TP Journal, make a plan of the school assembly and how each class is arranged. Listen to
prefects commanding the assembly for 10 minutes and record what they do. Mark on the plan
where the prefects stand. Observe how the pupils participate in the assembly, observe their dress
code and check to see whether girls are separated from the boys or mixed and why. Listen to how
announcements are made and observe how the closing ceremony is organised. DO
In your TP Journal, make a plan of the school assembly and how each class is arranged. Listen to
prefects commanding the assembly for 10 minutes and record what they do. Mark on the plan
where the prefects stand. Observe how the pupils participate in the assembly, observe their dress
code and check to see whether girls are separated from the boys or mixed and why. Listen to how
announcements are made and observe how the closing ceremony is organised. In your TP Journal, write down how the class teacher manages the children coming into and
leaving the assembly. Observation of leadership roles in the TP school • gained an awareness on the need for lesson note vetting by the head teache Y1 TP Activity 10.1: School leadership during assembly Student Teacher Handbook for Teaching Pratice REFLECT TOGETHER Share your findings with your TP partner. What did you both learn from reviewing the record of
information about the four pupils and their learning achievement? What other information should
be recorded to help the teacher assess the learning achievement of pupils in the class? What items
were on your checklist that the teacher did not keep a record of? How can this omission, if any,
affect pupils’ learning? Student teachers need to be provided with opportunities to practise writing on the white board or black board. Student teachers need to be provided with opportunities to practise writing on the white board or black board. Transforming Teacher Education and Learning 37 Transforming Teacher Education and Learning
www.t-tel.org 1 Year 1 - 3: Teaching Practice Y1 TP Topic 10: School Leadership
Purpose: In this Topic you will observe the ways in which the head teacher and teachers
demonstrate leadership skills
Activity 10.1: School leadership during assembly
Activity 10.2: School leadership for monitoring/supervision of teaching and learning
Activity 10.3: School leadership in vetting of teachers’ notes
Competencies: Student Teachers observe the teachers demonstrating:
(i) Gender stereotype: Competency 12(b and d); (ii) Leadership skills generalised to school level:
Competencies 6-13 (see Annex 1)
Observation of leadership roles in the TP school
When you have completed these Y1 Day 10 TP activities in your observation school you will have:
• observed the two main school assemblies;
• observed how teachers are involved in supervision activities in the school;
• gained an awareness on the need for lesson note vetting by the head teacher. Y1 TP Activity 10.1: School leadership during assembly
PLAN TOGETHER
In this activity you will observe how the two main school assemblies are organised every day in the
life of the school. These are the morning and afternoon assemblies. Discuss with your TP partner the
essence of assemblies, who are involved, what they do, and how it is organised
DO
In your TP Journal, make a plan of the school assembly and how each class is arranged. Listen to
prefects commanding the assembly for 10 minutes and record what they do. Mark on the plan
where the prefects stand. Observe how the pupils participate in the assembly, observe their dress
code and check to see whether girls are separated from the boys or mixed and why. Listen to how
announcements are made and observe how the closing ceremony is organised. REFLECT TOGETHER
hl REFLECT TOGETHER
With your TP partner reflect on these questions: • What is the main purpose of the morning and afternoon assemblies? • What new ideas have you learnt today about how school assemblies are organised and
managed? Student Teacher Handbook for Teaching Pratice 38 YEAR 1 • SCHOOL OBSERVATION TP Y1 TP Activity 10.2: School leadership for monitoring/supervision of teaching
and learning DO DO
Ask the teacher or head teacher for their advice on the things you should look for when observing
teaching and learning activities and checking pupils’ exercises. Ask the teacher or head teacher for their advice on the things you should look for when observing
teaching and learning activities and checking pupils’ exercises. • Choose four pupils (two girls and two boys) in your TP class; • Observe what they do while the teacher is giving instructions on the activity they are to do in
their exercise books; • Observe the four pupils while they complete the task in their exercise books; • Check the pupils’ exercise books at the end of the exercise. REFLECT TOGETHER
Reflect together with your TP partner on how the teacher monitors teaching and learning by
checking pupils’ exercise books. Discuss with your TP partner what new ideas you have learned today. PLAN TOGETHER In this activity Student Teachers will observe how teachers are involved in supervision activities in
the school by way of checking pupils’ exercises. In your TP Journal make a list of the activities that
you need to do when you check the pupils’ exercises in the class. These include checking how the
teacher gives instructions and checking the pupils’ exercise books. In this activity Student Teachers will observe how teachers are involved in supervision activities in
the school by way of checking pupils’ exercises. In your TP Journal make a list of the activities that
you need to do when you check the pupils’ exercises in the class. These include checking how the
teacher gives instructions and checking the pupils’ exercise books. Analysis, Review and Reflection on the School Observation • analysed the notes that you wrote in the TP Journal; • reviewed standards and understood competencies in teaching • acquired skills and knowledge on how to reflect on and assess observation tasks. NOTE on Y1 post-observation TP: the activities included in the preceding Y1 TP Topics 1-10 will
have been conducted by you, as Student Teachers, in the absence of your Tutors during the 10 days
of School Observation TP. Therefore, during the Y1 post-observation TP activities, your Tutor should
review and reflect together with the whole Student Teacher group, on your overall Y1 TP learning
experiences. NOTE on Y1 post-observation TP: the activities included in the preceding Y1 TP Topics 1-10 will
have been conducted by you, as Student Teachers, in the absence of your Tutors during the 10 days
of School Observation TP. Therefore, during the Y1 post-observation TP activities, your Tutor should
review and reflect together with the whole Student Teacher group, on your overall Y1 TP learning
experiences. Y1 Post-TP Activity 1: Analysis of the TP Journal notes Y1 TP Activity 10.3: School leadership in the vetting of teachers’ notes PLAN TOGETHER
In this activity you will have the opportunity to observe how the head teacher vets the teacher’s
lesson notes. You will need to have access to the class teacher’s lesson plans and be familiar with
the components that go into the plan. You should have access to the lesson plan chart posted in the
school office which shows the timetable for teachers to submit the lesson notes, the day and date,
those who submitted and those who didn’t, those vetted and those yet to be vetted. PLAN TOGETHER
In this activity you will have the opportunity to observe how the head teacher vets the teacher’s
lesson notes. You will need to have access to the class teacher’s lesson plans and be familiar with
the components that go into the plan. You should have access to the lesson plan chart posted in the
school office which shows the timetable for teachers to submit the lesson notes, the day and date,
those who submitted and those who didn’t, those vetted and those yet to be vetted. DO
Ask the head teacher for guidance on how the teachers’ notes are vetted. Ask the class teacher to give you three examples of the kinds of suggestions the head teacher gives
the teachers regarding the preparation of the lesson notes. DO
Ask the head teacher for guidance on how the teachers’ notes are vetted. Ask the class teacher to give you three examples of the kinds of suggestions the head teacher gives
the teachers regarding the preparation of the lesson notes. REFLECT TOGETHER
Compare what the head teacher says is checked with what the teacher says the head teacher checks
in the lesson plans. Discuss with your TP partner:
Why do you think vetting of lesson notes is important? Imagine you are the head teacher. What three things would you look for when you visit teachers’
classrooms to help them to improve the teaching and learning activities? Transforming Teacher Education and Learning
www.t-tel.org 39 1 Year 1 - 3: Teaching Practice Y1 Post-TP Topic: Post-Observation Activities (college-based)
Purpose: In this Topic you will review and reflect on your School Observation TP experience with
your Tutor and other Student Teachers
Post-TP Activity 1: Analysis of TP Observation Journal notes
Post-TP Activity 2: Review of Teaching Standards and Competencies for Year 1
Post-TP Activity 3: Reflect on skills and knowledge gained from School Observation TP Y1 Post-TP Activity 1: Analysis of the TP Journal notes PLAN TOGETHER
In this activity, you will discuss the outcomes of the Y1 TP observation with your TP group at college. Read through your TP Journal, think individually about what you have recorded, analyse what you
have observed throughout the 10 days observation period, and consider how you will incorporate or
use them in your teaching career in future. DO
Your Tutor will ask you to introduce yourself stating your name, the name of your TP Observation
School and to describe two facts about the TP school from your School Observation Checklist
(Y1 TP Activity 1.1) e.g. It is a rural village school. It has 4 teachers and 98 pupils and 20 of the pupils
are girls. Your Tutor will put you into groups to do a Think-Pair-Share activity on what you have observed in
the TP schools. THINK: What were the two most interesting activities and the two most difficult activities you
participated in during the 10-day school observation TP? PAIR: Discuss your TP observation experience with the Student Teacher sitting next to you in the
class. SHARE: Take turns round the class to tell an interesting activity or a difficult activity. When it is your
turn, tell another idea or add something to what other Student Teachers have said - don’t repeat
something another student has already said. DO
Your Tutor will ask you to introduce yourself stating your name, the name of your TP Observation
School and to describe two facts about the TP school from your School Observation Checklist
(Y1 TP Activity 1.1) e.g. It is a rural village school. It has 4 teachers and 98 pupils and 20 of the pupils
are girls. DO
Your Tutor will ask you to introduce yourself stating your name, the name of your TP Observation
School and to describe two facts about the TP school from your School Observation Checklist
(Y1 TP Activity 1.1) e.g. It is a rural village school. It has 4 teachers and 98 pupils and 20 of the pupils
are girls. Your Tutor will put you into groups to do a Think-Pair-Share activity on what you have observed in
the TP schools. THINK: What were the two most interesting activities and the two most difficult activities you
participated in during the 10-day school observation TP? PAIR: Discuss your TP observation experience with the Student Teacher sitting next to you in the
class. Y1 Post-TP Activity 1: Analysis of the TP Journal notes SHARE: Take turns round the class to tell an interesting activity or a difficult activity. When it is your
turn, tell another idea or add something to what other Student Teachers have said - don’t repeat
something another student has already said. SHARE: Take turns round the class to tell an interesting activity or a difficult activity. When it is your
turn, tell another idea or add something to what other Student Teachers have said - don’t repeat
something another student has already said. Student Teacher Handbook for Teaching Pratice 40 REFLECT TOGETHER REFLECT TOGETHER
With your TP class, discuss how the TP Journal helped you in the Observation TP and how you will
use the information from the Y1 TP Journal in future. Y1 Post-TP Activity 2: Review of standards and competencies in teaching. PLAN TOGETHER
In this activity you will reflect on and review your understanding of Teacher Competencies by
reading through the Teacher Competencies listed in Annex 1. Your Tutor will remind you of the four main competencies required of teachers: professional
knowledge, professional skills, professional values and commitment, and professional development. Brainstorm with your TP partner the main competencies required of teachers. DO
Which of the Teacher Competencies did you observe in your TP School? Look back through your TP
Journal and find any record you made of the Teacher Competencies you observed. REFLECT TOGETHER
Reflect together on the four main competencies that a beginner teacher needs to know and why. Y1 Post-TP Activity 3: Skills and knowledge gained from School Observation TP. PLAN TOGETHER
Now that you have completed your school Observation Teaching Practice, think about the
knowledge and skills you have gained from the experience. Think of all the situations you observed. Choose four main observation activities and write down the skills and knowledge you have acquired. DO
REFLECT TOGETHER
Review the skills and knowledge you gained and recorded in the Skills and Knowledge Observation
Record. Compare your Skills and Knowledge Observation Record with your TP partner or TP group. Finally, assess which two observation activities helped you to gain the most knowledge and skills. Explain why/how. OBSERVATION/SITUATION
SKILLS
KNOWLEDGE
observed a lesson
observed morning assembly
Make your Skills and Knowledge Observation Record like this in your TP Journal. With your TP class, discuss how the TP Journal helped you in the Observation TP and how you will
use the information from the Y1 TP Journal in future. Student Teacher Handbook for Teaching Pratice Year 2 On-Campus Teaching Practice Year 2 On-Campus Teaching Practice YEAR 2 - ON-CAMPUS TP YEAR 2 - ON-CAMPUS TP Y2 On-Campus Teaching Practice Activities •
Pre-On-Campus Teaching Practice activities to be undertaken by the Tutors in Semester 1,
On-Campus for all Year 2 Student Teachers; •
Pre-On-Campus Teaching Practice activities to be undertaken by the Tutors in Semester 1,
On-Campus for all Year 2 Student Teachers; On Campus for all Year 2 Student Teachers;
•
one topic for each of the three peer Teaching Practice weeks in Semester 1 with two to
three activities to be undertaken by the Student Teacher and recorded in their TP Journal
•
one topic for each of the three individual Teaching Practice weeks in Semester 2 with two
to three activities to be undertaken by the Student Teacher and recorded in their TP Journal
•
Post-On-Campus Teaching Practice activities to be undertaken by Tutors after completion
of the On-Campus Teaching Practice in Semester 2, for all Year 2 Student Teachers. On Campus for all Year 2 Student Teachers;
•
one topic for each of the three peer Teaching Practice weeks in Semester 1 with two to
three activities to be undertaken by the Student Teacher and recorded in their TP Journal
•
one topic for each of the three individual Teaching Practice weeks in Semester 2 with two
to three activities to be undertaken by the Student Teacher and recorded in their TP Journal
•
Post-On-Campus Teaching Practice activities to be undertaken by Tutors after completion
of the On-Campus Teaching Practice in Semester 2, for all Year 2 Student Teachers. •
Post-On-Campus Teaching Practice activities to be undertaken by Tutors after completion
of the On-Campus Teaching Practice in Semester 2, for all Year 2 Student Teachers. Y1 Post-TP Activity 2: Review of standards and competencies in teaching. PLAN TOGETHER
In this activity you will reflect on and review your understanding of Teacher Competencies by
reading through the Teacher Competencies listed in Annex 1. Your Tutor will remind you of the four main competencies required of teachers: professional
knowledge, professional skills, professional values and commitment, and professional development. Brainstorm with your TP partner the main competencies required of teachers. OBSERVATION/SITUATION
SKILLS
KNOWLEDGE
observed a lesson
observed morning assembly
Make your Skills and Knowledge Observation Record like this in your TP Journal. Make your Skills and Knowledge Observation Record like this in your TP Journal. REFLECT TOGETHER
Review the skills and knowledge you gained and recorded in the Skills and Knowledge Observation
Record. Compare your Skills and Knowledge Observation Record with your TP partner or TP group. Finally, assess which two observation activities helped you to gain the most knowledge and skills. Explain why/how. REFLECT TOGETHER
Review the skills and knowledge you gained and recorded in the Skills and Knowledge Observation
Record. Compare your Skills and Knowledge Observation Record with your TP partner or TP group. Finally, assess which two observation activities helped you to gain the most knowledge and skills. Explain why/how. 41 Transforming Teacher Education and Learning Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning
www.t-tel.org 2 Year 1 - 3: Teaching Practice Student Teacher Handbook for Teaching Pratice 42 TOPICS AND ACTIVITIES FOR YEAR 2 ON-CAMPUS TEACHING PRACTICE TOPICS AND ACTIVITIES FOR YEAR 2 ON-CAMPUS TEACHING PRACTICE Y2 Pre-TP Topic: Pre-On-Campus Activities (college-based)
Purpose: In this session you will familiarise yourself with the On-Campus Teaching Practice
Pre-TP Activity 1: TP Orientation - Teacher Competencies and Y2 TP Assessment Procedures
Pre-TP Activity 2: Using the TP Journal
Pre-TP Activity 3: Tutor and Student Teacher professional relationship and roles
TOPICS AND ACTIVITIES FOR YEAR 2 ON-CAMPUS TEACHING PRACTICE Y2 Pre-TP Topic: Pre-On-Campus Activities (college-based)
Purpose: In this session you will familiarise yourself with the On-Campus Teaching Practice
Pre-TP Activity 1: TP Orientation - Teacher Competencies and Y2 TP Assessment Procedures
Pre-TP Activity 2: Using the TP Journal
Pre-TP Activity 3: Tutor and Student Teacher professional relationship and roles Pre-TP Activity 3: Tutor and Student Teacher professional relationship and roles PLAN TOGETHER In your DBE programme your Tutor will have
introduced you to Teacher Competencies
(see Annex 1 and summary Text Box) and
Assessment Procedures that you will practise
in the Y2 TP (see Annex 4). Examples of Teacher Competencies to be
demonstrated and assessed in Y2 and Y3 TP
(see Annex 1):
• scheme of work and weekly forecast
• lesson planning
• lesson delivery
• classroom organisation, management and
control
• assessment of pupils’ learning needs
• use of TLMs
• Reflective Practice
• pedagogical content knowledge
• lesson closure. Your Tutor will assess how well you are able to
demonstrate each of these competencies and
together you will identify those competencies
you need to improve. Discuss with your TP group each Teacher
Competency (see Annex 1) included in the
assessment for Y2 TP. Discuss how they can be
demonstrated in the Y2 TP and how they are
included as part of your Y2 TP assessment. DO
Work with your TP partner. Look ahead in your Student Teacher Handbook and identify which
Teacher Competencies you will have to demonstrate each week of the Y2 TP. Y2 TP Week 1 Teacher Competencies
Y2 TP Week 2 Teacher Competencies
ETC.. In your TP Journal make a table showing the week-by-week Teacher Competencies, as below: Student Teacher Handbook for Tea
44
DO
Work with your TP partner. Look ahead in your Student Teacher Handbook and identify which
Teacher Competencies you will have to demonstrate each week of the Y2 TP. REFLECT TOGETHER
Compare your answers with your TP partner/TP group. Note the Teacher Competencies you
think are your strengths and those you feel less confident about. Reflect also on the Teacher
Competencies you observed in Y1 Observation TP. Y2 TP Week 1 Teacher Competencies
Y2 TP Week 2 Teacher Competencies
ETC.. In your TP Journal make a table showing the week-by-week Teacher Competencies, as below When you have completed these three Y2 Pre-TP Activities with your Tutor you will be: • familiar with the competencies and assessment for Y2 On-Campus Teaching Practice;
• able to use the TP Journal for Y2 TP; • aware of the Tutor and Student Teacher roles and relationships. Pre-TP Activity 1: Orientation – Teacher Competencies and Y2 TP assessment
procedures Purpose: In Year 2 On-Campus Teaching Practice: Student Teachers will be able to familiarise themselves with the competencies and skills of
classroom teaching and learning. The CoE Tutors will supervise and assess the competencies and skills acquired by the Student
Teachers and assist them where necessary. Teaching Practice Framework: Year 2 On-Campus Teaching Practice
Pre-On-Campus Teaching Practice Activities
Topic 1. Stimulating and Sustaining Pupils’ Interest – Peer Teaching in Lower Primary
Topic 2. Stimulating and Sustaining Pupils’ Interest – Peer Teaching in Upper Primary
Topic 3. Stimulating and Sustaining Pupils’ Interest – Peer Teaching in Junior High School
Topic 4. Lesson Sequence and Activities – Individual Teaching in Lower Primary
Topic 5. Lesson Sequence and Activities – Individual Teaching in Upper Primary
Topic 6. Lesson Sequence and Activities – Individual Teaching in Junior High School
Post-On-Campus Teaching Practice Activities Transforming Teacher Education and Learning Transforming Teacher Education and Learning
www.t-tel.org 43 2 Year 1 - 3: Teaching Practice DO Student Teacher Handbook for Teaching
Work with your TP partner. Look ahead in your Student Teacher Handbook and identify which
Teacher Competencies you will have to demonstrate each week of the Y2 TP. REFLECT TOGETHER
Compare your answers with your TP partner/TP group. Note the Teacher Competencies you
think are your strengths and those you feel less confident about. Reflect also on the Teacher
Competencies you observed in Y1 Observation TP. Y2 TP Week 1 Teacher Competencies
Y2 TP Week 2 Teacher Competencies
ETC.. In your TP Journal make a table showing the week-by-week Teacher Competencies, as below: ork with your TP partner. Look ahead in your Student Teacher Handbook and identify which
eacher Competencies you will have to demonstrate each week of the Y2 TP. Y2 TP Week 1 Teacher Competencies
Y2 TP Week 2 Teacher Competencies
ETC.. In your TP Journal make a table showing the week-by-week Teacher Competencies, as below: Y2 TP Week 1 Teacher Competencies
Y2 TP Week 2 Teacher Competencies
ETC.. In your TP Journal make a table showing the week-by-week Teacher Competencies, as below: REFLECT TOGETHER
Compare your answers with your TP partner/TP group. Note the Teacher Competencies you
think are your strengths and those you feel less confident about. Reflect also on the Teacher
Competencies you observed in Y1 Observation TP. REFLECT TOGETHER
Compare your answers with your TP partner/TP group. Note the Teacher Competencies you
think are your strengths and those you feel less confident about. Reflect also on the Teacher
Competencies you observed in Y1 Observation TP. Compare your answers with your TP partner/TP group. Note the Teacher Competencies you
think are your strengths and those you feel less confident about. Reflect also on the Teacher
Competencies you observed in Y1 Observation TP. Student Teacher Handbook for Teaching Pratice Student Teacher Handbook for Teaching Pratice Student Teacher Handbook for Teaching Pratice 44 Pre-TP Activity 2: Using the TP Journal How did the Tutor respond to the Student Teacher’s behaviour? After watching the role play, complete the table below in your TP Journal. REFLECT TOGETHER
Discuss with your Tutors and TP partner the actions to be taken if there are problems in the
relationship between Student Teacher and Tutor or problems in relationships among Student
Teachers (see Annex 5). Keep the discussion general and avoid personalising and naming of
individuals. Compare the DOs and DON’Ts from your discussions, with your TP group. Key Words: Positive Relationships between
Student Teacher and Tutor
• communication
• discipline
• professional support
• personal safety
Brainstorm with your TP group on each of the
key words in the textbox in the context of good
relationships between Student Teacher and
Tutor. Identify how to build good relationships
with your Tutors and think of some situations
that can result in poor relationships. Brainstorm
with your TP group on each of the key words in
the textbox in the context of good relationships
List examples of negative Student Teacher and
Tutor relationships
List examples of positive Student Teacher and
Tutor relationships
I
I
Ii
Ii REFLECT TOGETHER
Discuss with your TP partner your understanding of the importance of the TP Journal for your TP
assessment and how well you are completing the TP Journal. What do you need to improve in the
use of your own TP Journal? REFLECT TOGETHER
Discuss with your TP partner your understanding of the importance of the TP Journal for your TP
assessment and how well you are completing the TP Journal. What do you need to improve in the
use of your own TP Journal? Review and compare what you have recorded in your TP Journal since Y1. Remember your TP Journal will be used by your Tutor as part of your TP assessment, so it is
important to keep it up-to-date. You should be serious about keeping a good TP Journal. PLAN TOGETHER PLAN TOGETHER
between Student Teacher and Tutor. Identify how to build good relationships with your Tutors and
think of some situations that can result in poor relationships. Key Words: Positive Relationships between
Student Teacher and Tutor
• communication
• discipline
• professional support
• personal safety
Brainstorm with your TP group on each of the
key words in the textbox in the context of good
relationships between Student Teacher and
Tutor. Identify how to build good relationships
with your Tutors and think of some situations
that can result in poor relationships. Brainstorm
with your TP group on each of the key words in
the textbox in the context of good relationships between Student Teacher and Tutor. Identify how to build good relationships with your Tutors and
think of some situations that can result in poor relationships. Key Words: Positive Relationships between
Student Teacher and Tutor
• communication
• discipline
• professional support
• personal safety
Brainstorm with your TP group on each of the
key words in the textbox in the context of good
relationships between Student Teacher and
Tutor. Identify how to build good relationships
with your Tutors and think of some situations
that can result in poor relationships. Brainstorm
with your TP group on each of the key words in
the textbox in the context of good relationships DO Pre-TP Activity 2: Using the TP Journal YEAR 2 - ON-CAMPUS TP PLAN TOGETHER
Your Tutor will introduce you to the ways you will use the TP Journal during Y2 TP, starting with the
recording of Teacher Competencies (see Pre-TP Activity 1 above). Look back at Pre-TP Activity 1 and
the introductory section of this Handbook on how to use the TP Journal. PLAN TOGETHER
Your Tutor will introduce you to the ways you will use the TP Journal during Y2 TP, starting with the
recording of Teacher Competencies (see Pre-TP Activity 1 above). Look back at Pre-TP Activity 1 and
the introductory section of this Handbook on how to use the TP Journal. DO
Read the instructions given in the Introduction section of this handbook on how to use your
TP Journal. Review what you wrote in your TP Journal in Y1 Observation TP and how this record
was used by you and your Tutor to assess your Y1 Observation TP performance. DO
Read the instructions given in the Introduction section of this handbook on how to use your
TP Journal. Review what you wrote in your TP Journal in Y1 Observation TP and how this record
was used by you and your Tutor to assess your Y1 Observation TP performance. Transforming Teacher Education and Learning
www.t-tel.org
y y
y
y
p
REFLECT TOGETHER
Discuss with your TP partner your understanding of the importance of the TP Journal for your TP
assessment and how well you are completing the TP Journal. What do you need to improve in the
use of your own TP Journal? Review and compare what you have recorded in your TP Journal since Y1. Remember your TP Journal will be used by your Tutor as part of your TP assessment, so it is
important to keep it up-to-date. You should be serious about keeping a good TP Journal. Pre- TP Activity 3: Tutor and Student Teacher relationship
PLAN TOGETHER
between Student Teacher and Tutor. Identify how to build good relationships with your Tutors and
think of some situations that can result in poor relationships. DO
In your TP groups, prepare and act out a role play between a Tutor and a Student Teacher. First, role
play to show a poor relationship and then demonstrate a good relationship. Example Scenario: When a Tutor arrived at the classroom for supervision, the Student Teacher was
teaching without a lesson plan. Y2 TP Topic 1: “Stimulating and Sustaining Pupils’ Interest” – Peer Teaching in Lower Primary
(LP) Purpose: In this Topic you will practise a variety of activities that stimulate and sustain Lower
Primary pupils’ interest. Activity 1.1: Appropriate use of ICT and TLM in Lower Primary classes Activity 1.2: Group Work in Lower Primary classes Activity 1.3: Games and Songs in Lower Primary classes Competencies: Student Teacher demonstrates: (i) Use of ICT and TLMs: Competency 3(b-c);
(ii) Learning activities: Competency 4(c-g); (iii) Class management: Competency 13(a-d); (iv)
Classroom seating arrangement: Competency 14(a-c); (v) Lesson closure: Competency 15(a-c); (vi)
Lesson plan: Competency 16(a-e) (see Annex 1) DO Discuss with your Tutors and TP partner the actions to be taken if there are problems in the
relationship between Student Teacher and Tutor or problems in relationships among Studen
Teachers (see Annex 5). Keep the discussion general and avoid personalising and naming of
individuals. Compare the DOs and DON’Ts from your discussions, with your TP group. Discuss with your Tutors and TP partner the actions to be taken if there are problems in the
relationship between Student Teacher and Tutor or problems in relationships among Student
Teachers (see Annex 5). Keep the discussion general and avoid personalising and naming of
individuals. Compare the DOs and DON’Ts from your discussions, with your TP group. Transforming Teacher Education and Learning
www.t-tel.org 45 2 Year 1 - 3: Teaching Practice Y2 TP Week 1 - Summary of the Task When you have completed these three Y2 Week 1 activities with your Tutor you will have: • gained an awareness of the appropriate use of ICT and TLM in the Lower Primary (LP) classes;
• practised organising and teaching a group-work lesson using peer teaching in LP lessons;
• planned an LP lesson including games and songs and delivered the lesson. • practised organising and teaching a group-work lesson using peer teaching in LP lessons;
•
l
d
LP l
i
l di
d
d d li
d th l Y2 TP Activity 1.1: Appropriate use of ICT and TLM in Lower Primary classes
(link to STPD T6 and T11) Y2 TP Activity 1.2: Group Work (link to STPD T4 Group Work) Y2 TP Activity 1.2: Group Work (link to STPD T4 Group Work) YEAR 2 - ON-CAMPUS TP PLAN TOGETHER In this activity you and your TP partner will prepare a group-work activity. Choose a topic from the
LP English, maths or science curriculum. Think of a group-work activity, e.g. an open question such
as “which is your favourite animal and why?”, that pupils talk about in their group and decide how
to answer; or a simple picture and word matching game for each group. Make a lesson plan for the
group-work activity. Think about how much time you will have in the lesson and how much time the
pupils will need to complete the activity. Decide with your TP partner which parts of the lesson each
of you will teach. REMEMBER: you need to demonstrate ways of stimulating and sustaining
pupils’ interest – think about what the pupils will be doing in the group work activity, and not
only what you will be doing as the teacher. In this activity you and your TP partner will prepare a group-work activity. Choose a topic from the
LP English, maths or science curriculum. Think of a group-work activity, e.g. an open question such
as “which is your favourite animal and why?”, that pupils talk about in their group and decide how
to answer; or a simple picture and word matching game for each group. Make a lesson plan for the
group-work activity. Think about how much time you will have in the lesson and how much time the
pupils will need to complete the activity. Decide with your TP partner which parts of the lesson each
of you will teach. REMEMBER: you need to demonstrate ways of stimulating and sustaining
pupils’ interest – think about what the pupils will be doing in the group work activity, and not
only what you will be doing as the teacher. DO
Teach the lesson with your TP partner according to the lesson plan. DO
Teach the lesson with your TP partner according to the lesson plan. Y2 TP Activity 1.3: Games and Songs (link to STPD T1 Creative Approaches) PLAN TOGETHER
Use the STPD Creative Approaches Theme T1 that you studied with your Tutor. Review the ideas
from T1 and choose an activity from the Games and Songs Teaching Strategies to try out in your
next peer teaching practice. Prepare a lesson plan for the chosen activity with your TP partner
remembering to allocate some parts of the lesson to each of you. Remember to focus your lesson
on ways of stimulating and sustaining pupils’ interest – think about what the pupils will be doing
during the games and songs activities, and not only what you will be doing as the teacher. DO
Teach the lesson with your TP partner according to the lesson plan. REFLECT TOGETHERl REFLECT TOGETHER
In your TP group, reflect on the modalities for grouping, group dynamics, class control, etc. and
how challenging and interesting these were for the pupils. Listen to the feedback and constructive comments from your Tutor and other Student Teachers in
the class to identify which parts of the lesson went well and which parts could be improved on. REFLECT TOGETHER
In your TP group, reflect on the modalities for grouping, group dynamics, class control, etc. and
how challenging and interesting these were for the pupils. Listen to the feedback and constructive comments from your Tutor and other Student Teachers in
the class to identify which parts of the lesson went well and which parts could be improved on. Record in your TP Journal two points on what went well and two points on what could be
improved. PLAN TOGETHER For the peer teaching practice activity this week, working with your TP partner, plan an LP English,
maths or science lesson that includes the use of one type of ICT and use of TLMs. Choose a topic
from the Grade 1 or Grade 2 curriculum and prepare a lesson plan. In the lesson plan describe the
ICT and TLM you will use and decide with your TP partner which parts of the lesson each of you
will teach. For the peer teaching practice activity this week, working with your TP partner, plan an LP English,
maths or science lesson that includes the use of one type of ICT and use of TLMs. Choose a topic
from the Grade 1 or Grade 2 curriculum and prepare a lesson plan. In the lesson plan describe the
ICT and TLM you will use and decide with your TP partner which parts of the lesson each of you
will teach. DO
Teach the lesson with your TP partner according to the lesson plan. REMEMBER – you need to demonstrate ways of stimulating and sustaining pupils’ interest
through the use of ICT and TLMs – think about what the pupils will be doing with the materials,
and not only what you will be doing as the teacher. REFLECT TOGETHER
After the TP lesson your Tutor may ask the other Student Teachers in the class to give some
feedback and your Tutor may also provide you with some constructive comments. Discuss with your TP partner which parts of the lesson went well and which parts could be improved. What you will be doing as the teacher
What the pupils will be doing
•
•
•
•
•
•
Record this table in your TP Journal. Write three bullet points in each column. Record in your TP Journal two points for each. Student Teacher Handbook for Teaching Pratice 46 Y2 TP Topic 2: “Stimulating and Sustaining Pupils’ Interest” Peer Teaching in Upper Primary
(UP) Activity 2.3: Role Play and Demonstration in Upper Primary classes Competencies: Student Teacher demonstrates: (i) Use of ICT and TLMs: Competency 3 and 5 (a and d); (ii) Group/pair work: Competency 7
(a and d); (iii) Classroom seating arrangements: Competency 14 (a-c); (iv) Learning activities:
Competency 4(f) (see Annex 1) STUDENT TEACHER SELF-ASSESSMENT QUESTION: Transforming Teacher Education and Learning
www.t-tel.org 47 2 Year 1 - 3: Teaching Practice Y2 TP Week 2 - Summary of the Task When you have completed these three Y2 Week 2 activities with your Tutor you will have: • gained an awareness of the appropriate use of ICT and TLMs to promote questioning and
discussion in Upper Primary (UP) classes; • practised organising and teaching pair work and group work in UP lessons;
• planned a UP lesson including role play and demonstration using peer teachin • practised organising and teaching pair work and group work in UP lessons; • practised organising and teaching pair work and group work in UP lessons;
• planned a UP lesson including role play and demonstration using peer teaching • practised organising and teaching pair work and group work in UP lessons;
• planned a UP lesson including role play and demonstration using peer teaching. Y2 TP Activity 2.1: Appropriate use of ICT and use of TLMs in UP classes
(link to STPD T2 Questioning) PLAN TOGETHER In your subject specialist classes and STPD sessions, your Tutor will have introduced some ideas
for using ICT and TLM in the UP classes. Working with your TP partner, plan a UP English, maths or
science lesson that includes the use of one type of ICT and use of TLMs. Choose a lesson from the
Grade 4 or Grade 5 curriculum and prepare a lesson plan in which you describe the ICT and TLMs you
will use. Decide with your TP partner which part of the lesson each of you will teach. REMEMBER- you need to demonstrate ways of stimulating and sustaining pupils’ interest through
the use of the materials - so you must think about what the pupils will be doing as well as what
you will be doing. PLAN TOGETHER PLAN TOGETHER
Prepare a pair- and group-work activity. Review the ideas that were presented in the STPD T4
Group Work Theme. Choose a topic from the UP English, maths or science curriculum and think of a
pair- and group-work activity. Think of an open question relevant to your chosen lesson, for example
‘Why do we wash our hands before eating?’ Make a lesson plan for the pair- and group-work activity. Decide with your TP partner which part of the lesson each of you will teach. Remember to focus on ways of stimulating and sustaining pupils’ interest through group work
activities - think about what the pupils will be doing as well as what you will be doing. DO
Teach the lesson with your TP partner according to the lesson plan. REFLECT TOGETHER YEAR 2 - ON-CAMPUS TP YEAR 2 - ON-CAMPUS TP REFLECT TOGETHER
After the TP lesson the other Student Teachers in the class will give some feedback and your Tutor
will also provide some constructive comments specific to the competencies you have practised. Reflect on the difference if any, between the LP and UP activities in relation to the competencies
you practised. Discuss with your TP partner which part of the lesson went well and which part could
be improved. REFLECT TOGETHER Listen to the feedback and constructive comments from your Tutor and other Student Teachers
in the class on this activity. Reflect on how different the group dynamics, class control, etc. in LP
classes are from those in UP classes. Discuss with your TP partner which part of the lesson went
well and which part needs improvement. Record in your TP Journal two points for each one. Y2 TP Activity 2.3: Role play; Demonstration (link to STPD T1 Creative Approaches) PLAN TOGETHER
Review the STPD T1 Creative Approaches, Role Play Teaching Strategy that you have studied with
your Tutor. Choose an activity from the Role Play Teaching Strategy and prepare a lesson plan with
your TP partner. Allocate some part of the lesson to each of you. Remember to focus on ways of
stimulating and sustaining pupils’ interest through role play and demonstration - think about
what the pupils will be doing as well as what you will be doing. Complete the following table in your TP Journal: What the pupils (or Student Teachers acting
as pupils) will be doing: What the pupils (or Student Teachers acting
as pupils) will be doing: DO
Teach the lesson with your TP partner according to the lesson plan. Your Tutor will try to arrange for
your peer-teaching practices to be delivered in neighbouring schools when possible so that you can
gain experience of teaching pupils in a real classroom situation. Student Teacher Handbook for Teaching Pratice 48 Activity 3.3: Formative Assessment in JHS classes Activity 3.3: Formative Assessment in JHS classes Competencies: Student Teacher demonstrates: Competencies: Student Teacher demonstrates:
(i) Clear explanations: Competency 2(e) and 5(b); (ii) Organisation of group/pair work: Competen-
cy 7(d); (iii) Teach girls and boys equally: Competency 11(c); (iv) Assessment of pupil understand-
ing: Competency 8(a and e); (v) Lesson closure: Competency 15(a-c); (vi) Lesson plan: Competency
16(a-e) (see Annex 1) Y2 TP Week 3 - Summary of the Task When you have completed these three Y2 Week 3 activities with your Tutor you will have: • gained an understanding of and practice in Direct Instruction and Enquiry-Based learning in
Junior High School (JHS) teaching; • practised Think-Pair-Share; • planned a JHS lesson using formative assessment Y2 TP Activity 3.1: Using Direct Instruction and Enquiry-Based Learning Record in your TP Journal two points to improve your lessons. Student Teacher Self-Assessment Question: to guide your learning and improve your professional skills,
ask yourself the following questions. Write brief answers in your TP Journal and give yourself a score for
each question:
• How well-prepared were you for the three TP peer-teaching activities? • How suitable do you think the activities were for Upper Primary pupils? • How did the pupils/Student Teachers react to your lessons? (enjoyed, bored, active, etc)
Score: needs improvement = 1; satisfactory = 2; good performance = 3 Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 49 2 Year 1 - 3: Teaching Practice PLAN TOGETHER Direct Instruction: the teacher stands at
the front of the class and the lesson is
teacher-centred
Enquiry-Based learning: involves the learner
in posing questions and problem solving and
the lesson is learner-centred Your Tutor will tell you which subject to
teach from the JHS English, maths or science
curriculum. Choose a topic in the given subject. Discuss and make two lesson plans with your
TP partner, one on the use of direct instruction
and another lesson plan to practise
enquiry-based learning (see Text Box). Decide with your TP partner which part of the lesson each of you will teach and prepare everything
you need for the lesson. REMEMBER: you need to determine ways of stimulating and sustaining
pupils’ interest through the use of direct instruction and enquiry-based learning. DO
Teach the two lessons with your TP partner according to the lesson plans. Teach the two lessons with your TP partner according to the lesson plans. Y2 TP Topic 3: “Stimulating and Sustaining Pupils’ Interest” Peer Teaching in Junior High
School (JHS) Purpose: In this Topic you will peer-teach three JHS lessons, one for each activity. Activity 3.1: Direct Instruction – Verbal Exposition and Enquiry-Based Learning in JHS classes Activity 3.1: Direct Instruction – Verbal Exposition and Enquiry-Based Learning in JHS classes
Activity 3 2: Think Pair Share (Group and Pair Work); group formation and differentiated/same Activity 3.2: Think–Pair–Share (Group- and Pair-Work); group formation and differentiated/same
task activities in JHS classes Y2 TP Activity 3.2: Think-Pair-Share YEAR 2 - ON-CAMPUS TP REFLECT TOGETHER Compare the effectiveness of the two lessons - Direct Instruction and Enquiry-Based learning. Discuss with your TP partner whether the teaching methodology was appropriate to the topic and
the class. Identify which parts of the lessons went well and which parts need to be improved. Compare the effectiveness of the two lessons - Direct Instruction and Enquiry-Based learning. Discuss with your TP partner whether the teaching methodology was appropriate to the topic and
the class. Identify which parts of the lessons went well and which parts need to be improved. In your TP Journal record two points that were successful and two points for
improvement in future. Student Teacher Handbook for Teaching Pratice 50 PLAN TOGETHER Ways of Organising Pupils for
Pair- and Group- Work
• mixed ability pairs/groups
• same ability pairs/groups
• girls only and boys only pairs/groups
• mixed girls and boys pairs / groups
• pupils choose their own pairs / groups With your TP partner, prepare a Think-Pair-
Share activity for a JHS lesson. First, your Tutor
should tell you which subject you will teach
(English, maths or science). Then choose the
topic to teach. Think of the best way to
organise the pupils into pairs and the most
appropriate pair-work activity for your topic
(see Text Boxes). Include activities in your lesson plan for each of the three steps of Think-Pair-Share. (
) For example: REFLECT TOGETHER REFLECT TOGETHER
Which of the formative assessment strategies have you observed in your peer-teaching lessons
during Y2 Week 3 TP? Which strategies worked well? Discuss with your TP group why some
strategies were more successful than others. For example: Step 1 THINK: ask the pupils to think about their responses to an open question or a problem such
as ‘solve this algorithm in your head: 18 x 5 =?; then write how you worked out the answer. Step 2 PAIR: discuss with your partner how you worked out the answer. Can you think of other ways
to solve the algorithm? Step 2 PAIR: discuss with your partner how you worked out the answer. Can you think of other ways
to solve the algorithm? Step 3 SHARE: share with the rest of the class the most interesting solution you found. NOTE: Include the Formative Assessment activity (Y2 TP Activity 3.3) in your lesson plan. Step 3 SHARE: share with the rest of the class the most interesting solution you found. NOTE: Include the Formative Assessment activity (Y2 TP Activity 3.3) in your lesson plan. Ideas for Group Work Activities
• Brainstorm
• Make a list
• Discuss an open question
• Prepare a short presentation
• Prepare a role play
DO
Prepare your lesson using Think-Pair-Share
incorporating the formative assessment
activity (Y2 TP Activity 3.3, below), Teach the
lesson with your TP partner using peer-teaching
as planned. Examples of Formative Assessment
• checking pupils’ work
• asking probing questions and listening to
pupils answers
• observing pupils during group work and
project work
REFLECT TOGETHER
Reflect with your TP partner on what was
successful in the Think-Pair-Share activity. What needs to be improved? Write in your TP Journal two ideas for improving your classroom organisation. Ideas for Group Work Activities
• Brainstorm
• Make a list
• Discuss an open question
• Prepare a short presentation
• Prepare a role play
DO
Prepare your lesson using Think-Pair-Share
incorporating the formative assessment
activity (Y2 TP Activity 3.3, below), Teach the
lesson with your TP partner using peer-teaching
as planned. Examples of Formative Assessment
• checking pupils’ work
• asking probing questions and listening to
pupils answers
• observing pupils during group work and
project work
REFLECT TOGETHER
Reflect with your TP partner on what was
successful in the Think-Pair-Share activity. What needs to be improved? Write in your TP Journal two ideas for improving your classroom organisation. Write in your TP Journal two ideas for improving your classroom organisation Transforming Teacher Education and Learning Transforming Teacher Education and Learning
www.t-tel.org 51 2 2 Year 1 - 3: Teaching Practice Student Teacher Handbook for Teaching Pratice PLAN TOGETHER PLAN TOGETHER
Prepare a lesson activity that includes a formative assessment of pupils’ learning achievement
(see Text Box). Include the formative assessment activity at the end of the Think-Pair-Share lesson
(Y2 TP Activity 3.2). Review your DBE programme notes on Formative Assessment for ideas you can
incorporate in the lesson. DO
Conduct the formative assessment lesson activities as part of the Think-Pair-Share lesson
(Y2 TP Activity 3.2). In the lesson, assess pupils’ learning and provide them with feedback on their
performance. Y2 TP Topic 4: “Lesson Sequence and Activities” Individual Teaching in Lower Primary (LP) YEAR 2 - ON-CAMPUS TP YEAR 2 - ON-CAMPUS TP Purpose: In this Topic you will practise individual teaching of a lesson focusing on sequence and
activities at Lower Primary. Activity 4.1: Lesson preparation and lesson introduction in LP classes Activity 4.2: Lesson activities in LP classes Competencies: Student Teacher demonstrates:
(i) Lesson opening: Competency 1(d); (ii) Use of TLMs: Competency 3; (iii) Learning activities:
Competency 4(a, c, g and i); (iv) Assess pupils’ understanding: Competency 8(a,e); (v) Constructive
feedback: Competency 9(a-c); (vi) Lesson closure: Competency 15(a-c); (vii) Lesson plan:
Competency 16(a-e) (see Annex 1) Student Teacher Self-Assessment Question: Student Teacher Self-Assessment Question:
To guide your learning and to help you improve your professional skills, ask yourself these ques-
tions. Write brief answers in your TP Journal and give yourself a score for each question:
• How well-prepared were you for the LP, UP and JHS TP peer-teaching activities? • How suitable do you think the activities were for JHS pupils? • How did the pupils or Student Teachers react in your lessons? (e.g. enjoyed, bored, active, etc)
Score: needs improvement = 1; satisfactory = 2; good performance = 3 Score: needs improvement = 1; satisfactory = 2; good performance = 3 52 Student Teacher Handbook for Teaching Pratice PLAN TOGETHER Plan these LP lessons together with your TP partner. In this individual teaching practice lesson you
will plan a lesson and practise how to introduce the lesson in a LP class in English, maths or science
(see Competency 1, Annex 1). Prepare in advance everything you will need to teach the lesson. Make or organise real or improvised TLMs that are appropriate for the lesson topic (see Competency 3, Annex 1). Consider whether there are enough TLMs for the pupils to use. DO
Teach the LP English, maths or science lesson introduction to your peer Student Teachers who will
be acting as LP pupils, using real or improvised TLMs that you have prepared. Teach the LP English, maths or science lesson introduction to your peer Student Teachers who will
be acting as LP pupils, using real or improvised TLMs that you have prepared. Y2 TP Week 4 - Summary of the Task When you have completed these three Y2 Week 4 individual teaching activities you will have: • practised lesson preparation and lesson introduction in the Lower Primary (LP) classes;
• delivered an individual lesson using games and songs, group work and questioning;
• conducted formative assessment including written/oral assessment and pupil feedback Y2 TP Activity 4.1: Lesson preparation and lesson introduction REFLECT TOGETHER Listen to comments from your peer Student Teachers acting as LP pupils on what they observed
about your lesson preparation and lesson introduction: what went well and what requires
improvement. Refer to Competencies 1 and 3 (Annex 1) when you reflect on the activity. Your Tutor
should also evaluate what you, as an individual teacher, did in your TP and provide comments and
constructive feedback on ways of improving your lesson preparation and lesson introduction. 53 Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 2 2 Year 1 - 3: Teaching Practice Y2 TP Activity 4.2: Games and Songs, Group Work and Q&A
(link to STPD T1 Creative Approaches; T4 Group Work; T2 Questioning) DO Teach the LP English, maths or science lesson according to your lesson plan. Include in the lesson (i)
a game or song, (ii) a group-work activity and (iii) different types of questions. Teach the LP English, maths or science lesson according to your lesson plan. Include in the lesson (i)
a game or song, (ii) a group-work activity and (iii) different types of questions. Question Types Question Types Closed Questions require a one-word
answer or single response e.g. Yes/No
answers and multiple choice. Open Questions allow for longer answers
and different answers from each pupil. The
answers can be more creative and can reveal
a pupil’s understanding. In your lesson planning think of how you will pose different types of questions (closed, open and
probing) and indicate the types of questions and strategies you will use to elicit information from
pupils in a LP class when teaching. PLAN TOGETHER In this activity you as a Student Teacher will
use the STPD T1 Creative Approaches Theme. Your Tutor will review with you the ideas from
T1 and guide you to choose an activity from
the ‘Games and Songs’ Teaching Strategies to
try out in your individual teaching. Choose a topic from the LP English, maths
or science curriculum and prepare a lesson
including group-work activities (see Text
Boxes on ‘Ways of Organising Pupils for Pair
and Group Work’ and ‘Ideas for Group-Work
Activities’, Y2 TP Activity 3.2 and STPD T4
Group Work). In your lesson planning think of how you will pose different types of questions (closed, open and
probing) and indicate the types of questions and strategies you will use to elicit information from
pupils in a LP class when teaching. In this activity you as a Student Teacher will
use the STPD T1 Creative Approaches Theme. Your Tutor will review with you the ideas from
T1 and guide you to choose an activity from
the ‘Games and Songs’ Teaching Strategies to
try out in your individual teaching. Choose a topic from the LP English, maths
or science curriculum and prepare a lesson
including group-work activities (see Text
Boxes on ‘Ways of Organising Pupils for Pair
and Group Work’ and ‘Ideas for Group-Work
Activities’, Y2 TP Activity 3.2 and STPD T4
Group Work). Question Types
Closed Questions require a one-word
answer or single response e.g. Yes/No
answers and multiple choice. Open Questions allow for longer answers
and different answers from each pupil. The
answers can be more creative and can reveal
a pupil’s understanding. Probing Questions allow pupils to answer
more fully. Student Teacher Handbook for Teaching Pratice PLAN TOGETHER In this activity you as a Student Teacher will
use the STPD T1 Creative Approaches Theme. Your Tutor will review with you the ideas from
T1 and guide you to choose an activity from
the ‘Games and Songs’ Teaching Strategies to
try out in your individual teaching. Choose a topic from the LP English, maths
or science curriculum and prepare a lesson
including group-work activities (see Text
Boxes on ‘Ways of Organising Pupils for Pair
and Group Work’ and ‘Ideas for Group-Work
Activities’, Y2 TP Activity 3.2 and STPD T4
Group Work). In your lesson planning think of how you will pose different types of questions (closed, open and
probing) and indicate the types of questions and strategies you will use to elicit information from
pupils in a LP class when teaching. DO
Teach the LP English, maths or science lesson according to your lesson plan. Include in the lesson (i)
a game or song, (ii) a group-work activity and (iii) different types of questions. REFLECT TOGETHER
Listen to comments from your peer Student Teachers who either observed you teaching pupils in a
nearby school or who acted as pupils in the CoE classroom. They should tell you what went well
and what needs improvement in your use of questions and answers and group work. Your Tutor
will evaluate your use of questions and answers and group work, and will give you comments and
constructive feedback to help you to improve your teaching skills and competencies. In this activity you as a Student Teacher will
use the STPD T1 Creative Approaches Theme. Your Tutor will review with you the ideas from
T1 and guide you to choose an activity from
the ‘Games and Songs’ Teaching Strategies to
try out in your individual teaching. Choose a topic from the LP English, maths
or science curriculum and prepare a lesson
including group-work activities (see Text
Boxes on ‘Ways of Organising Pupils for Pair
and Group Work’ and ‘Ideas for Group-Work
Activities’, Y2 TP Activity 3.2 and STPD T4
Group Work). Question Types
Closed Questions require a one-word
answer or single response e.g. Yes/No
answers and multiple choice. Open Questions allow for longer answers
and different answers from each pupil. The
answers can be more creative and can reveal
a pupil’s understanding. Probing Questions allow pupils to answer
more fully. Y2 TP Activity 4.3: Formative Assessment; written and oral assessment,
feedback on pupils’ work
(link to STPD T7 Assessment for Learning) Y2 TP Activity 4.3: Formative Assessment; written and oral assessment,
feedback on pupils’ work
(link to STPD T7 Assessment for Learning) YEAR 2 - ON-CAMPUS TP DO DO
Conduct a formative assessment of English, maths or science in an LP class using written and oral
assessment strategies and providing feedback to pupils when appropriate. You may incorporate the
formative assessment activity at the end of the Y2 week 4 Activity 4.2 lesson. Conduct a formative assessment of English, maths or science in an LP class using written and oral
assessment strategies and providing feedback to pupils when appropriate. You may incorporate the
formative assessment activity at the end of the Y2 week 4 Activity 4.2 lesson. PLAN TOGETHER Examples of Formative Assessment
Activities
•
written exercises
•
asking oral questions
•
marking pupils’ corrections before,
during and after lesson delivery
Remind Student Teachers to pay attention
to pupils’ individual academic needs during
teaching. Examples of Formative Assessment
Activities
•
written exercises
•
asking oral questions
•
marking pupils’ corrections before,
during and after lesson delivery
Remind Student Teachers to pay attention
to pupils’ individual academic needs during
teaching. In this activity you will use the STPD
Assessment for Learning Theme T7 on
Formative Assessment to conduct formative
assessment in LP classes in English, maths or
science. Plan to use both written and oral
assessment strategies when teaching pupils
and provide feedback to them accordingly. For example plan the written exercises and
oral questions to ask, and mark pupils’
exercises before, during and after lesson
delivery (see Annex 1, Teacher Competencies
8 and 9). Pay attention to pupils’ individual
academic needs when planning. REFLECT TOGETHER Listen to comments from your peer Student Teachers who either observed you teaching pupils in a
nearby school or who acted as pupils in the CoE classroom. They should tell you what went well
and what needs improvement in your use of questions and answers and group work. Your Tutor
will evaluate your use of questions and answers and group work, and will give you comments and
constructive feedback to help you to improve your teaching skills and competencies. Listen to comments from your peer Student Teachers who either observed you teaching pupils in a
nearby school or who acted as pupils in the CoE classroom. They should tell you what went well
and what needs improvement in your use of questions and answers and group work. Your Tutor
will evaluate your use of questions and answers and group work, and will give you comments and
constructive feedback to help you to improve your teaching skills and competencies. 54 Student Teacher Handbook for Teaching Pratice Y2 TP Topic 5: “Lesson Sequence and Activities” Individual Teaching in Upper Primary (UP) Purpose: In this Topic you will practise individual teaching of a lesson focusing on sequence and
activities at Upper Primary. Activity 5.1: Lesson preparation and lesson introduction in UP classes Activity 5.1: Lesson preparation and lesson introduction in UP classes Activity 5.2: Lesson activities in UP classes Activity 5.3: Assessment for learning in UP classes (formative assessment) Competencies: Student Teacher demonstrates: (i) Lesson opening: Competency 1(d); (ii) Use
of TLMs: Competency 3; (iii) Learning activities: Competency 4(a, c, g and i); (iv) Assess pupils’
understanding: Competency 8(a and e); (v) Constructive feedback: Competency 9(a-c); (vi)
Lesson closure: Competency 15(a-c); (vii) Lesson plan: Competency 16(a-e) (see Annex 1) DO Teach the UP English, maths or science lesson introduction incorporating TLMs you have prepared in
advance of the lesson, reducing teacher talk time and ensuring that both girls and boys participate
equally. Y2 TP Week 5 - Summary of the Task When you have completed these three Y2 Week 5 activities you will have: When you have completed these three Y2 Week 5 activities you will have: • practised lesson preparation and lesson introduction in Upper Primary (UP) classes; • practised delivering an individual lesson in the UP using brainstorming and Q&A; p
g
g
g
Q
;
• conducted formative assessment for learning in UP classes with reflection on what has
been learnt. REFLECT TOGETHER REFLECT TOGETHER
Your peer Student Teachers will give comments on your formative assessment activity. Your Tutors
will provide constructive feedback on your use of formative assessment to help you to improve your
teaching skills. Your peer Student Teachers will give comments on your formative assessment activity. Your Tutors
will provide constructive feedback on your use of formative assessment to help you to improve your
teaching skills. Record in your TP Journal two points you need to improve in conducting formative
assessment. Transforming Teacher Education and Learning 55 Transforming Teacher Education and Learning
www.t-tel.org 2 Year 1 - 3: Teaching Practice PLAN TOGETHER In the DBE programme you will have been introduced to lesson sequence and activities in teaching. For the individual teaching exercise this week, you will need to prepare a lesson to be delivered in
UP English, maths or science classes focusing on lesson introduction. You will need to prepare or
improvise TLMs that are appropriate for the lesson. Review the Teacher Competencies for lesson
preparation and lesson introduction (see Annex 1, Competency 3). Plan how you will reduce teacher
talk time and allow plenty of time for both girls and boys (female and male peer Student Teachers)
to participate equally in the lesson. The delivery of the lesson will need to be conducted in the CoE using peer Student Teachers
because only the introduction of the lesson is practised. So this cannot be taught very easily in a
school teaching practice situation. In the DBE programme you will have been introduced to lesson sequence and activities in teaching. For the individual teaching exercise this week, you will need to prepare a lesson to be delivered in
UP English, maths or science classes focusing on lesson introduction. You will need to prepare or
improvise TLMs that are appropriate for the lesson. Review the Teacher Competencies for lesson
preparation and lesson introduction (see Annex 1, Competency 3). Plan how you will reduce teacher
talk time and allow plenty of time for both girls and boys (female and male peer Student Teachers)
to participate equally in the lesson. The delivery of the lesson will need to be conducted in the CoE using peer Student Teachers
because only the introduction of the lesson is practised. So this cannot be taught very easily in a
school teaching practice situation. The delivery of the lesson will need to be conducted in the CoE using peer Student Teachers
because only the introduction of the lesson is practised. So this cannot be taught very easily in a
school teaching practice situation. Y2 TP Activity 5.2: Lesson activities: Brainstorming-Thought Shower; Q&A
(link to STPD T2 Questioning) YEAR 2 - ON-CAMPUS TP YEAR 2 - ON-CAMPUS TP REFLECT TOGETHER Your peer Student Teachers who act as UP pupils will give comments on what they have observed
in the lesson introduction and will give suggestions for improvement. Your Tutor will also evaluate
your lesson introduction and provide comments and constructive feedback on ways you can improve
your lesson preparation and introduction. Your peer Student Teachers who act as UP pupils will give comments on what they have observed
in the lesson introduction and will give suggestions for improvement. Your Tutor will also evaluate
your lesson introduction and provide comments and constructive feedback on ways you can improve
your lesson preparation and introduction. Student Teacher Handbook for Teaching Pratice 56 REFLECT TOGETHER Allow your peer Student Teachers who either observed you teaching pupils in a nearby school or
who acted as pupils in the CoE classroom to make comments on what they observed during your
lesson. They should provide feedback to you on what went well and what needs improvement in
the use of brainstorming, open and probing questions. Your Tutor will evaluate your use of
brainstorming, and use of open and probing questions. They will give comments and constructive
feedback to help you to improve your teaching skills and competencies. PLAN TOGETHER In this activity you will use the STPD T2 Questioning Theme. Your Tutor will review with you the
ideas from T2 and guide you to choose an activity from the Brainstorming approach to try out in
your individual teaching lesson. Choose a topic from the UP English, maths or science curriculum and
identify an appropriate brainstorming approach for the lesson. Think of how you will pose different
types of questions (closed, open and probing – see Text Box in Y2 TP Activity 4.2) and indicate the
types of questions and strategies you will use to elicit information from pupils in the UP class, for
example, probing when teaching. Prepare a lesson plan and any TLMs needed to teach the selected
activity in your individual teaching practice. In this activity you will use the STPD T2 Questioning Theme. Your Tutor will review with you the
ideas from T2 and guide you to choose an activity from the Brainstorming approach to try out in
your individual teaching lesson. Choose a topic from the UP English, maths or science curriculum and
identify an appropriate brainstorming approach for the lesson. Think of how you will pose different
types of questions (closed, open and probing – see Text Box in Y2 TP Activity 4.2) and indicate the
types of questions and strategies you will use to elicit information from pupils in the UP class, for
example, probing when teaching. Prepare a lesson plan and any TLMs needed to teach the selected
activity in your individual teaching practice. DO
Teach your lesson using the brainstorming approach that you have planned. Remember to organise
the pupils for the brainstorming activity and use open and probing questioning approaches. Y2 TP Topic 6: “Lesson Sequence and Activities” Individual Teaching in Junior High School
(JHS) Purpose: In this Topic you will practise individual teaching of a lesson focusing on sequence and
activities at Junior High School. Activity 6.1: Pre-lesson preparation and lesson introduction in JHS classes Activity 6.1: Pre-lesson preparation and lesson introduction in JHS classes Activity 6.2: Lesson Activities in JHS classes Activity 6.3: Assessment for learning in JHS classes (formative assessment) Competencies: Student Teacher demonstrates: (i) Lesson opening: Competency 1(d); (ii) Use
of TLMs: Competency 3; (iii) Learning activities: Competency 4(a, c, g and i); (iv) Assess pupils’
understanding: Competency 8(a and e); (v) Constructive feedback: Competency 9(a-c); (vi) Lesson
closure: Competency 15(a-c); (vii) Lesson plan: Competency 16(a-e) (see Annex 1) Y2 TP Activity 6.1: Lesson preparation and lesson introduction Y2 TP Activity 6.1: Lesson preparation and lesson introduction PLAN TOGETHER PLAN TOGETHER
In this activity you will use the STPD Assessment for Learning Theme T7 that you studied with your
Tutor to help you plan how to practise the skills and knowledge of classroom assessment in an UP
class. In your preparation, reflect on what you have learnt and practised in your assessment lessons
in your DBE programme. (See Text Box in Y2 TP Activity 4.3 for examples of Formative Assessment
Activities). Pay attention to pupils’ individual academic needs during your planning. In this activity you will use the STPD Assessment for Learning Theme T7 that you studied with your
Tutor to help you plan how to practise the skills and knowledge of classroom assessment in an UP
class. In your preparation, reflect on what you have learnt and practised in your assessment lessons
in your DBE programme. (See Text Box in Y2 TP Activity 4.3 for examples of Formative Assessment
Activities). Pay attention to pupils’ individual academic needs during your planning. DO
Conduct a formative assessment of English, maths or science in an UP class using written and oral
assessment strategies and providing feedback to pupils when appropriate. You may incorporate the
formative assessment activity at the end of Y2 week 5 Activity 5.2. DO
Conduct a formative assessment of English, maths or science in an UP class using written and oral
assessment strategies and providing feedback to pupils when appropriate. You may incorporate the
formative assessment activity at the end of Y2 week 5 Activity 5.2. REFLECT TOGETHER
Your peer Student Teachers may point out what went well or needs improvement when conducting
assessment activities. Listen to the overall comments and constructive feedback from your Tutor and peer
Student Teachers observations and record in your TP Journal two points that need
improvement when conducting pupil assessment. REFLECT TOGETHER
Your peer Student Teachers may point out what went well or needs improvement when conducting
assessment activities. Listen to the overall comments and constructive feedback from your Tutor and peer
Student Teachers observations and record in your TP Journal two points that need
improvement when conducting pupil assessment. 57 Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 2 Year 1 - 3: Teaching Practice When you have completed these three Y2 Week 6 activities you will have: • practised lesson preparation and lesson introduction in the JHS classes;
• delivered an individual lesson in JHS using brainstorming, and use of ICT and games;
• practised conducting assessment for learning in JHS classes. • delivered an individual lesson in JHS using brainstorming, and use of ICT and games;
• practised conducting assessment for learning in JHS classes. g
g
• practised conducting assessment for learning in JHS classes. PLAN TOGETHER Prepare the JHS English, maths or science lesson plan and any TLMs you will need in advance of
teaching the lesson. In your lesson preparation you will need to prepare or improvise TLMs that are
appropriate for the lesson to be delivered. In the individual teaching practice lesson you will practise
how to introduce an English, maths or science lesson in a JHS class. Review the Teacher Competencies for lesson preparation and lesson introduction (see Annex 1,
Competency 1) and use of TLMs (Competency 3), as an indication of what is expected of you. The lesson will need to be conducted in the CoE using peer Student Teachers, as only the introduction
of the lesson is practised. This cannot be taught very easily in a school classroom situation. Prepare the JHS English, maths or science lesson plan and any TLMs you will need in advance of
teaching the lesson. In your lesson preparation you will need to prepare or improvise TLMs that are
appropriate for the lesson to be delivered. In the individual teaching practice lesson you will practise
how to introduce an English, maths or science lesson in a JHS class. Review the Teacher Competencies for lesson preparation and lesson introduction (see Annex 1,
Competency 1) and use of TLMs (Competency 3), as an indication of what is expected of you. The lesson will need to be conducted in the CoE using peer Student Teachers, as only the introduction
of the lesson is practised. This cannot be taught very easily in a school classroom situation. DO
Teach the introduction to the JHS lesson using the activities/materials in a way that will engage,
stimulate and sustain the pupils’ interest. Reduce teacher talk time and allow girls and boys to
participate fully and equally in the lesson. REFLECT TOGETHER REFLECT TOGETHER
Your peer Student Teachers who act as JHS pupils will give comments on what they observed in
the lesson introduction and will give suggestions for improvement. Your Tutor should also evaluate
your lesson introduction and provide comments and constructive feedback on ways in which you can
improve your lesson preparation and introduction. Your peer Student Teachers who act as JHS pupils will give comments on what they observed in
the lesson introduction and will give suggestions for improvement. Your Tutor should also evaluate
your lesson introduction and provide comments and constructive feedback on ways in which you can
improve your lesson preparation and introduction. Your peer Student Teachers who act as JHS pupils will give comments on what they observed in
the lesson introduction and will give suggestions for improvement. Your Tutor should also evaluate
your lesson introduction and provide comments and constructive feedback on ways in which you can
improve your lesson preparation and introduction. DO Teach the English, maths or science lesson according to your lesson plan, using thought shower
activities, questions, games and puzzles. Remember to encourage equal participation of girls
and boys. REFLECT TOGETHER Listen to comments from your peer Student Teachers who either observed you teaching pupils in
a nearby school or acted as pupils in the CoE classroom. They should describe what went well and
suggest what needs improvement. Your Tutor will evaluate your use of thought shower, questioning
and games, and will give comments and constructive feedback to help you to improve your Teacher
Competencies and skills. Listen to comments from your peer Student Teachers who either observed you teaching pupils in
a nearby school or acted as pupils in the CoE classroom. They should describe what went well and
suggest what needs improvement. Your Tutor will evaluate your use of thought shower, questioning
and games, and will give comments and constructive feedback to help you to improve your Teacher
Competencies and skills. Y2 TP Activity 6.2: Lesson Activities: Brainstorm; Q&A: ICT; Games
(link to STPD T2 Questioning; T11 Use of ICT; STPD T1 Creative Approaches) In this activity, you will use the STPD Questions; STPD T11 Digital Technology for Learning and STPD
T1 Creative Approaches on Games Teaching Strategies you studied with your Tutor. In this activity, you will use the STPD Questions; STPD T11 Digital Technology for Learning and STPD
T1 Creative Approaches on Games Teaching Strategies you studied with your Tutor. Student Teacher Handbook for Teaching Pratice 58 Your Tutor will review with you the ideas from T11 and T1 and guide you to choose an activity
from brainstorming, digital technology for learning, or Games and Puzzles to try out in your
individual teaching. YEAR 2 - ON-CAMPUS TP Choose a topic from the JHS English, maths or science curriculum and prepare a brainstorming
activity which will encourage girls and boys to participate equally, making reference to STPD
T11 and STPD T1 Themes. In your lesson planning think of how you will let pupils brainstorm and pose different types of
questions (closed, open and probing – see Text Box in Y2 TP Activity 4.2) and indicate the types
of questions and strategies you will use to elicit information from pupils in a JHS class during the
teaching practice. Prepare a lesson plan for English, maths or science in your chosen topic and
include an activity using ICT. Y2 Post TP Topic: Post-On-Campus Teaching Practice Activities (college-based) ): Peer and
Individual Teaching Purpose: In this Topic you will reflect on the On-Campus TP activities in LP, UP and JHS classes
Post-TP Activity 1: Review of Teaching Standards and Competencies for Year 2
Post-TP Activity 2: Reflection and assessment of the On-Campus TP tasks Y2 Post-TP Activities - Summary of the Task When you have completed these two Y2 Post-On-Campus activities with your Tutor you will have: • reviewed and understood the teaching standards and Teacher Competencies; • reviewed and understood the teaching standards and Teacher Competencies;
• reflected on and assessed your On-Campus TP tasks. • reflected on and assessed your On-Campus TP tasks. Y2 Post-TP Activity 1: Review of Teaching Standards and Competencies for Year 2 PLAN TOGETHER PLAN TOGETHER
In this activity you will use the STPD Theme T7 Assessment for Learning to plan how to conduct an
assessment of pupils’ learning in JHS classes in English, maths or science. Use both written and oral
assessment strategies in the TP lesson (see Text Box in Y2 TP Activity 4.3 for Examples of Formative
Assessment Activities). Pay attention to pupils’ individual learning needs when planning. Revisit the Teacher Competencies for formative assessment (see Annex 1, Competencies 8 and 9). PLAN TOGETHER
In this activity you will use the STPD Theme T7 Assessment for Learning to plan how to conduct an
assessment of pupils’ learning in JHS classes in English, maths or science. Use both written and oral
assessment strategies in the TP lesson (see Text Box in Y2 TP Activity 4.3 for Examples of Formative
Assessment Activities). Pay attention to pupils’ individual learning needs when planning. Revisit the Teacher Competencies for formative assessment (see Annex 1, Competencies 8 and 9). DO
Conduct a formative ‘assessment for learning’ activity for English, maths or science in a JHS class
using written and oral assessment strategies. Provide feedback to pupils as appropriate. Incorporate
the formative assessment activity at the end of the Y2 week 6 Activity 6.2 lesson. REFLECT TOGETHER
Listen to your peer Student Teachers’ comments on what went well or needs improvement while
conducting assessment for learning of the pupils. Also listen to the overall comments and
constructive feedback from your Tutor. Record in your TP Journal two points that you need to
improve when conducting formative assessment. STUDENT TEACHER SELF-REFLECTION
Ask yourself the following questions:
1. What new knowledge/skill have I learnt during the Y3 TP Weeks 4 to 6? 2. How can I use this in my classroom teaching in future? 3. What can I do differently to improve my teaching in future? Transforming Teacher Education and Learning
www.t-tel.org 59 2 Year 1 - 3: Teaching Practice Student Teacher Handbook for Teaching Pratice PLAN TOGETHER PLAN TOGETHER
Your Tutor will discuss the outcomes of the Y2 TP with you in the Post-On-Campus TP activities. A review of your TP Journal and the activities you have undertaken in Y2 TP will bring out some of
the Teacher Competencies you have developed during the Y2 On-Campus TP. You will be able to
identify those Teacher Competencies you found more difficult and the competencies you found
easier to demonstrate, and at which levels of LP, UP and JHS. Your Tutor will discuss each of the
Teacher Competencies (see Annex 1) and how they are demonstrated, observed and assessed in the
teaching practice situation. Your Tutor will discuss the outcomes of the Y2 TP with you in the Post-On-Campus TP activities. A review of your TP Journal and the activities you have undertaken in Y2 TP will bring out some of
the Teacher Competencies you have developed during the Y2 On-Campus TP. You will be able to
identify those Teacher Competencies you found more difficult and the competencies you found
easier to demonstrate, and at which levels of LP, UP and JHS. Your Tutor will discuss each of the
Teacher Competencies (see Annex 1) and how they are demonstrated, observed and assessed in the
teaching practice situation. DO
Review your TP Journal log of feedback and reflection on each lesson and identify from your notes
all the skills and competencies that you need to improve, e.g. notes from Y2 TP Activities 5.1, 5.2 and
5.3. Summarise in your TP Journal the Teacher Competencies that you need to work on and improve
in Y3 TP. Note the levels (LP, UP or JHS) and the subjects in which these were most challenging. REFLECT TOGETHER REFLECT TOGETHER
Your Tutor may ask you how you fared during the On-Campus Teaching Practice. Reflect on any
observable differences between the participation and involvement in class activities of the girls and
the boys in the schools where you taught. Were there differences in the TP experience for female
Student Teachers compared to male Student Teachers? Your Tutor may ask you how you fared during the On-Campus Teaching Practice. Reflect on any
observable differences between the participation and involvement in class activities of the girls and
the boys in the schools where you taught. Were there differences in the TP experience for female
Student Teachers compared to male Student Teachers? REFLECT TOGETHER REFLECT TOGETHER
Your Tutor will guide you to think or reflect on those Teacher Competencies you need to improve. Discuss with your TP partner/TP group how to improve on these Teacher Competencies. Your Tutor will guide you to think or reflect on those Teacher Competencies you need to improve. Discuss with your TP partner/TP group how to improve on these Teacher Competencies. 60 Student Teacher Handbook for Teaching Pratice DO DO
Discuss first with your TP partner/TP group and then with your Tutor the highlights of your
experiences during the On-Campus Teaching Practice – the most challenging activity and why; the
most enjoyable activity (for you/for the pupils) and why; the easiest activity (for you/for the pupils)
and why. Y2 Post-TP Activity 2: Reflection and assessment of the On-Campus tasks YEAR 2 - ON-CAMPUS TP YEAR 2 - ON-CAMPUS TP PLAN TOGETHER
Plan with your Tutor the criteria you will use to reflect on and assess your performance during the
On-Campus TP, considering the following points: PLAN TOGETHER
Plan with your Tutor the criteria you will use to reflect on and assess your performance during the
On-Campus TP, considering the following points: • your involvement during the Y2 teaching practice lessons; • your involvement during the Y2 teaching practice lessons;
• the number of times you taught in the class or the school;
• the feedback you had from your Tutor and TP partner/group;
• preparation and assessment of a portfolio of your own teaching learning materials;
• your TP Journal. • the number of times you taught in the class or the school; the number of times you taught in the class or the school;
• the feedback you had from your Tutor and TP partner/group;
• preparation and assessment of a portfolio of your own teaching DO
Discuss first with your TP partner/TP group and then with your Tutor the highlights of your
experiences during the On-Campus Teaching Practice – the most challenging activity and why; the
most enjoyable activity (for you/for the pupils) and why; the easiest activity (for you/for the pupils)
and why. Student Teacher Handbook for Teaching Pratice STUDENT TEACHER SELF-REFLECTION Finally, in your TP Journal write two examples of teaching activities that you did with pupils
during the Y2 On-Campus Teaching Practice - one example that you felt was your most
successful activity and explain why; a second example that you felt was least successful and
explain why and how you would improve it in future. Transforming Teacher Education and Learning Transforming Teacher Education and Learning
www.t-tel.org 61 3 Year 1 - 3: Teaching Practice Student Teacher Handbook for Teaching Pratice 62 YEAR 3 - OFF-CAMPUS TP Y3 - Off Campus Teaching Practice Activities •
Pre-Off-Campus Teaching Practice activities to be undertaken by the Tutors at the start of
Semester 1, in the CoE for all Year 3 Student Teachers; •
Pre-Off-Campus Teaching Practice activities to be undertaken by the Tutors at the start of
Semester 1, in the CoE for all Year 3 Student Teachers; f •
one topic for each of the 12 Off-Campus Teaching Practice weeks in Semester 1 with two
to three activities to be undertaken EACH WEEK by the Student Teacher with their Lead
Mentor and Mentor and recorded in their TP Journal; •
Post-Off-Campus Teaching Practice activities to be undertaken by Tutors and Student
Teachers after completion of the Off-Campus TP at the end of Semester 1, in the CoE for all
Year 3 Student Teachers. Purpose: In Year 3 Off-Campus Teaching Practice: Student Teachers will be able to demonstrate questioning and communication skills in their
lesson delivery. They will exhibit knowledge of appropriate use of TLMs and be able to assess
their pupils’ learning effectively after teaching. The Student Teachers should arrange to meet with their mentor each morning for planning and
each evening for review and exchange of information gathered. At the end of each week, Reflective Practice meetings will be scheduled on a regular basis
throughout the Y3 TP period. The Lead Mentors/Head Teachers will be aware of their supervisory roles. The Mentors will be aware of their responsibilities to support Student Teachers in working to The Lead Mentors/Head Teachers will be aware of their supervisory roles. Th M
ill b
f h i
ibili i
S
d
T
h
i
ki The Mentors will be aware of their responsibilities to support Student Teachers in working to
achieve the goals of the Off-Campus TP programme. The Mentors will be aware of their responsibilities to support Student Teachers in working to
achieve the goals of the Off-Campus TP programme. The CoE Tutors will visit TP schools to supervise, evaluate and support Student Teachers.fifi The CoE Tutors will visit TP schools to supervise, evaluate and support Student Teachers. The TPC will be the liaison officer of the CoE, District Education Office and TP schools and will
organise training for Lead Mentors, Mentors and Link Tutors. The CoE Tutors will visit TP schools to supervise, evaluate and support Student Teachers. The TPC will be the liaison officer of the CoE, District Education Office and TP schools and will
organise training for Lead Mentors, Mentors and Link Tutors. The TPC will be the liaison officer of the CoE, District Education Office and TP schools and will
organise training for Lead Mentors, Mentors and Link Tutors. The GEO and CS will represent the District Directorate through visits to TP schools to monitor the
activities of Student Teachers and Mentors. The GEO takes a particular interest in female Student
Teachers in the schools. Teaching Practice Framework: Year 3 Off-Campus Teaching Practice You will be assessed based on your
performance against the Teacher
Competencies given in Annex 1 with some
specific examples shown in the Text Box. Examples of Y3 TP skills and competencies
to be assessed
The Student Teacher uses effective
strategies:
•
to open the lesson
•
to give pupils explanations for new
knowledge, skills and concepts
•
to facilitate learning through use of TLMs
List in your TP Journal ten key competencies on which you will be assessed
(Annex 1 Teacher Competencies). Y3 Pre-TP Topic: Pre-Off-Campus TP Activities (college-based)
Purpose: This Topic will prepare you for the 12-week Off-Campus Teaching Practice
Pre-TP Activity 1: TP Orientation - Teacher Competencies and Y3 TP Assessment Procedures
Pre-TP Activity 2: Using the TP Journal
Pre-TP Activity 3: Lead Mentor, Mentor and Student Teacher professional relationship and roles
TOPICS AND ACTIVITIES FOR YEAR 3 OFF-CAMPUS TEACHING PRACTICE Y3 Pre-TP Topic: Pre-Off-Campus TP Activities (college-based)
Purpose: This Topic will prepare you for the 12-week Off-Campus Teaching Practice
Pre-TP Activity 1: TP Orientation - Teacher Competencies and Y3 TP Assessment Procedures
Pre-TP Activity 2: Using the TP Journal
Pre-TP Activity 3: Lead Mentor, Mentor and Student Teacher professional relationship and roles
TOPICS AND ACTIVITIES FOR YEAR 3 OFF-CAMPUS TEACHING PRACTICE TOPICS AND ACTIVITIES FOR YEAR 3 OFF-CAMPUS TEACHING PRACTICE DO DO
Attend the orientation to prepare for your Y3 TP
In your TP Journal write a summary of how you will be assessed in Y3 TP by your Tutor, and your
school-based Lead Mentor and Mentor. Attend the orientation to prepare for your Y3 TP
In your TP Journal write a summary of how you will be assessed in Y3 TP by your Tutor, and your
school-based Lead Mentor and Mentor. List in your TP Journal ten key competencies on which you will be assessed
(Annex 1 Teacher Competencies). List in your TP Journal ten key competencies on which you will be assessed
(Annex 1 Teacher Competencies). When you have completed these three Y3 Pre-TP activities with your Tutor, you will have: When you have completed these three Y3 Pre-TP activities with your Tutor, you will have: • acquired more insight into the use of the TP Journal for Y3 TP; • clarified the roles of the Lead Mentor, Mentor and the Student Teacher. Y3 Pre-Off-Campus TP Activity 1: What are the specific skills and Teacher
Competencies to be practised in Y3 TP and how are they assessed? PLAN TOGETHER Before you begin your Off-Campus Teaching
Practice, you will go through a formal
orientation with your Tutor. You are already
familiar with the use of P-D-R, and this will
feature across all activities. With your
Tutor and TP group review the Teacher
Competencies (see Annex 1) and the summary
information about how you will be assessed
during your Y3 TP as part of your final grading
(see Annex 4 Assessment Procedures). You will be assessed based on your
performance against the Teacher
Competencies given in Annex 1 with some
specific examples shown in the Text Box. Teaching Practice Framework: Year 3 Off-Campus Teaching Practice With your
Tutor and TP group review the Teacher
Competencies (see Annex 1) and the summary
information about how you will be assessed
during your Y3 TP as part of your final grading
(see Annex 4 Assessment Procedures). You will be assessed based on your
performance against the Teacher
Competencies given in Annex 1 with some
specific examples shown in the Text Box. Examples of Y3 TP skills and competencies
to be assessed
The Student Teacher uses effective
strategies:
•
to open the lesson
•
to give pupils explanations for new
knowledge, skills and concepts
•
to facilitate learning through use of TLMs
List in your TP Journal ten key competencies on which you will be assessed
(Annex 1 Teacher Competencies). Y3 Pre-TP Topic: Pre-Off-Campus TP Activities (college-based)
Purpose: This Topic will prepare you for the 12-week Off-Campus Teaching Practice
Pre-TP Activity 1: TP Orientation - Teacher Competencies and Y3 TP Assessment Procedures
Pre-TP Activity 2: Using the TP Journal
Pre-TP Activity 3: Lead Mentor, Mentor and Student Teacher professional relationship and roles
When you have completed these three Y3 Pre-TP activities with your Tutor, you will have:
• information about specific Teacher Competencies and skills, and assessment procedures;
• acquired more insight into the use of the TP Journal for Y3 TP;
• clarified the roles of the Lead Mentor, Mentor and the Student Teacher. Y3 Pre-Off-Campus TP Activity 1: What are the specific skills and Teacher
Competencies to be practised in Y3 TP and how are they assessed? PLAN TOGETHER
DO
Attend the orientation to prepare for your Y3 TP
In your TP Journal write a summary of how you will be assessed in Y3 TP by your Tutor, and your
school-based Lead Mentor and Mentor. REFLECT TOGETHER
i h
d P
Y3 Pre-Off-Campus TP - Summary of the Task
TOPICS AND ACTIVITIES FOR YEAR 3 OFF-CAMPUS TEACHING PRACTICE
Before you begin your Off-Campus Teaching
Practice, you will go through a formal
orientation with your Tutor. You are already
familiar with the use of P-D-R, and this will
feature across all activities. With your
Tutor and TP group review the Teacher
Competencies (see Annex 1) and the summary
information about how you will be assessed
during your Y3 TP as part of your final grading
(see Annex 4 Assessment Procedures). Teaching Practice Framework: Year 3 Off-Campus Teaching Practice Pre-Off Campus Teaching Practice Activities (college-based)
Topic 1. Introduction to Teaching Practice by Lead Mentor/Mentor
Topic 2. Introducing a lesson
Topic 3. Deliver the lesson and manage the class
Topic 4. Practise questioning and close the lesson
Topic 5. Practise questioning and communication skills
Topic 6. Use TLMs to assess students’ learning: Formative Assessment
Topic 7. Identify learning needs and plan a teaching activity: Formative Assessment
Topic 8. Conduct an assessment and use results in teaching: Formative Assessment
Topic 9. Identify learning needs and provide appropriate support: Formative Assessment
Topic 10. Prepare a test and marking scheme: Summative Assessment
Topic 11. Administer the test, score and record test results: Summative Assessment
Topic 12. Interpret test results: Formative and Summative Assessment
Post-Off-Campus Teaching Practice Activities (college-based) Transforming Teacher Education and Learning
www.t-tel.org 63 3 Year 1 - 3: Teaching Practice Y3 Pre-TP Topic: Pre-Off-Campus TP Activities (college-based)
Purpose: This Topic will prepare you for the 12-week Off-Campus Teaching Practice
Pre-TP Activity 1: TP Orientation - Teacher Competencies and Y3 TP Assessment Procedures
Pre-TP Activity 2: Using the TP Journal
Pre-TP Activity 3: Lead Mentor, Mentor and Student Teacher professional relationship and roles
When you have completed these three Y3 Pre-TP activities with your Tutor, you will have:
• information about specific Teacher Competencies and skills, and assessment procedures;
• acquired more insight into the use of the TP Journal for Y3 TP;
• clarified the roles of the Lead Mentor, Mentor and the Student Teacher. Y3 Pre-Off-Campus TP Activity 1: What are the specific skills and Teacher
Competencies to be practised in Y3 TP and how are they assessed? PLAN TOGETHER
DO
Attend the orientation to prepare for your Y3 TP
In your TP Journal write a summary of how you will be assessed in Y3 TP by your Tutor, and your
school-based Lead Mentor and Mentor. REFLECT TOGETHER
With your Tutor and TP group:
Y3 Pre-Off-Campus TP - Summary of the Task
TOPICS AND ACTIVITIES FOR YEAR 3 OFF-CAMPUS TEACHING PRACTICE
Before you begin your Off-Campus Teaching
Practice, you will go through a formal
orientation with your Tutor. You are already
familiar with the use of P-D-R, and this will
feature across all activities. Y3 Pre-TP Activity 3: How the Mentor and Student Teacher work together PLAN TOGETHER
Before you begin your Y3 TP, your Tutor will help you to become familiar with your roles and
responsibilities as a Student Teacher and the roles and responsibilities of your Mentor. This will also
include an explanation of the roles and responsibilities of the Lead Mentor. PLAN TOGETHER
Before you begin your Y3 TP, your Tutor will help you to become familiar with your roles and
responsibilities as a Student Teacher and the roles and responsibilities of your Mentor. This will also
include an explanation of the roles and responsibilities of the Lead Mentor. Your Tutor will guide you to work together with your TP group to review the roles and
responsibilities of Mentors and Student Teachers, outlined in the Student Teacher Handbook
Introduction section. Consider how you will work together with your Mentor during the Y3 TP. DO
Fill in the blank spaces in each column selecting from the bullet points below the table, to show the
roles or responsibilities for each designated position. Some bullet pointed roles may apply to more
than one designated position. Copy the blank ‘Checklist of Roles and Responsibilities’ (below) into your TP Journal. Your Tutor will guide you to work together with your TP group to review the roles and
responsibilities of Mentors and Student Teachers, outlined in the Student Teacher Handbook
Introduction section. Consider how you will work together with your Mentor during the Y3 TP. REFLECT TOGETHER With your Tutor and TP group: • review the Teacher Competencies listed and identify what new knowledge about Teacher
Competencies you have learned in Y1 Observation TP and Y2 On-Campus TP; • ask for clarifications on the information you have gathered on Teacher Competencies and
assessment of Y3 TP. What do you think are the two strongest competencies you possess and which two competencies do
you need to work hardest to improve? Student Teacher Handbook for Teaching Pratice 64 Y3 Pre-TP Activity 2: Using the TP Journal REFLECT TOGETHER State three examples of information you previously recorded in your TP Journal in Y1 and Y2 TP that
you find most useful and interesting to re-read now. State three activities you think you will be recording in the TP Journal during Y3, after your
discussion with your TP partner. Review with your TP partner the first two TP Journal activities for Y3 TP Week 1 that you have
identified. Ask your Tutor for clarification if the instructions for those two activities are not clear. Y3 Pre-TP Activity 3: How the Mentor and Student Teacher work together PLAN TOGETHER Your Tutor and TPC will provide you with guidance and insight into the recording of your daily
activities. It is important for you to remember that the TP Journal forms part of your assessment. You must therefore keep it updated in line with the TP activities. You must always carry your
TP Journal with you whenever you are in school, so that it can be shared with your TP partner,
Mentor or Tutor. DO
First, review all the information you have recorded in your TP Journal during Y1 and Y2 TP. Then study the Y3 section of the TP Handbook to find out what you will have to record in your
TP Journal. Discuss with your TP partner, what you might record in the TP Journal in Y3. With reference to your Student Teacher Handbook identify the first two activities you will have to
record in your TP Journal during Y3 TP Week 1. Copy the blank ‘Checklist of Roles and Responsibilities’ (below) into your TP Journal. Student Teacher Handbook for Teaching Pratice 66 YEAR 3 - OFF-CAMPUS TP Y3 TP Topic 1: Introduction to Teaching Practice School by Lead Mentor/Mentor
Purpose: In this Topic you will observe school routines, school rules and management of
day-to-day affairs in the school
Activity 1.1: Prepare a Plan for the 12-week Teaching Practice period
Activity 1.2: Prepare School Profile Report based on the TP Y1 Day 1 Activity “Familiarisation
with School Organisation”
Competencies Student Teacher demonstrates: (i) Skills of lesson planning: Competency 16(f);
(ii) Use of different interactive methods to source information: Competency 4(a) (see Annex 1) Y3 TP Topic 1: Introduction to Teaching Practice School by Lead Mentor/Mentor
Purpose: In this Topic you will observe school routines, school rules and management of
day-to-day affairs in the school
Activity 1.1: Prepare a Plan for the 12-week Teaching Practice period
Activity 1.2: Prepare School Profile Report based on the TP Y1 Day 1 Activity “Familiarisation
with School Organisation”
Competencies Student Teacher demonstrates: (i) Skills of lesson planning: Competency 16(f);
(ii) Use of different interactive methods to source information: Competency 4(a) (see Annex 1) Y3 TP Topic 1: Introduction to Teaching Practice School by Lead Mentor/Mentor
Purpose: In this Topic you will observe school routines, school rules and management of
day-to-day affairs in the school
Activity 1.1: Prepare a Plan for the 12-week Teaching Practice period
Activity 1.2: Prepare School Profile Report based on the TP Y1 Day 1 Activity “Familiarisation
with School Organisation”
Competencies Student Teacher demonstrates: (i) Skills of lesson planning: Competency 16(f);
(ii) Use of different interactive methods to source information: Competency 4(a) (see Annex 1) Competencies Student Teacher demonstrates: (i) Skills of lesson planning: Competency 16(f);
(ii) Use of different interactive methods to source information: Competency 4(a) (see Annex 1) Y3 TP Week 1 - Summary of the Task When you have completed these two activities for Y3 TP Week 1 with your Mentor you will have: • prepared a 12-week Teaching Practice Plan with your Mentor; • prepared and recorded a School Profile Report; Y3 TP Activity 1.1: Mentor and Student Teachers prepare 12-week TP Plan PLAN TOGETHER
During your Off-Campus Teaching Practice, you will be working with your Mentor and TP partner
for 12 weeks following a programme in which you gradually take on more and more responsibility
for teaching the class. At the beginning, you will need time to become familiar with the school and
classroom routines, and you will begin learning from observing lessons with your Mentor. An important part of the final teaching practice will be your 12-week Teaching Practice Plan. You
will need to work with your Mentor and TP partner to think carefully about the tasks that will be
undertaken each week. In this activity, you will prepare the first version of the Teaching Practice
Plan. You and your Mentor may decide that some changes need to be made later on but this first
draft will help you to map out in your mind all the activities that will be covered during the 12-week
TP. By mapping out the activities in this way you will be able to see the developmental stages in
learning from observing your Mentor; you will practise each teaching skill and reflect with your
Mentor on the progress that you will make each week. When you have made your 12-week Teaching Practice Plan, you will find it helpful as a guide to
writing your weekly Teaching Practice Lesson Plans. Copy the blank ‘Checklist of Roles and Responsibilities’ (below) into your TP Journal. Fill in the blank spaces in each column selecting from the bullet points below the table, to show the
roles or responsibilities for each designated position. Some bullet pointed roles may apply to more
than one designated position. Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 65 3 Year 1 - 3: Teaching Practice CHECKLIST OF ROLES AND RESPONSIBILITIES
MENTOR/LEAD MENTOR
STUDENT TEACHER
TUTOR
•
•
•
•
•
•
•
•
•
•
Visits Student Teachers on routine school
supervision
•
Prepares TP lesson plans
•
Reflects on daily activities
•
Records daily experiences in the TP
Journal
•
Marks pupils’ exercises
•
Marks daily attendance of pupils
•
Provides feedback on pupils’ work
•
Meets parents of individual pupils
•
Controls pupils in class
•
Vets Student Teachers’ prepared lesson plans
•
Supervises Student Teachers whilst teaching
•
Observes lessons
•
Takes pupils on learning trips
•
Assesses pupils after teaching
•
Gives pupils homework
•
Supports Student Teachers whilst teaching
•
Helps Student Teachers to prepare TLMs
•
Searches for Realia to support teaching CHECKLIST OF ROLES AND RESPONSIBILITIES REFLECT TOGETHER
With your Tutor and TP group, reflect together on the following questions: • What new knowledge have you acquired from the Y3 TP orientation progr
• What are the next steps in preparing for Y3 TP? • What is the first thing you should do on arrival at your TP school? • What should you do each day during your Y3 TP? • How will you work with your TP partner during the Y3 TP? • What new knowledge have you acquired from the Y3 TP orientation programme? • What are the next steps in preparing for Y3 TP? • What is the first thing you should do on arrival at your TP school? • What should you do each day during your Y3 TP? • How will you work with your TP partner during the Y3 TP? Student teachers must encourage female pupils to develop their interest and participation in science lessons. Student teachers must encourage female pupils to develop their interest and participation in science lessons. Prepare a 12-week TP Plan Review the list of topics covered in the Y3 TP Framework. Use this to prepare a12-week Teaching
Practice Plan, including the resources and information you might need and 2-3 bullet points of
what you will do and what your Mentor will do against each week/topic. You may add more bullet
points later. You could set out your Teaching Practice Plan like this: Transforming Teacher Education and Learning
www.t-tel.org 67 Transforming Teacher Education and Learning 3 Year 1 - 3: Teaching Practice Make a note of any points you need to check with your Mentor when you meet. TOPIC
What I will do …
What my Mentor will do …
Resources/Information
Week 1
TP
Orientation
•
Observe the
school routines
•
Observe a boy
and a girl in the
TP class
•
Make a classroom
seating plan
•
Introduce me to the
class
•
Share the class rules
and timetable
•
TP Journal and
notebook
•
Notes about what to
observe in the
classroom
Week 2
Lesson
Introduction
•
Prepare a lesson
plan for five
lesson introduc-
tions (one each
day) – working
with TP partner
•
Observe and
make notes when
the Mentor
introduces
lessons
•
Help me to choose a
lesson (one for each
day)
•
Discuss with me what
should be included in
the lesson introduction. •
Class timetable
•
Textbooks/syllabus/
teacher’s guide for
grade/subjects
•
How the children are
grouped
Week 3
Week 4
Week 5 etc Make a note of any points you need to check with your Mentor when you meet. Make a note of any points you need to check with your Mentor when you meet. REFLECT TOGETHER
Arrange a time when you can share your completed 12-week Teaching Practice Plan with your
Mentor and TP partner. REFLECT TOGETHER
Arrange a time when you can share your completed 12-week Teaching Practice Plan with your
Mentor and TP partner. Arrange a time when you can share your completed 12-week Teaching Practice Plan with you
Mentor and TP partner. Discuss your Teaching Practice Plan and make a note of any changes that are suggested during the
discussion. Remember to ask any questions that you thought about during the planning process. Y3 TP Activity 1.2: Prepare a School Profile Report DO
Make a School Profile Checklist DO Note 1: your Tutor may have helped you to prepare, during the Y3 Pre-TP activities or as part of your
Y1 TP activities, a Checklist of information that you will need to gather about your TP school - if so,
refer to that Checklist and update or develop it as necessary. Systematically gather and organise the information for your School Profile Report and record it in
your TP Journal. Note 2: There will be too much information to record everything. Decide which information you
will need to refer to frequently, for example the times the school starts and closes, names of key
members of staff, and number of pupils in each class showing number of girls and number of boys,
etc. Decide which information you do not need to record in detail. Record where the information can
be found, for example the class register and the school rules. Check the accuracy and correctness of information with your TP partner and also with your Mentor. REFLECT TOGETHER
Think about whether there are any important gaps in the information you have gathered for your
School Profile by reviewing the checklist and comparing the information you have recorded with
that of your TP colleagues. scuss with your Mentor and ask for any clarifications on the information you have gathered. Discuss with your Mentor and ask for any clarifications on the information you have gathered. Discuss with your Mentor and the other TP Student Teachers the information you found out about
the number of boys and girls in each class. What are the interesting points about this information? Discuss with your Mentor and the other TP Student Teachers the information you found out about
the number of boys and girls in each class. What are the interesting points about this information? Y3 TP Activity 1.2: Prepare a School Profile Report PLAN TOGETHER
During your Year 1 School Observation TP, you will have gathered information about the school you
visited, such as daily routines in the school and in the classroom, school rules and seating plans. In
the Year 1 section of the Teaching Practice Handbook for Student Teachers, the activities for Day
1 describe the task of “Familiarisation with School Organisation”. Look back at that task and use the
same ideas for preparing a School Profile for your current TP school. This task will help you to gather
all the important information you will need to refer to throughout your 12-week teaching practice. Plan with the other Student Teachers in your TP school and, if necessary, divide out the tasks
between your TP colleagues; then share information that you gather. Arrange to meet together
each morning for planning and each evening for review and exchange of information gathered. Your Mentor and Lead Mentor (Head Teacher) are there to help you so remember to ask them if you
cannot find the information you need. They will also provide you with an orientation of the school. Student Teacher Handbook for Teaching Pratice 68 STUDENT TEACHER SELF-ASSESSMENT EXERCISE: • In which classes are there more boys than girls and in which classes are there more
girls than boys? What do you think this information tells you about the gender balance
in your school? What might explain any differences in the gender balance in different
classes? • What is the gender balance among the teachers in your school? What are your
observations on this point? • Have you found any information about pupils with disabilities during your information
gathering? If not can you find out some information to complete this part of the school
profile. If you have found out some information on disabilities in the school what are
the three key points you have observed? Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 69 3 Year 1 - 3: Teaching Practice Y3 TP Topic 2: Introduce a lesson
Student Teacher observes the Mentor and they co-teach a lesson
Purpose: In this Topic you will gain an awareness of stages of the lesson and lesson delivery
Activity 2.1: Prepare an observation checklist to focus on stages of the lesson and lesson delivery
Activity 2.2: Mentor and Student Teacher co-teach a lesson each day – the Student Teacher
delivers the lesson introduction
Activity 2.3: Mentor and Student Teacher reflect together on both the lesson observed and the
co-taught lesson each day
Competencies: Student Teacher demonstrates: (i) Use of strategies to open the lesson:
Competency 1( a-e); (ii) Use of strategies to close the lesson: Competency 15(a-c) (see Annex 1) Y3 TP Activity 2.1: Prepare and use an observation checklist for stages of the lesson
and lesson delivery Y3 TP Activity 2.1: Prepare and use an observation checklist for stages of the lesson
and lesson delivery Student Teacher Handbook for Teaching Pratice Y3 TP Week 2 - Summary of the Task • reflected on lesson observed and the co-taught lesson. PLAN TOGETHER Throughout your 12-week Off-Campus Teaching Practice, you will plan your lessons with the support
of your Mentor. It will be necessary for you to observe a whole lesson delivery by your Mentor whilst
you take notes on various steps that take place. You will discuss with your Mentor the lesson you are
going to observe. As you prepare to teach your first lesson introduction (see Activity 2.2), your
Mentor will discuss with you what should go into the introduction. You will need to study the class timetable, syllabus, textbooks and teacher’s guide/handbook
which are the major sources/reference books. You should also look for other approved
supplementary materials. 70 Student Teacher Handbook for Teaching Pratice REFLECT TOGETHER
Lesson Observation Activity: Tick each example of Good Teaching that you
observed
TICK
1. Discusses the topic that is going to be taught
2. Explains difficult things clearly
3. Allows pupils to ask questions
4. Brings pictures or objects to help teach the lesson and organises them well
5. Uses activities like games, songs, stories or role play to help teach the lesson
6. Asks questions about what pupils think about the topic
7. Asks analytical questions like, ‘Why do you think this?’
8. Encourages quiet pupils to speak
9. Organises pupils well to work together in groups or pairs
10. Allows pupils to mark/review each other’s work
11. Distributes questions evenly to girls and boys (gender sensitive)
12. Tells pupils how they can improve and encourages them in their work
13. Gives extra help to pupils who are having trouble understanding
14. Encourages girls to take leadership roles
15. Treats boys and girls equally and fairly
16. Uses signals and symbols including songs to control the class
17. Makes sure the boys and girls are mixed in the classroom seating arrangement
18. Gets pupils to stop talking or pay attention without shouting
19. Goes over the important things he/she has taught
20. Makes the subject interesting and easy to understand
DO
Prepare an observation checklist and observe your Mentor. In the box below there are some observation points on good teaching. Use them to make a simple checklist in your TP Journal for the lesson observation. YEAR 3 - OFF-CAMPUS TP REFLECT TOGETHER REFLECT TOGETHER
Make notes of any issues you need to discuss with your Mentor and arrange a time to meet. Discuss with your Mentor your findings on the lesson observation. Ask questions about approaches
and methods that you found interesting and those that were new to you. Make notes of any issues you need to discuss with your Mentor and arrange a time to meet. Discuss with your Mentor your findings on the lesson observation. Ask questions about approaches
and methods that you found interesting and those that were new to you. Make notes of any issues you need to discuss with your Mentor and arrange a time to meet. Discuss with your Mentor your findings on the lesson observation. Ask questions about approaches
and methods that you found interesting and those that were new to you. 71 Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 3 Year 1 - 3: Teaching Practice Year 1 - 3: Teaching Practice Y3 TP Activity 2.2: Mentor and Student Teacher co-teach a lesson each day - plan a
lesson together and Student Teacher delivers the lesson introduction DO
Teach the Lesson Introduction Teach the lesson introduction following the lesson introduction plans while your Mentor and TP
partner observe you and make notes. REFLECT TOGETHER
After each lesson, discuss with your Mentor and TP partner the steps and methodology that you
used in the lesson introduction. This will help you to become more effective in your teaching. Y3 TP Activity 2.3: Reflect Together on co-taught lessons observed each day. PLAN TOGETHER
Plan together with your Mentor and TP partner on how you will reflect together after observing
the Mentor’s lesson and your teaching of the lesson introduction. Each of you, Mentor and Student
Teachers, should review the checklist for the lesson observation and the lesson introduction plans. DO
Student Teachers Reflect Together on the co-taught lesson observed Y3 TP Activity 2.2: Mentor and Student Teacher co-teach a lesson each day - plan a
lesson together and Student Teacher delivers the lesson introduction PLAN TOGETHER
You, your TP partner and your Mentor will study the Scheme of Work and select the appropriate
topics to be taught in the lessons each day of TP Week 2. The Mentor will discuss with you what
should be included in each lesson introduction that you will teach. PLAN TOGETHER
You, your TP partner and your Mentor will study the Scheme of Work and select the appropriate
topics to be taught in the lessons each day of TP Week 2. The Mentor will discuss with you what
should be included in each lesson introduction that you will teach. Make a plan for the lesson introductions using the Lesson Plan Format (see Annex 2). Your Mentor
will support you to organise all materials needed for the lesson, for example charts, real objects,
textbooks, exercise books, chalk, crayons, etc. You may use a story, song, a question, and/or an
activity to connect with the lesson objective for a good introduction. DO
Student Teachers Reflect Together on the co-taught lesson observed Consider the following question in discussion with your TP partner:
How can these elements of a good lesson be observed and demonstrated? (a) Lesson objectives, (b)
relevant previous knowledge (RPK), (c) organisation of TLMs, (d) good introductory activities, (e)
logical sequence for the lesson presentation, (f) effective group activities, (g) assessment
procedures including good questioning skills and (h) definitive closure. REFLECT TOGETHER
After the co-taught lesson each day you, your Mentor and TP partner will reflect on the co-taught
lesson with the following points as a guide: Purpose: In this Topic you will practise lesson delivery and classroom management Purpose: In this Topic you will practise lesson delivery and classroom management Activity 3.1: Mentor and Student Teacher plan a lesson each day – the Student Teacher practises
lesson delivery and classroom management competencies Activity 3.2: The Student Teacher and Mentor co-teach the lesson together each day
Activity 3.3: Mentor and Student Teacher reflect together on the co-taught lessons Competencies: Student Teacher demonstrates: (i) Use of strategies to effectively manage a
class: Competency 13(a-d); (ii) Use of strategies to provide clear explanations for new concepts,
knowledge and skills: Competency 2(a-e) (see Annex 1) Y3 TP Week 3 - Summary of the Task When you have completed these three activities for Y3 TP Week 3 with your Mentor, you will have: • co-taught a planned lesson; • reflected on the co-taught lesson. Y3 TP Activity 3.1: Plan a lesson to co-teach each day to practise lesson delivery and
classroom management competencies PLAN TOGETHER
Last week (Y3 TP Week 2), you practised teaching the lesson introduction. In this week’s co-taught
lessons you will teach the main part of the lesson and your Mentor will teach the introduction and
closure of the lesson. You will practise classroom management skills while teaching your part of
each lesson. PLAN TOGETHER
Last week (Y3 TP Week 2), you practised teaching the lesson introduction. In this week’s co-taught
lessons you will teach the main part of the lesson and your Mentor will teach the introduction and
closure of the lesson. You will practise classroom management skills while teaching your part of
each lesson. Plan the lesson together with your Mentor and your TP partner, selecting the lesson topics you will
co-teach each day and concentrating on the lesson delivery section that you will teach. Plan the lesson together with your Mentor and your TP partner, selecting the lesson topics yo
co-teach each day and concentrating on the lesson delivery section that you will teach. DO
Prepare your lesson plans to be co-taught by you and your Mentor. DO
Prepare your lesson plans to be co-taught by you and your Mentor. Prepare your lesson plans to be co-taught by you and your Mentor. In planning this week’s lessons, consider the lesson delivery and classroom management skills that
make a good lesson. These are some of the main elements you should include in your lesson delivery
plan and demonstrate in your lesson delivery: • linking the activities to pupils’ RPK; • using the activity method of teaching, including discussion, demonstration, songs, stories, etc.;
• effective use of questioning skills, i.e. using probing questions, distributing questions
evenly, etc.; • being sensitive to gender issues during lesson delivery; • clear and audible voice together with legible handwriting on the chalkboard; • effective use of chalkboard, which helps pupils to follow the lesson sequence. As much as possible, plan your lesson bringing in most of the elements that have been mentioned
above. REFLECT TOGETHER • Discuss the good practice that you observed. • What did not go so well and why?f • What should be done differently next time? Identify two strong points and two teaching points from your lesson introduction for improvement. Identify two strong points and two teaching points from your lesson introduction for improvement. Record the salient points in your TP Journal for future reference. Student Teacher Handbook for Teaching Pratice Student Teacher Handbook for Teaching Pratice 72 YEAR 3 - OFF-CAMPUS TP Y3 TP Topic 3: Deliver the lesson and manage the class - Student Teacher co-teaches with the
Mentor Y3 TP Topic 3: Deliver the lesson and manage the class - Student Teacher co-teaches with the
Mentor Co-teach the planned lessons with your Mentor Co-teach the planned lessons with your Mentor Co teach the planned lessons with your Mentor
Co-teach the lesson with your Mentor, focusing on your part of the lesson - delivery of the main
section of the lesson and classroom management skills – applying the elements of good teaching
practice that you identified in Activity 3.1. p
y
Co-teach the lesson with your Mentor, focusing on your part of the lesson - delivery of the main
section of the lesson and classroom management skills – applying the elements of good teaching
practice that you identified in Activity 3.1. Y3 TP Week 3 - Summary of the Task Ask your Mentor to support you with the preparation of materials, classroom management
approaches or other suggestions that will help you to succeed. As much as possible, plan your lesson bringing in most of the elements that have been mentioned
above. Ask your Mentor to support you with the preparation of materials, classroom management
approaches or other suggestions that will help you to succeed. Transforming Teacher Education and Learning
www.t-tel.org 73 3 Year 1 - 3: Teaching Practice Year 1 - 3: Teaching Practice DO DO
Co-teach the planned lessons with your Mentor
Co-teach the lesson with your Mentor, focusing on your part of the lesson - delivery of the main
section of the lesson and classroom management skills – applying the elements of good teaching
practice that you identified in Activity 3.1. REFLECT TOGETHER With the co-teaching activity in mind, answer the following questions with your Mentor and
TP partner for reflection on each day’s lesson preparation: With the co-teaching activity in mind, answer the following questions with your Mentor and
TP partner for reflection on each day’s lesson preparation: • How can the lesson plan and lesson preparation be improved? • How can the lesson plan and lesson preparation be improved? Record your improved lesson plan in your TP Journal. Y3 TP Activity 3.2: Each day the Student Teacher and Mentor teach the lesson to PLAN TOGETHER
In Activity 3.1 you will have prepared the lesson plans for the lessons you will teach with your
mentor, in which you will practise lesson delivery and classroom management. As a final preparation
before commencing each of the co-taught lessons, review your lesson plan and check that you have
prepared everything you will need. Consider the following: PLAN TOGETHER
In Activity 3.1 you will have prepared the lesson plans for the lessons you will teach with your
mentor, in which you will practise lesson delivery and classroom management. As a final preparation
before commencing each of the co-taught lessons, review your lesson plan and check that you have
prepared everything you will need. Consider the following: • where you will stand and how you will move around the classroom during the lesson; • how you will organise the pupils during the lesson and ensure that both girls and boys
participate actively in the lessons; • how you will use strategies to practise good classroom discipline without shouting or losing
the attention and enjoyment of the pupils. Discuss some of these points with your TP partner and ask your Mentor for tips and suggestions on
any points where you may have doubts. Discuss some of these points with your TP partner and ask your Mentor for tips and suggestions on
any points where you may have doubts. REFLECT TOGETHER Reflect back on the notes you made in your TP Journal during Week 2 Activity 2.3 on how to
improve your teaching. Discuss with your Mentor and TP partner whether the teaching points for
improvement encountered during the previous co-taught lessons have been improved in this week’s
co-taught lessons. 74 Student Teacher Handbook for Teaching Pratice Y3 TP Activity 3.3: Reflect Together on each day’s co-taught lessons Y3 TP Activity 3.3: Reflect Together on each day’s co-taught lesson PLAN TOGETHER PLAN TOGETHER
At the end of Week 3 plan a Reflective Practice activity to cover all the co-taught lessons. This activity will take the form of a meeting involving the Lead Mentor, all Mentors and all your
TP partners in the school, and other concerned officials. You will plan the Reflective Practice meeting such that it will not coincide with any lessons in the
classroom for the day. The meeting should be conveniently held during break periods or the first
half-hour after classes. You will have to arrange all logistics needed for the meeting in collaboration
with your Mentor. At the Reflective Practice meeting your Mentors will encourage you to express yourselves freely,
to enable you to elicit the maximum exchange of ideas and support from your Mentors. You will organise the meetings in a classroom to accommodate all participants conveniently
DO
Participate in a Reflective Practice meeting. The convener of the Reflective Practice meeting will be the Lead Mentor. Any Tutor, TLA, SPA or TPC
who is visiting the school at the time of the meeting will also participate PLAN TOGETHER
At the end of Week 3 plan a Reflective Practice activity to cover all the co-taught lessons. This activity will take the form of a meeting involving the Lead Mentor, all Mentors and all your
TP partners in the school, and other concerned officials. At the end of Week 3 plan a Reflective Practice activity to cover all the co-taught lessons. his activity will take the form of a meeting involving the Lead Mentor, all Mentors and all your
P partners in the school, and other concerned officials. You will plan the Reflective Practice meeting such that it will not coincide with any lessons in the
classroom for the day. The meeting should be conveniently held during break periods or the first
half-hour after classes. You will have to arrange all logistics needed for the meeting in collaboration
with your Mentor. At the Reflective Practice meeting your Mentors will encourage you to express yourselves freely,
to enable you to elicit the maximum exchange of ideas and support from your Mentors. y
g
pp
y
You will organise the meetings in a classroom to accommodate all participants conveniently DO Participate in a Reflective Practice meeting. The convener of the Reflective Practice meeting will be the Lead Mentor. Any Tutor, TLA, SPA or TPC
who is visiting the school at the time of the meeting will also participate. You will bring your TP Journal to the Reflective Practice meeting and will keep a record of
the main discussion points. FRAMING QUESTIONS a) Ask ‘remembering’ and recall questions e.g. What do you remember about…? How do you
define….? What is….? b) Ask questions that require analysis and evaluation e.g. How can you classify…? Why do you
think…? Can you compare with ….? What are the advantages/disadvantages of …? What is
the most important…? Is there a better solution than…? Can you suggest an alternative? Can you predict the outcome if…? PLAN TOGETHER In Y3 TP Week 2, you co-taught the lesson introduction and observed the lesson sequence with your
Mentor. In Week 3, you co-taught the lesson delivery and practised classroom management skills. This week, you will prepare lessons to practise questioning skills and lesson closure competencies. Once again, you will assemble all relevant materials in the lesson planning stage with help, if
needed, from your Mentor. The table below shows ways of framing questions for effective learning. Use these as examples for
your lesson preparation. REFLECT TOGETHER At the end of the Reflective Practice meeting, identify with your TP partner two teaching points
from this week’s co-taught lessons on which you need to focus attention to further improve your
teaching skills and competencies. As you did at the end of Week 2, make a record in your TP Journal of the two teaching points to be
improved from this week’s Reflect Together activity, for future reference. Transforming Teacher Education and Learning 75 Transforming Teacher Education and Learning
www.t-tel.org 3 Year 1 - 3: Teaching Practice Y3 TP Topic 4: Practise questioning and close the lesson
Student Teacher observes the Mentor and they co-teach a lesson
Purpose: In this Topic you will practise questioning skills and lesson closure
Activity 4.1: Mentor and Student Teacher plan a lesson each day for the Student Teachers to
practise questioning skills and lesson closure competencies
Activity 4.2: The Student Teacher and Mentor co-teach the lesson together each day
Activity 4.3: Mentor and Student Teacher reflect together on the co-taught lessons
Competencies: Student Teacher demonstrates: (i) Use of a range of questions during the lesson:
Competency 5(a, b and d) (see Annex 1) Y3 TP Week 4 - Summary of the Task When you have completed these three activities for Y3 TP Week 4 with your Mentor you will have: • planned a lesson together with your Mentor, to practise questioning skills and lesson closure
competencies; • co-taught the lesson;l • reflected on the co-taught lessons together. Y3 TP Activity 4.1: Plan a lesson to co-teach each day to practise questioning skills and
lesson closure competencies YOU MAY USE THESE TEACHER QUESTIONING SKILLS: Student Teacher Handbook for Teaching Pratice 76 YEAR 3 - OFF-CAMPUS TP Hints on effective lesson closure: Hints on effective lesson closure: • assign homework;
• administer short/flash tests;
• summarise the lesson by way of reviewing;
• sometimes ask pupils to summarise;
• leave core points on the blackboard;
• in practical lessons like fine art/natural science, tidy up the classroom;
• you may close the lesson with a song or rhyme. • assign homework;
• administer short/flash tests;
• summarise the lesson by way of reviewing;
• sometimes ask pupils to summarise;
• leave core points on the blackboard;
• in practical lessons like fine art/natural science, tidy up the classroom;
• you may close the lesson with a song or rhyme. With your Mentor, select the lessons and topics you will co-teach each day. DO
Prepare co-teaching lesson plans on questioning skills and lesson closure. Incorporate some of the ideas given above into your lesson plans for each day to practise one or two
questioning skills and to practise the key steps in lesson closure. The questions can be incorporated
into the closure part of the lesson. Ensure that you have planned how much time you will have for
the lesson closure. Familiarise yourself with the Mentor’s lesson plan for each day so that you
can make a smooth link between the main delivery part of the lesson and your closure which will
summarise what the pupils have been learning. PAY ATTENTION TO THESE GENDER ISSUES: a) Give equal chance to girls and boys to ask/answer questions
b) Assign leadership roles equally to girls and boys in lesson activities
c) Make sure girls and boys participate equally in activities
d) Allow sufficient time for girls and boys who may be shy or afraid to speak out
e) Check if both girls and boys equally understand the lesson
f) Provide constructive feedback to both girls and boys in class Co-teach the planned lessons with your Mentor Co-teach the lesson with your Mentor using questioning as part of the lesson closure. Your
Mentor will observe you teaching and will give feedback after the lesson to help you to improve
your performance. Co-teach the lesson with your Mentor using questioning as part of the lesson closure. Your
Mentor will observe you teaching and will give feedback after the lesson to help you to improve
your performance. Student Teacher Handbook for Teaching Pratice REFLECT TOGETHER With your Mentor and TP partner reflect on your lesson preparation for the co-teaching lessons
using the following questions as prompts: With your Mentor and TP partner reflect on your lesson preparation for the co-teaching lessons
using the following questions as prompts: • Do your planned lesson closure activities link well with the Mentor’s lesson delivery plan? • Do your planned lesson closure activities link well with the Mentor’s lesson delivery plan? • Have you prepared questions that will require pupils to recall, analyse and evaluate? • How will you ensure your questioning skills are gender responsive? y
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• Have you prepared questions that will require pupils to recall, analyse and evaluate? • How will you ensure your questioning skills are gender responsive? • Have you prepared questions that will require pupils to recall, analyse and evaluate? • How will you ensure your questioning skills are gender responsive? Record in your TP Journal two new ideas you gain from the Reflect Together activity. Record in your TP Journal two new ideas you gain from the Reflect Together activity. Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 77 3 Year 1 - 3: Teaching Practice Y3 TP Activity 4.2: Each day the Student Teacher and Mentor teach the lesson Y3 TP Activity 4.2: Each day the Student Teacher and Mentor teach the lesson together PLAN TOGETHER
In Activity 4.1, you will have prepared your lesson plans for the week and checked them with your
Mentor. Before you co-teach the lesson, practise asking the questions you are going to use in the
lesson. Practise by asking your TP partner each question you plan to ask the pupils. Either your
TP partner tries to answer each question as though they are one of the pupils in the class, or you
discuss together the kinds of responses the pupils might give in answer to each of the questions. Use this opportunity to check whether any of the questions need to be revised. PLAN TOGETHER
In Activity 4.1, you will have prepared your lesson plans for the week and checked them with your
Mentor. Before you co-teach the lesson, practise asking the questions you are going to use in the
lesson. Practise by asking your TP partner each question you plan to ask the pupils. Either your
TP partner tries to answer each question as though they are one of the pupils in the class, or you
discuss together the kinds of responses the pupils might give in answer to each of the questions. Use this opportunity to check whether any of the questions need to be revised. DO
Co-teach the planned lessons with your Mentor
Co-teach the lesson with your Mentor using questioning as part of the lesson closure. Your
Mentor will observe you teaching and will give feedback after the lesson to help you to improve
your performance. Participate in a Reflective Practice meetingl Bring your TP Journal to the Reflective Practice meeting and keep a record of the main discussion
points. Discuss freely and frankly any issues that will help to improve your professional practice. Y3 TP Activity 4.3: Reflect Together on each day’s co-taught lessons Y3 TP Activity 4.3: Reflect Together on each day’s co-taught lessons PLAN TOGETHER At the end of Week 4 plan a Reflective Practice meeting to cover all the co-taught lessons this week. This will be a repeat of the Reflective Practice meeting conducted last week in Activity 3.3. You and
your TP group will plan the Reflective Practice meeting to review the questioning skills and lesson
closure competencies practised this week. The Lead Mentor, all Mentors and all your TP partners in the school, and other concerned officials
should attend. You and the TP group members will plan the meeting such that it will not coincide
with any lessons in the classroom for the day. The meeting should be conveniently held during break
periods or the first half-hour after classes. You will arrange all logistics needed for the meeting in
collaboration with your Mentor. REFLECT TOGETHER REFLECT TOGETHER
With your Mentor and TP partner, reflect on the notes you made in your TP Journal at the end of
Week 3 Activity 3.3 on how to improve your teaching. Discuss with your Mentor and TP partner
whether the teaching points for improvement that you identified last week have been improved in
this week’s co-taught lessons. Discuss the following questions in your TP group or TP partner: With your Mentor and TP partner, reflect on the notes you made in your TP Journal at the end of
Week 3 Activity 3.3 on how to improve your teaching. Discuss with your Mentor and TP partner
whether the teaching points for improvement that you identified last week have been improved in
this week’s co-taught lessons. Discuss the following questions in your TP group or TP partner: • What went well in the lesson? • What challenges did you encounter? • If you had another chance to teach the same lesson, what would you do differently? • If you had another chance to teach the same lesson, what would you do differently? • What suggestions did your mentor make? • How will you use suggestions made by your Mentor to improve your professional practice? 78 Student Teacher Handbook for Teaching Pratice REFLECT TOGETHER At the end of the Reflective Practice meeting identify with your TP partner two teaching points
from this week’s co-taught lessons on which you need to focus attention to further improve your
teaching skills and competencies. At the end of the Reflective Practice meeting identify with your TP partner two teaching points
from this week’s co-taught lessons on which you need to focus attention to further improve your
teaching skills and competencies. As you did at the end of Week 3, make a record in your TP Journal of the two teaching
points to be improved from this week’s Reflect Together exercise, for future reference. Transforming Teacher Education and Learning
www.t-tel.org 79 Transforming Teacher Education and Learning 3 Year 1 - 3: Teaching Practice Activity 5.3: Mentor and Student Teachers reflect together on the co-taught lessons Competencies: Student Teacher demonstrates: (i) Question and answer: Competency 4(a) and
8(a); (ii) Use of constructive feedback in response to pupils’ answers, work or effort: Competency
9(a-d) (see Annex 1) Y3 TP Week 5 - Summary of the Task When you have completed these three activities for Y3 TP Week 5 with your Mentor you will • planned a co-teaching lesson together with your Mentor, to practise questioning and
communication skills and demonstrate PCK; • co-taught the lesson using appropriate subject knowledge;l • co-taught the lesson using appropriate subject knowledge
• reflected on the co taught lessons together • reflected on the co-taught lessons together. • reflected on the co-taught lessons together. Y3 TP Activity 5.1: Plan a lesson to co-teach each day to practise questioning and
communication skills and to demonstrate PCK Y3 TP Activity 5.1: Plan a lesson to co-teach each day to practise questioning and
communication skills and to demonstrate PCK Student Teacher Handbook for Teaching Pratice Y3 TP Topic 5: Practise questioning and communication skills
Student Teachers co-teach a single or double period Purpose: In this Topic you will practise questioning and communication skills, and pedagogical
content knowledge (PCK) Activity 5.1: Mentor and Student Teachers plan a lesson each day for the Student Teachers
to co-teach – to practise questioning and communication skills and PCK Activity 5.2: Student Teachers co-teach the single or double period lesson each day and the
Mentor observes Prepare your co-teaching lesson plans together. You will need to plan the co-teaching lessons carefully with your TP partner to ensure that you
include opportunities for both of you to practise questioning and communication skills and to
demonstrate PCK. Prepare the co-teaching lesson plans for each day clearly indicating which parts
of the lesson each of you will teach. Consider how you will gain and sustain the interest of your
pupils whilst ensuring equal attention is given to boys and girls and avoiding any gender
stereotyping. You will need to plan the co-teaching lessons carefully with your TP partner to ensure that you
include opportunities for both of you to practise questioning and communication skills and to
demonstrate PCK. Prepare the co-teaching lesson plans for each day clearly indicating which parts
of the lesson each of you will teach. Consider how you will gain and sustain the interest of your
pupils whilst ensuring equal attention is given to boys and girls and avoiding any gender
stereotyping. You will have to demonstrate appropriate use of pedagogical approaches (e.g. role play, songs,
games, sorting out letters/words, story-telling, questions and answers, etc.) for the topic being
taught and the age group of the pupils. You may want to practise teaching the lesson together before you teach it to the class. You may want to practise teaching the lesson together before you teach it to the class PLAN TOGETHER For the next two weeks of your Y3 TP you will co-teach lessons with your TP partner. This week you
will focus on questioning and communication skills and PCK. Your Mentor will provide support and
guidance during the planning and reflection activities, and will observe you co-teaching the lessons. In planning the Y3 Week 5 lessons, review the previous lessons you co-taught with your Mentor in
Y3 Week 4 on the use of questioning. Choose two to three examples of question types from the
framework of questions in Activity 4.1that you did not use in Week 4 to practise in your co-teaching
lessons in Week 5. You should also review last week’s TP Journal record on teaching points to
improve. Include in your lesson plan for this week some activities to practise and improve those
teaching skills. Practise the following teaching strategies during your co-teaching to attract the attention of your
pupils as you interact with them. 80 Student Teacher Handbook for Teaching Pratice • speak clearly – loud enough, not too fast, face
the class, avoid mannerisms; YEAR 3 - OFF-CAMPUS TP ‘Content Knowledge’ means the topics
that you will teach in English, maths or
science. You will need to agree the
topics with your Mentor and then study
the scheme of work and textbooks to
check the detail of the content
knowledge to be covered in the lessons. • have suitable non-verbal communication skills
– the use of appropriate gestures and facial
expressions, moving around the
classroom, whole class eye contact; • speak understandably – use words that your
pupils will be able to understand and explain; • use the right speed in your speech – your pupils
must be able to absorb the material that is
presented; ’Pedagogical Content Knowledge’
means the teaching approaches you will
use which are appropriate to the topic
and the age group of the pupils, for ex-
ample use of songs, stories, group work,
games, role play and so on. • encourage two-way communication – you should
regularly check if your pupils are following your
presentation; you should allow them space to
express themselves and ask questions, too; • emphasise important knowledge – make clear
what is important and new to the pupils; • knowledge must be presented in a logical
manner so that it becomes easy to learn. PLAN TOGETHER In the co-teaching lessons, you will also need to demonstrate your skills in ‘PCK’ (see Text Box Year 1 - 3: Teaching Practice Year 1 - 3: Teaching Practice Y3 TP Activity 5.2: Each day the Student Teachers co-teach a single or double period
lesson using appropriate PCK and the Mentor observes Co-teach the planned lesson with your TP partner Co-teach the planned lessons with your TP partner each day, demonstrating questioning and
communication skills and appropriate teaching and learning activities (PCK). Your Mentor wil
observe the lesson while you teach. Co-teach the planned lessons with your TP partner each day, demonstrating questioning and
communication skills and appropriate teaching and learning activities (PCK). Your Mentor will
observe the lesson while you teach. Student Teacher Handbook for Teaching Pratice PLAN TOGETHER PLAN TOGETHER
In Activity 5.1, you will have prepared your lesson plans for the week and checked them with your
Mentor. Before you co-teach the lesson, ensure that you have all the TLMs you are going to use in
the lesson. Discuss with your TP partner how you will organise the pupils for the activities you plan
to use in the lesson. Check on the timing of the activities in the lesson, especially if you are teaching
a double period lesson. You will need to vary the activities to sustain the interest of the pupils. You
might want to think of one or two activities that you can give to any pupils who complete their work
quickly, and to give all the pupils time to complete their work. You should neither rush through the
activities nor spend too long on any activity. Ask your Mentor for advice if you are not sure about
any of these points. In Activity 5.1, you will have prepared your lesson plans for the week and checked them with your
Mentor. Before you co-teach the lesson, ensure that you have all the TLMs you are going to use in
the lesson. Discuss with your TP partner how you will organise the pupils for the activities you plan
to use in the lesson. Check on the timing of the activities in the lesson, especially if you are teaching
a double period lesson. You will need to vary the activities to sustain the interest of the pupils. You
might want to think of one or two activities that you can give to any pupils who complete their work
quickly, and to give all the pupils time to complete their work. You should neither rush through the
activities nor spend too long on any activity. Ask your Mentor for advice if you are not sure about
any of these points. Your Mentor will be observing as you teach and may be assessing your teaching performance
following the CoE teaching practice assessment guidelines (see Annex 4). DO
Co-teach the planned lesson with your TP partner
Co-teach the planned lessons with your TP partner each day, demonstrating questioning and
communication skills and appropriate teaching and learning activities (PCK). Your Mentor will
observe the lesson while you teach. REFLECT TOGETHER You and your TP partner should reflect together with your Mentor on the co-teaching lesson
plans for each day. The reflection may focus on the questioning and communication skills, and
pedagogical approaches that are planned. For example; • Which pedagogical approaches do you plan to use, and are they appropriate to the topic and
the age group of the class? • How will you divide the teaching in each lesson between you and your TP partner? h
d
k ll
ll
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f ll • What questioning and communication skills will you practise to ensure the pupils’ full
participation during the lesson? Transforming Teacher Education and Learning
www.t-tel.org 81 Transforming Teacher Education and Learning 3 REFLECT TOGETHER REFLECT TOGETHER
You and your TP partner should reflect on the co-taught lessons and find out whether you have
improved the teaching skills that you identified in your TP Journal entries from previous weeks. Discuss the following questions in your TP group: You and your TP partner should reflect on the co-taught lessons and find out whether you ha
improved the teaching skills that you identified in your TP Journal entries from previous wee
Discuss the following questions in your TP group: • What went well in the lesson? • What challenges did you encounter? • If you had another chance to teach the same lesson, what would you do differently? • How will you use suggestions made by your Mentor to improve your professional practice? 82 Student Teacher Handbook for Teaching Pratice 3 TP Activity 5.3: Mentor and Student Teachers reflect together on the co-taught PLAN TOGETHER
With your Lead Mentor, Mentor, TP partner and other TP group members, schedule a convenient
time for a Reflective Practice meeting on the co-taught lessons following the same arrangements as
last week (Activity 4.3). A classroom will be an appropriate venue and you must remember to bring your TP Journal
with you for the meeting. PLAN TOGETHER With your Lead Mentor, Mentor, TP partner and other TP group members, schedule a convenient
time for a Reflective Practice meeting on the co-taught lessons following the same arrangements as
last week (Activity 4.3). DO
Participate in a Reflective Practice meeting
Attend a Reflective Practice meeting with your Lead Mentor and Mentor. Reflect together on
the week’s co-teaching practice focusing on the main improvements you have made as Student
Teachers. Identify the aspects of your teaching where further practice in skills and competencies
is needed. REFLECT TOGETHER
With your Mentor agree the main challenges you encountered during the delivery of the co-taught
lesson and how you will address these challenges in next week’s teaching practice. Feel free to ask questions that are on your mind as far as your professional training is concerned. A classroom will be an appropriate venue and you must remember to bring your TP Journal
with you for the meeting. Record the action points from the discussion in your TP Journal. DO
Participate in a Reflective Practice meeting
Attend a Reflective Practice meeting with your Lead Mentor and Mentor. Reflect together on
the week’s co-teaching practice focusing on the main improvements you have made as Student
Teachers. Identify the aspects of your teaching where further practice in skills and competencies
is needed. REFLECT TOGETHER REFLECT TOGETHER
With your Mentor agree the main challenges you encountered during the delivery of the co-taught
lesson and how you will address these challenges in next week’s teaching practice. Feel free to ask questions that are on your mind as far as your professional training is concerned. ith your Mentor agree the main challenges you encountered during the delivery of the co-taug
sson and how you will address these challenges in next week’s teaching practice. y
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eel free to ask questions that are on your mind as far as your professional training is concerned Record the action points from the discussion in your TP Journal. Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 83 3 Year 1 - 3: Teaching Practice Y3 TP Topic 6: Use TLMs to assess pupils’ learning
Student Teachers co-teach a single or double lesson
Purpose: In this Topic you will practise the appropriate use of TLMs and assessment of learning
Activity 6.1: Mentor and Student Teachers plan together a lesson for each day for the Student
Teachers to co-teach; practise appropriate use of TLMs and assessment of learning
Activity 6.2: Student Teachers co-teach the single or double period lesson each day using TLMs
and assessment of learning and the Mentor observes
Activity 6.3: Mentor and Student Teachers reflect together on the co-taught lessons
Competencies: Student Teacher demonstrates: (i) Use of different TLMs to facilitate learning:
Competency 3(a-g); (ii) Assessment of learning: Competencies 8 and 9; (see Annex 1) Y3 TP Week 6 - Summary of the Task When you have completed these three activities for Y3 Week 6 with your Mentor you will hav • planned a co-teaching lesson with your Mentor, practised the appropriate use of TLMs and
assessment of learning; • co-taught the lesson together using TLMs and assessment of learning; • reflected together on the co-taught lessons. Y3 TP Activity 6.1: Plan a lesson to co-teach for each day to practise the appropriate
use of TLMs and assessment of learning PLAN TOGETHER This week, you will co-teach with your TP partner as you did last week. You will be looking at ways of
handling TLMs appropriately in your lessons and also, you will begin to practise how to assess your
pupils’ learning progress and achievement. This week, you will co-teach with your TP partner as you did last week. You will be looking at ways of
handling TLMs appropriately in your lessons and also, you will begin to practise how to assess your
pupils’ learning progress and achievement. Plan with your Mentor and TP partner the English, maths or science topics you will co-teach this
week. TEACHING TIPS: ask yourself these questions as you plan your co-teaching lesson on use of T • Which TLMs will be most appropriate for the topic I am teaching and the age group of the
pupils (for example story books, videos, games, flashcards, etc.)? TIP: Choose one or two TLMs
to include in each co-teaching lesson. • At what point in the lesson will I introduce the TLMs? TIP: introducing the TLMs too early in the
lesson may distract pupils’ attention. • How many TLMs will I need for the class? TIP: if you make a class chart, it should be written
clearly and displayed so that it can be seen and read by all pupils. If you make sets of TLMs for
group work, think about how long it will take to make the sets and consider how you will store
them for future use. Student Teacher Handbook for Teaching Pratice 84 You will need to prepare and search for suitable
TLMs to use in your planned co-teaching lessons. YEAR 3 - OFF-CAMPUS TP Examples of types of classroom
assessment
• reading tests
• mental maths tests
• short comprehension tests
• spelling drills/tests
• dictation
• True/False questions Assessment of pupils’ learning is a very important
skill for all teachers to acquire. In planning for
this week’s co-teaching lessons, you will begin by
recalling what you learned in your DBE programme
on how to assess your pupils’ learning. There are
multiple formats and assessment types that can
be used to paint an accurate picture of pupil
achievement and understanding (see Text Box
examples). You will be practising some of these in
the following weeks of your teaching practice. DO Prepare your co-teaching lesson plan together on the use of TLMs and assessment of learning
You will need to plan the co-teaching lessons carefully with your TP partner to ensure that you
include opportunities for both of you to practise the use of TLMs and assessment of pupils’ learning. Prepare the co-teaching lesson plans for each day clearly indicating which parts of the lesson each of
you will teach. Consider how you will gain and sustain the interest of your pupils ensuring that equal
attention is given to boys and girls and avoiding any gender stereotyping. You may want to organise
micro teaching with the support of your Mentor where you will practise the assessment tasks you
have planned. You will have to demonstrate appropriate use of pedagogical approaches (e.g. role play, songs,
games, sorting out letters/words, story-telling, questions and answers, etc.) for the topic being
taught and the age group of the pupils. You may want to practise teaching the lesson together before you teach it to the class PLAN TOGETHER For this first week of incorporating pupil assessment in teaching, choose one assessment type that
you are familiar with and plan a simple test for the end of each day’s co-teaching lesson (see Text
Box examples). Y3 TP Activity 6.2: Each day the Student Teachers co-teach a single or double period
lesson using TLMs and assessment of learning as the Mentor observes PLAN TOGETHER
In Activity 6.1, you will have prepared your lesson plans for the week and checked them with your
Mentor. Before you co-teach the lesson, ensure that you have all the TLMs ready. Decide with your
TP partner how you will organise the pupils for the activities you plan to use in the lesson. Check on
the timing of the activities in the lesson, especially if you are teaching a double period lesson. You
will need to vary the activities to sustain the interest of the pupils. PLAN TOGETHER
In Activity 6.1, you will have prepared your lesson plans for the week and checked them with your
Mentor. Before you co-teach the lesson, ensure that you have all the TLMs ready. Decide with your
TP partner how you will organise the pupils for the activities you plan to use in the lesson. Check on
the timing of the activities in the lesson, especially if you are teaching a double period lesson. You
will need to vary the activities to sustain the interest of the pupils. Towards the end of the lesson, you will give the pupils the assessment task and mark it to find out
what the pupils have learned from your lesson. Student Teacher Handbook for Teaching Pratice REFLECT TOGETHER l REFLECT TOGETHER
Reflect with your TP partner and your Mentor on the reasons why TLMs should be used in
teaching, for example: TLMs help pupils to solve problems and develop concepts; TLMs help pupils
enjoy the lesson. Reflect with your TP partner and your Mentor on the reasons why TLMs should be used in
teaching, for example: TLMs help pupils to solve problems and develop concepts; TLMs help pupils
enjoy the lesson. Brainstorm and give five more reasons why TLMs should be used in teaching and record
them in your TP Journal. Transforming Teacher Education and Learning Transforming Teacher Education and Learning
www.t-tel.org 85 3 Year 1 - 3: Teaching Practice Y3 TP Activity 6.2: Each day the Student Teachers co-teach a single or double period
lesson using TLMs and assessment of learning as the Mentor observes Y3 TP Activity 6.3: Mentor and Student Teachers reflect together on the co-taught
lessons YEAR 3 - OFF-CAMPUS TP YEAR 3 - OFF-CAMPUS TP Co-teach the planned lesson with your TP partner Co-teach the planned lessons with your TP partner each day demonstrating the appropriate use of
TLM and assessment of pupils’ learning. Your Mentor will observe the lesson while you teach and help you if necessary with the pupil
assessment activities. REFLECT TOGETHER
You, your Mentor and TP partner should reflect on the co-taught lessons from last week and this
week and identify the teaching skills and competencies that you have improved. Discuss the following questions in your TP group after each lesson: • What went well in the lesson? • What challenges did you encounter? • Did the assessment help to show pupils’ understanding of the lesson? • If you had another chance to teach the same lesson, what would you have done differently • If you had another chance to teach the same lesson, what would you have done differently? • How will you use suggestions made by your Mentor to improve your professional practice? • How will you use suggestions made by your Mentor to improve your professional practice? Student Teacher Handbook for Teaching Pratice 86 Y3 TP Activity 6.3: Mentor and Student Teachers reflect together on the co-taug
l PLAN TOGETHER PLAN TOGETHER
With your Lead Mentor, Mentor, TP partner and other TP group members, schedule a convenient
time for a Reflective Practice meeting on the co-taught lessons on the appropriate use of TLMs
and assessment of learning activities, following the same arrangements as in the previous weeks
(Activities 4.3 and 5.3). With your Lead Mentor, Mentor, TP partner and other TP group members, schedule a convenient
time for a Reflective Practice meeting on the co-taught lessons on the appropriate use of TLMs
and assessment of learning activities, following the same arrangements as in the previous weeks
(Activities 4.3 and 5.3). DO DO
Participate in a Reflective Practice meeting
In this week’s Reflective Practice meeting, review the planned lessons covering the use of TLMs
and assessment of learning activities. You will conduct a brainstorming activity among the Student
Teacher group to identify the four main challenges encountered in the lesson planning and delivery. The Lead Mentors and Mentors should provide suggestions on how each of the challenges can be
overcome to help you to improve your teaching skills. In the Reflective Practice meeting, discuss freely and frankly to bring out all issues which will help STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE:
Answer these questions based on the lessons taught in Week 6. Teaching skill/competence
Score
Teaching skill/competence
Score
• Was I organised? • Did I use a clear voice? • Were my objectives clear? • Did I explain the lesson effectively? • Did I remain focused on my topic? • Did I show confidence? Score: needs improvement = 1; satisfactory = 2; good performance = 3 STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE: Transforming Teacher Education and Learning Transforming Teacher Education and Learning
www.t-tel.org 87 3 Year 1 - 3: Teaching Practice Y3 TP Week 7 - Summary of the Task When you have completed these three activities for Y3 Week 7 with your Mentor, you will have: • developed a lesson plan to identify and plan for individual learning needs; • taught the single or double lessons individually; • evaluated the individual lesson with your Mentor. Y3 TP Activity 7.1: The Student Teachers and Mentor plan together a lesson for each
day for the Student Teachers to practise identifying and planning for individual
learning needs Student Teacher Handbook for Teaching Pratice PLAN TOGETHER After the lesson, you will plan one or two
activities that you will use in the next lesson to help the pupils according to their individual
learning needs. Teach the lesson individually Teach the lesson ensuring that you focus on the subject knowledge that you have planned to cover. You will also identify the learning needs of two girls and two boys in the class. After you have
identified the individual learning needs of the two girls and two boys, you will plan specifically for
their identified learning needs in the next lesson. You will also be able to use the planned learning
needs activities with other pupils in the class who have similar learning needs. Teach the lesson ensuring that you focus on the subject knowledge that you have planned to cover. You will also identify the learning needs of two girls and two boys in the class. After you have
identified the individual learning needs of the two girls and two boys, you will plan specifically for
their identified learning needs in the next lesson. You will also be able to use the planned learning
needs activities with other pupils in the class who have similar learning needs. Make a seating plan of the classroom and circle all the seats where you noticed that children
struggled with their learning during your lessons. PLAN TOGETHER For the next two weeks, you will teach a full
lesson individually, each day. Work with your
Mentor to choose one English, maths or
science lesson to teach each day, and plan with
your Mentor which strategy you will practise
each day to identify the learning needs of
pupils in the class (see Text Box). At the end
of the lesson, reflect with your Mentor on the
progress that you have made each day. Strategies for identifying learner needs
•
Question and answer (where students
also ask questions of the teacher)
•
Whole class dialogue (e.g. discussion,
debate, brainstorming)
•
Small group work (e.g. discussion,
debate, brainstorming)
•
Class assignment
•
Student presentation
•
Role-play As part of the planning stage discuss with your
Mentor the pupils’ prior learning and their
level of understanding of the concepts you will
be teaching in the lesson. You should also plan
with your Mentor the pupils you might select
in the activity to identify their learning needs. 88 Student Teacher Handbook for Teaching Pratice DO
Prepare the lesson plans for individual teaching and identification of pupils’ learning needs
In your lesson preparation, incorporate strategies (see the text box) that will help you identify
learner needs. Each day, identify the learning needs of two girls and two boys who are struggling
with the lesson in the class. It will be easier to focus on the learning needs of a few pupils initially
but later, as you gain experience and competence in teaching, you will be able to identify and plan
for the learning needs of all the pupils in your class. After the lesson, you will plan one or two
activities that you will use in the next lesson to help the pupils according to their individual
learning needs. DO
Prepare the lesson plans for individual teaching and identification of pupils’ learning needs
In your lesson preparation, incorporate strategies (see the text box) that will help you identify
learner needs. Each day, identify the learning needs of two girls and two boys who are struggling
with the lesson in the class. It will be easier to focus on the learning needs of a few pupils initially
but later, as you gain experience and competence in teaching, you will be able to identify and plan
for the learning needs of all the pupils in your class. PLAN TOGETHER Now that you have prepared your lesson plans in Activity 7.1, prepare everything you will need
to teach the lessons individually using appropriate subject knowledge and skills. Your Mentor will
observe your individual lessons and will provide you with some specific feedback at the end of each
lesson on the skills you are practicing this week – how to identify and plan for the individual learning
needs of pupils. Now that you have prepared your lesson plans in Activity 7.1, prepare everything you will need
to teach the lessons individually using appropriate subject knowledge and skills. Your Mentor will
observe your individual lessons and will provide you with some specific feedback at the end of each
lesson on the skills you are practicing this week – how to identify and plan for the individual learning
needs of pupils. REFLECT TOGETHER ith your Mentor and TP partner, review the lesson plans for each day and answer these questio • Is the subject knowledge appropriate for the lesson and the age group/ability of all
the pupils in the class? • Do your lesson plans cover enough or too much ground for the lesson? • Do your lesson plans cover enough or too much ground for the lesson? • Are the planned activities realistic and achievable in identifying individual learning needs? Do your lesson plans cover enough or too much ground for the lesson? Are the planned activities realistic and achievable in identifying individual learning needs? y
p
g
g
• Are the planned activities realistic and achievable in identifying individual learning needs? • Are the planned activities realistic and achievable in identifying individual lea Y3 TP Activity 7.2: Each day the Student Teachers teach the single or double lesson
individually using appropriate subject knowledge as the Mentor observes 3 TP Activity 7.2: Each day the Student Teachers teach the single or double lesson
dividually using appropriate subject knowledge as the Mentor observes Y3 TP Activity 7.3: Mentor and Student Teachers reflect together on the individual
lessons PLAN TOGETHER
The weekly Reflective Practice meetings that were arranged in Week 4 to Week 6 will continue
to the end of the Y3 TP period. Schedule the meeting for this week as you have done in
previous weeks. REFLECT TOGETHER Discuss with your Mentor how the lesson was presented. Discuss with your Mentor the skill used to
identify the individual learning need. The Student Teacher should be able to explain the learning
need and how it could be catered for. Discuss with your Mentor how the lesson was presented. Discuss with your Mentor the skill used to
identify the individual learning need. The Student Teacher should be able to explain the learning
need and how it could be catered for. Share your seating plan with your TP partner and Mentor. What do you think this information tells
you about the gender balance in class? Share your seating plan with your TP partner and Mentor. What do you think this information tells
you about the gender balance in class? Discuss what you have observed about the seating plan and make a record in your
TP Journal of two points that you have observed. Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 89 3 Year 1 - 3: Teaching Practice Y3 TP Activity 7.3: Mentor and Student Teachers reflect together on the individual
lessons Participate in a Reflective Practice meeting pl
g
In this week’s Reflective Practice meeting, review the planned lessons covering the identification of,
and planning for, pupils individual learning needs. List the various learning needs you identified and
discuss how they can be addressed. Brainstorm with your Student Teacher group to identify the four
main challenges encountered in the lesson planning and delivery. In this week’s Reflective Practice meeting, review the planned lessons covering the identification of,
and planning for, pupils individual learning needs. List the various learning needs you identified and
discuss how they can be addressed. Brainstorm with your Student Teacher group to identify the four
main challenges encountered in the lesson planning and delivery. The Lead Mentors and Mentors should provide suggestions on how each of the challenges can be
overcome to help you to improve your teaching skills. In the Reflective Practice meeting, discuss freely and frankly to bring out all issues which will help
improve your professional practice. In the Reflective Practice meeting, discuss freely and frankly to bring out all issues which will help
improve your professional practice. REFLECT TOGETHER
Ask your Mentor and TP co-teaching partner about your overall performance for the week, including
a review of your performance in identifying the individual learning needs and in addressing
individual learning needs. Then complete the Student Teacher Self-Assessment Questionnaire. STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE:
Answer these questions based on the lessons taught in Week 6. Teaching skill/competence
Score
• Were my questions fairly distributed during the lesson? • Were my questions able to differentiate and cater for individual learning needs? • Did I identify and plan adequately for individual learning needs? Score: needs improvement = 1; satisfactory = 2; good performance = 3
Record your self-assessment score in your TP Journal. REFLECT TOGETHER
Ask your Mentor and TP co-teaching partner about your overall performance for the week, including
a review of your performance in identifying the individual learning needs and in addressing
individual learning needs. Then complete the Student Teacher Self-Assessment Questionnaire. STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE:
Answer these questions based on the lessons taught in Week 6. Teaching skill/competence
Score
• Were my questions fairly distributed during the lesson? • Were my questions able to differentiate and cater for individual learning needs? • Did I identify and plan adequately for individual learning needs? PLAN TOGETHER The weekly Reflective Practice meetings that were arranged in Week 4 to Week 6 will continue
to the end of the Y3 TP period. Schedule the meeting for this week as you have done in
previous weeks. Student Teacher Handbook for Teaching Pratice Y3 TP Topic 8: Conduct an assessment and use results in teaching
Student Teachers individually teach a single or double period Purpose: In this Topic you will practise using results of formative assessment showing pupils’
progress and achievement for teaching/learning Activity 8.1: Mentor and Student Teachers plan together a lesson for each day for the Student
Teachers to practise using formative assessment results for teaching/learning Activity 8.2: Student Teachers teach the single or double formative assessment lesson
individually each day and the Mentor observes Activity 8.3: Mentor and Student Teachers reflect together on the individually taught lessons Competencies: Student Teacher demonstrates: (i) Use of formative assessment of learners’
progress to evaluate achievement, use results for teaching and report results to learners:
Competencies 8(a-e) and, 9(a-c) (see Annex 1) Y3 TP Week 8 - Summary of the Task When you have completed these three activities for Y3 TP Week 8 with your Mentor, you will have • planned a lesson together with support from your Mentor to teach each day focusing on
formative assessment of learners’ progress and achievement; • taught each day the single or double lessons focusing on formative assessment of individual
pupils; • reflected together on the single or double lessons taught individually by you. Y3 TP Activity 8.1: The Student Teachers and Mentor plan together a lesson for each
day for the Student Teachers to practise using formative assessment results for
teaching/learning
PLAN TOGETHER
In Week 8, you will continue to practise
formative assessment of pupils’ progress
and achievement. Last week, you practised
identifying and planning for individual learning
needs. This week, the focus is on using the
results of formative assessment showing
pupils’ progress and achievement for
teaching and learning. Work with your Mentor
to choose an English, maths or science topic
to teach each day. Plan a lesson for each day
using a formative assessment tool that is
appropriate for the lesson and the age group/
ability of the pupils (see Text Box). Strategies/tools for formative assessment
•
Question and answer (where students
also ask questions of the teacher)
•
Whole-class dialogue (e.g. discussion,
debate, brainstorming)
•
Class assignment
•
Songs
•
Role-play
The lesson plans should describe how you plan to use the results of the formative assessment in
the next lesson. You should also think about how you will give constructive feedback to pupils on
the results of the formative assessment. For example, choose two girls and two boys who are
struggling with their learning. Identify a particular challenge they are facing in the lesson and
prepare an activity to encourage and motivate them to succeed in the next lesson. Your Mentor
will assist you in planning the lessons. Y3 TP Activity 8.1: The Student Teachers and Mentor plan together a lesson for each
day for the Student Teachers to practise using formative assessment results for
teaching/learning Participate in a Reflective Practice meeting Score: needs improvement = 1; satisfactory = 2; good performance = 3
Record your self-assessment score in your TP Journal. Then complete the Student Teacher Self Assessment Questionnaire. STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE:
Answer these questions based on the lessons taught in Week 6. Teaching skill/competence
Score
• Were my questions fairly distributed during the lesson? • Were my questions able to differentiate and cater for individual learning needs? • Did I identify and plan adequately for individual learning needs? Score: needs improvement = 1; satisfactory = 2; good performance = 3
Record your self-assessment score in your TP Journal. Then complete the Student Teacher Self Assessment Questionnaire. STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE:
Answer these questions based on the lessons taught in Week 6. Teaching skill/competence
Score
• Were my questions fairly distributed during the lesson? • Were my questions able to differentiate and cater for individual learning needs? • Did I identify and plan adequately for individual learning needs? Score: needs improvement = 1; satisfactory = 2; good performance = 3
Record your self-assessment score in your TP Journal. 90 Student Teacher Handbook for Teaching Pratice YEAR 3 - OFF-CAMPUS TP Prepare the lesson plans for individual teaching and use of pupils’ formative assessment
results You will need to prepare the chosen formative assessment activities that you will use in your lessons
each day. You will need to plan how you will include the formative assessment activities in each
lesson – at the beginning of the lesson, during the main part of the lesson or at the end of the
lesson. Will you do the activity with the whole class or only some of the pupils? If only some of the
pupils will be involved, which pupils will do the formative assessment activity and what will the
other pupils do at that time? Identify two girls and two boys in the class who are struggling with the lesson and closely observe
their performance in the assessment activity. This will be easier than trying to observe the
performance of all the pupils in the class. For this exercise, focus on the same four pupils
throughout the week but ensure that you give equal attention to all the pupils in the class while
conducting the teaching and learning activities. You will need to be prepared to provide all the
pupils with regular feedback and very clear explanations of each exercise and assignment. The
exercises should be neatly marked and feedback provided to the pupils as an important part of the
formative assessment process. REFLECT TOGETHER As a final preparation, with your Mentor and TP partner, reflect on the lesson plan and the activities
to be taught and answer the following questions: • How will you involve girls and boys in the lesson and ensure that they participate equally? • How do you plan to mark the pupils’ exercises and what feedback do you plan to provide
to pupils? • How will you manage the feedback session with the whole class and the four pupils who are
struggling with the lesson? Y3 TP Activity 8.2: Each day the Student Teachers teach the single or double formative
assessment lesson individually using appropriate subject knowledge and the Mentor
observes PLAN TOGETHER In Week 8, you will continue to practise
formative assessment of pupils’ progress
and achievement. Last week, you practised
identifying and planning for individual learning
needs. This week, the focus is on using the
results of formative assessment showing
pupils’ progress and achievement for
teaching and learning. Work with your Mentor
to choose an English, maths or science topic
to teach each day. Plan a lesson for each day
using a formative assessment tool that is
appropriate for the lesson and the age group/
ability of the pupils (see Text Box). In Week 8, you will continue to practise
formative assessment of pupils’ progress
and achievement. Last week, you practised
identifying and planning for individual learning
needs. This week, the focus is on using the
results of formative assessment showing
pupils’ progress and achievement for
teaching and learning. Work with your Mentor
to choose an English, maths or science topic
to teach each day. Plan a lesson for each day
using a formative assessment tool that is
appropriate for the lesson and the age group/
ability of the pupils (see Text Box). The lesson plans should describe how you plan to use the results of the formative assessment in
the next lesson. You should also think about how you will give constructive feedback to pupils on
the results of the formative assessment. For example, choose two girls and two boys who are
struggling with their learning. Identify a particular challenge they are facing in the lesson and
prepare an activity to encourage and motivate them to succeed in the next lesson. Your Mentor
will assist you in planning the lessons. Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 91 3 Year 1 - 3: Teaching Practice Teach the lesson individually y
Teach the lesson according to the lesson plan while your Mentor observes. Your Mentor will make
notes on the formative assessment activities and the ways in which you use the results of the pupils’
assessment to assist their future learning. REFLECT TOGETHER Reflect with your Mentor and TP partner on the formative assessment activities and feedback, and
discuss these three questions: Reflect with your Mentor and TP partner on the formative assessment activities and feedback, and
discuss these three questions: • What strategies should a teacher use if some pupils are struggling to complete a formative
assessment task? Give two examples. • What strategies should a teacher use if some pupils find the formative assessment task too
easy? Give two examples. • How should feedback be given when some children are identified as having special learning
needs? Record the four examples in your TP Journal for future reference. Y3 TP Activity 8.3: Mentor and Student Teachers reflect together on the individual
l PLAN TOGETHER In this lesson, you will focus on the use of
formative assessment results of pupils’
progress and achievement for teaching and
learning. You will need to ensure that you have
prepared the teaching materials and assess-
ment activities in advance. For the formative
assessment activity, consider how you will
organise the pupils, what instructions you will
need to give them, what they will be doing and
how you might offer the necessary help to any
pupils who are struggling with the assessment
task. Ask your Mentor for advice on this last
point about helping pupils who are struggling
with an assessment task. What will your Mentor
be observing during this lesson (see Text Box)? In this lesson, you will focus on the use of
formative assessment results of pupils’
progress and achievement for teaching and
learning. You will need to ensure that you have
prepared the teaching materials and assess-
ment activities in advance. For the formative
assessment activity, consider how you will
organise the pupils, what instructions you will
need to give them, what they will be doing and
how you might offer the necessary help to any
pupils who are struggling with the assessment
task. Ask your Mentor for advice on this last
point about helping pupils who are struggling
with an assessment task. What will your Mentor
be observing during this lesson (see Text Box)? Student Teacher Handbook for Teaching Pratice Student Teacher Handbook for Teaching Pratice 92 DO
Teach the lesson individually
Teach the lesson according to the lesson plan while your Mentor observes. Your Mentor will make
notes on the formative assessment activities and the ways in which you use the results of the pupils’
assessment to assist their future learning. DO Participate in a Reflective Practice meeting • How did I provide feedback to the pupils in my lessons? • Were the assessment tasks appropriate? REFLECT TOGETHER
Reflect with your TP group on the importance of regularly assessing your pupils’ progress and
learning achievement. PLAN TOGETHER At the end of Week 8, you will again arrange a Reflective Practice meeting with all the Mentors and
Student Teachers in your TP school. During Weeks 7 and 8, you will have taught an individual lesson each day. In preparation for the
Reflective Practice meeting, check that your TP Journal is up to date and read through the entries
for the two weeks to remind yourself of the highlights and learning achievements you experienced,
especially in relation to the use of formative assessment. At the end of Week 8, you will again arrange a Reflective Practice meeting with all the Mentors and
Student Teachers in your TP school. During Weeks 7 and 8, you will have taught an individual lesson each day. In preparation for the
Reflective Practice meeting, check that your TP Journal is up to date and read through the entries
for the two weeks to remind yourself of the highlights and learning achievements you experienced,
especially in relation to the use of formative assessment. DO
Participate in a Reflective Practice meeting
Discuss the individual teaching lessons with your Mentor and TP Partners during the Reflective
Practice meeting using the following questions: STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE: Y3 TP WEEK 9 - Summary of the Task Y3 TP Topic 9: Identify learning needs and teach to provide appropriate support
Student Teachers individually teach all periods Purpose: In this Topic you will practise identifying individual learning needs and teach to provide
appropriate support Activity 9.1: Mentor and Student Teachers plan together all lessons for each day for the Student
Teachers to practise identifying individual learning needs and teaching to provide appropriate
support Activity 9.2: Student Teachers teach all lessons individually each day and the Mentor observes Competencies: Student Teacher demonstrates: (i) Equal treatment of all learners and shows
respect; handles pupils’ responses well and uses strategies to assess pupils’ understanding:
Competency 11(a-f) (see Annex 1) Y3 TP WEEK 9 - Summary of the Task When you have completed these three Y3 Week 9 activities with your Mentor, you will hav • planned together with your Mentor all lessons for each day for the Student Teachers to prac
identifying learning needs and teaching to provide appropriate support; • taught all lessons individually using appropriate subject knowledge and the Mentor observes al
lessons; • reflected together with the Mentor on the individual lessons • reflected together with the Mentor on the individual lessons Y3 TP Activity 9.1: Plan all lessons for each day to practise identifying individual
learning needs and teach to provide appropriate support STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE: Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 93 3 Year 1 - 3: Teaching Practice Y3 TP Topic 9: Identify learning needs and teach to provide appropriate support
Student Teachers individually teach all periods
Purpose: In this Topic you will practise identifying individual learning needs and teach to provide
appropriate support
Activity 9.1: Mentor and Student Teachers plan together all lessons for each day for the Student
Teachers to practise identifying individual learning needs and teaching to provide appropriate
support
Activity 9.2: Student Teachers teach all lessons individually each day and the Mentor observes
Activity 9.3: Mentor and Student Teachers reflect together on the individual lessons
Competencies: Student Teacher demonstrates: (i) Equal treatment of all learners and shows
respect; handles pupils’ responses well and uses strategies to assess pupils’ understanding:
Competency 11(a-f) (see Annex 1)
When you have completed these three Y3 Week 9 activities with your Mentor, you will have:
• planned together with your Mentor all lessons for each day for the Student Teachers to practise
identifying learning needs and teaching to provide appropriate support;
• taught all lessons individually using appropriate subject knowledge and the Mentor observes all
lessons;
• reflected together with the Mentor on the individual lessons
Y3 TP Activity 9.1: Plan all lessons for each day to practise identifying individual
learning needs and teach to provide appropriate support
PLAN TOGETHER
During Week 9, you and your TP partner will teach all the lessons each day. With the support of
your Mentor and working closely with your TP partner, plan all the lessons for each day. In these
lessons, you will again be practising how to identify individual learning needs and teach to provide
appropriate support. Choose a topic for each day from the science, maths or English curriculum and think of an activity
for each lesson which will help you to identify and work with the high-achieving pupils in the class as
well as those who are struggling to learn. REMEMBER you will need to identify individual learning needs first and then teach to provide
appropriate support. PLAN TOGETHER During Week 9, you and your TP partner will teach all the lessons each day. With the support of
your Mentor and working closely with your TP partner, plan all the lessons for each day. In these
lessons, you will again be practising how to identify individual learning needs and teach to provide
appropriate support. Choose a topic for each day from the science, maths or English curriculum and think of an activity
for each lesson which will help you to identify and work with the high-achieving pupils in the class as
well as those who are struggling to learn. REMEMBER you will need to identify individual learning needs first and then teach to provide
appropriate support. REMEMBER you will need to identify individual learning needs first and then teach to provide
appropriate support. Student Teacher Handbook for Teaching Pratice DO DO
Teach all the lessons individually each day
Teach all the lessons that have been allocated
to you this week according to the lesson plans
while your Mentor observes. Remember to
treat pupils equitably and with respect, be
mindful of individual abilities and appreciate
individual efforts (see Text Boxes above). DO
Prepare all lesson plans for each day with
activities to identify and support individual
learning needs Prepare all lesson plans for each day with
activities to identify and support individual
learning needs
Agree with your TP partner on the lessons each
of you will teach each day. Prepare the lesson
plans for your lessons ensuring that you focus
on the subject knowledge and the activities that
will help you to identify and support individual learning needs (see Text Box). Remember pupils
learn in a variety of different ways. When you have identified individual pupils’ learning needs you
may want to group them for different activities by same/mixed ability groups, or by gender for
example. You should explain how you plan to group the pupils for each activity and explain why you
are choosing that arrangement. As a Student Teacher you should
remember to:
•
treat pupils equitably and with respect
•
be mindful of individual abilities
•
appreciate individual efforts Y3 TP Activity 9.2: Teach all lessons individually using appropriate subject knowledge
while your Mentor observes Y3 TP Activity 9.2: Teach all lessons individually using appropriate subject knowledge
while your Mentor observes REMEMBER you will need to identify individual learning needs first and then teach to provide
appropriate support. 94 Student Teacher Handbook for Teaching Pratice YEAR 3 - OFF-CAMPUS TP PLAN TOGETHER Review with your Mentor the lesson plans and
activities you have prepared for the week. Your Mentor will help you to clarify the ways in
which you plan to organise the pupils for each
activity so that you are able to provide
appropriate support to the pupils according
to their learning needs. (link to STPD T4
Group Work). Review with your Mentor the lesson plans and
activities you have prepared for the week. Your Mentor will help you to clarify the ways in
which you plan to organise the pupils for each
activity so that you are able to provide
appropriate support to the pupils according
to their learning needs. (link to STPD T4
Group Work). REFLECT TOGETHER l Reflect together with your TP partner on your lesson plans and ensure there is good continuity
between the lessons and the activities. Reflect with your Mentor on any aspects of this week’s
teaching practice on which you may need further guidance and advice. You must be fully prepared
to teach all the lessons this week. Revise your lesson plans and formative assessment activities if necessary after receiving feedback
and constructive comments from your Mentor and TP partner. Revise your lesson plans and formative assessment activities if necessary after receiving feedback
and constructive comments from your Mentor and TP partner. Student Teacher Handbook for Teaching Pratice REFLECT TOGETHER At the end of each day, discuss the lessons with your TP partner and your Mentor. Compare what
you observed about the learning needs of individual pupils and how you provided them with
support. The following questions can serve as a guide:f • Did your TP partner make similar or different observations about the learning needs of
individual pupils? • Did your TP partner provide similar support to pupils with individual learning needs? Transforming Teacher Education and Learning
www.t-tel.org 95 3 Year 1 - 3: Teaching Practice Y3 TP Activity 9.3: Reflect together with your Mentor on the individual lesson PLAN TOGETHER
Arrange a convenient time for this week’s Reflective Practice meeting in which the topic for
discussion will be “How the teacher can identify individual learning needs and provide appropriate
support for individual pupils”. Come to the meeting ready to discuss the successes and challenges you experienced in this week’s
teaching practice. DO
Participate in the Reflective Practice meeting
Present one example of a successful activity from this week’s lessons in which you identified and
provided support for individual learning needs of the pupils in your class. Present one example of a less successful activity from this week’s lessons and explain briefly why it
was less successful in identifying and providing support for individual learning needs of the pupils in
your class. REFLECT TOGETHER
Complete the following Self-Assessment Questionnaire and record your score in your TP Journal:
STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE:
Answer the following questions based on the lessons presented in Week 9. Teaching skill/competence
Score
a) How prepared was I for the lessons? b) How well did I assess the learning needs of individual pupils? c) How effective were my activities to support individual learning needs? d) Was my organisation/grouping of the pupils suitable for the activities? Score: needs improvement = 1; satisfactory = 2; good performance = 3
Make a brief record (bullet points) in your TP Journal of the successful and unsuccessful
examples you presented at the Reflective Practice meeting. PLAN TOGETHER PLAN TOGETHER
Arrange a convenient time for this week’s Reflective Practice meeting in which the topic for
discussion will be “How the teacher can identify individual learning needs and provide appropriate
support for individual pupils”. Come to the meeting ready to discuss the successes and challenges you experienced in this week’s
teaching practice. Participate in the Reflective Practice meeting Format of summative assessment
Selected response questions
• Multiple choice
• True/false
• Matching
Short answer questions
• Fill in the blank
• One or two sentence response
• Extended written response
Recall what you learned in the DBE programme about test construction and marking scheme
preparation. Refer to the college notes you made during that part of the DBE programme. Agree with your Mentor on the specific periods you will have to teach and the lessons in which you
will conduct the summative assessment. Discuss with your Mentor the topics you should cover in the
summative assessment tests you will prepare for English, maths and science. NOTE: the summative assessment tests can be short, especially as you practise this skill for the first
i
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i
l d
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i
i
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h
i
d
i i Y3 TP Topic 10: Prepare a test and marking scheme
Student Teachers individually teach specific periods
Purpose: In this Topic you will practise the use of summative assessment (a) test construction and
(b) marking scheme design
Activity 10.1: Mentor and Student Teachers plan together a lesson for each day for the Student
Teachers to practise the use of summative assessment (a) test construction and (b) marking
scheme design
Activity 10.2: Student Teachers teach the summative assessment lesson individually each day and
the Mentor observes
Activity 10.3: Mentor and Student Teachers reflect together on the individual lessons
Competencies: Student Teacher demonstrates: (i) Use of strategies to assess pupils using
summative assessment: Competency 8(h) (see Annex 1) Y3 TP Topic 10: Prepare a test and marking scheme
Student Teachers individually teach specific periods
Purpose: In this Topic you will practise the use of summative assessment (a) test construction and
(b) marking scheme design
Activity 10.1: Mentor and Student Teachers plan together a lesson for each day for the Student
Teachers to practise the use of summative assessment (a) test construction and (b) marking
scheme design
Activity 10.2: Student Teachers teach the summative assessment lesson individually each day and
the Mentor observes
Activity 10.3: Mentor and Student Teachers reflect together on the individual lessons
Competencies: Student Teacher demonstrates: (i) Use of strategies to assess pupils using
summative assessment: Competency 8(h) (see Annex 1) YEAR 3 - OFF-CAMPUS TP Y3 TP Week 10 - Summary of the Task When you have completed these 3 exercises you will have: • planned together with your Mentor a lesson for each day to help you to practise the use of
summative assessment (a) test construction; (b) marking scheme design; • taught the summative assessment lesson individually using appropriate subject knowledge and
the Mentor observes; ;
• reflected together (both Mentor and Student Teacher) on the individual lessons Y3 TP Activity 10.1: Plan a lesson for each day to practise the use of summative
assessment (a) test construction and (b) marking scheme design Participate in the Reflective Practice meeting Complete the following Self-Assessment Questionnaire and record your score in your TP Journal:
STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE:
Answer the following questions based on the lessons presented in Week 9. Teaching skill/competence
Score
a) How prepared was I for the lessons? b) How well did I assess the learning needs of individual pupils? c) How effective were my activities to support individual learning needs? d) Was my organisation/grouping of the pupils suitable for the activities? Score: needs improvement = 1; satisfactory = 2; good performance = 3 96 Student Teacher Handbook for Teaching Pratice Y3 TP Topic 10: Prepare a test and marking scheme
Student Teachers individually teach specific periods
Purpose: In this Topic you will practise the use of summative assessment (a) test construction and
(b) marking scheme design
Activity 10.1: Mentor and Student Teachers plan together a lesson for each day for the Student
Teachers to practise the use of summative assessment (a) test construction and (b) marking
scheme design
Activity 10.2: Student Teachers teach the summative assessment lesson individually each day and
the Mentor observes
Activity 10.3: Mentor and Student Teachers reflect together on the individual lessons
Competencies: Student Teacher demonstrates: (i) Use of strategies to assess pupils using
summative assessment: Competency 8(h) (see Annex 1)
When you have completed these 3 exercises you will have:
• planned together with your Mentor a lesson for each day to help you to practise the use of
summative assessment (a) test construction; (b) marking scheme design;
• taught the summative assessment lesson individually using appropriate subject knowledge and
the Mentor observes;
• reflected together (both Mentor and Student Teacher) on the individual lessons. Y3 TP Activity 10.1: Plan a lesson for each day to practise the use of summative
assessment (a) test construction and (b) marking scheme design
PLAN TOGETHER
Y3 TP Week 10 - Summary of the Task
In the past three weeks of Y3 TP, you
have been learning how to use formative
assessment in your teaching practice lessons. This week, you will start learning to use
summative assessment by practising the skills
of test construction and marking scheme
preparation (see Text Box). Discuss with your Mentor and TP partner the
differences between formative and summative
assessment. PLAN TOGETHER Discuss with your Mentor how to teach the summative assessment lesson using appropriate
subject knowledge. Decide whether the test will be set formally or given to the pupils as a
regular classroom activity. If the test is set formally, pupils may become anxious, whereas if the test
is blended into the regular classroom activity it is less stressful, especially for younger pupils. Take the advice of the Mentor as it is important to conduct the tests in a way that will be familiar
to the pupils. Discuss with your Mentor how to teach the summative assessment lesson using appropriate
subject knowledge. Decide whether the test will be set formally or given to the pupils as a
regular classroom activity. If the test is set formally, pupils may become anxious, whereas if the test
is blended into the regular classroom activity it is less stressful, especially for younger pupils. Take the advice of the Mentor as it is important to conduct the tests in a way that will be familiar
to the pupils. Discuss with your Mentor how to teach the summative assessment lesson using appropriate
subject knowledge. Decide whether the test will be set formally or given to the pupils as a
regular classroom activity. If the test is set formally, pupils may become anxious, whereas if the test
is blended into the regular classroom activity it is less stressful, especially for younger pupils. Take the advice of the Mentor as it is important to conduct the tests in a way that will be familiar
to the pupils. DO
Teach the lesson incorporating the summative assessment test and use the marking scheme as
the Mentor observes
Teach the lesson individually according to the lesson planned each day. Administer the summative
assessment as agreed with your Mentor. The Mentor will observe the lesson, paying particular
attention to your test and marking scheme. PLAN TOGETHER Recall what you learned in the DBE programme about test construction and marking scheme
preparation. Refer to the college notes you made during that part of the DBE programme. Agree with your Mentor on the specific periods you will have to teach and the lessons in which you
will conduct the summative assessment. Discuss with your Mentor the topics you should cover in the
summative assessment tests you will prepare for English, maths and science. NOTE: the summative assessment tests can be short, especially as you practise this skill for the first
time. For example, the test may include only five to six questions or take a short time to administer,
and be no longer than five to ten minutes. During this Week 10 TP, you will construct five short tests,
one for a selected lesson each day. Transforming Teacher Education and Learning
www.t-tel.org 97 Transforming Teacher Education and Learning 3 Year 1 - 3: Teaching Practice DO
Prepare all lesson plans incorporating information on the planned summative assessment tests
and marking scheme with the Mentor’s support
Under the supervision of your Mentor, plan the lesson for each day in which you will include a
summative assessment activity. You will have to agree with your Mentor the kind of test you will
construct (see Text Box) as well as marking scheme preparation. Develop the test questions and the
marking scheme you will use for the summative assessment following guidance from your Mentor. REFLECT TOGETHER REFLECT TOGETHER
Review with your Mentor and TP partner the lessons and test questions you have prepared, and the
marking scheme you have developed. If possible, find an opportunity to trial the test questions to
check whether they work as you expect them to and whether the marking scheme works. Y3 TP Activity 10.2: Teach the summative assessment lesson individually using
appropriate subject knowledge, with the test and marking scheme, as the Mentor
observes REFLECT TOGETHER
Review with your Mentor and TP partner the lessons and test questions you have prepared, and the
marking scheme you have developed. If possible, find an opportunity to trial the test questions to
check whether they work as you expect them to and whether the marking scheme works. Y3 TP Activity 10.2: Teach the summative assessment lesson individually using
i
bj
k
l d
i h h
d
ki
h
h
M REFLECT TOGETHER
Review with your Mentor and TP partner the lessons and test questions you have prepared, and the
marking scheme you have developed. If possible, find an opportunity to trial the test questions to
check whether they work as you expect them to and whether the marking scheme works. Y3 TP Activity 10.2: Teach the summative assessment lesson individually using
appropriate subject knowledge, with the test and marking scheme, as the Mentor
observes Student Teacher Handbook for Teaching Pratice REFLECT TOGETHER REFLECT TOGETHER
Together with your Mentor and TP partner discuss the lessons taught and the competencies you
demonstrated regarding test construction and marking scheme development. Together with your Mentor and TP partner discuss the lessons taught and the competencies you
demonstrated regarding test construction and marking scheme development. 98 Student Teacher Handbook for Teaching Pratice YEAR 3 - OFF-CAMPUS TP Y3 TP Activity 10.3: Mentor and Student Teachers reflect together on the individual
lessons incorporating test construction and marking scheme development PLAN TOGETHER Plan with the Mentors and TP group in your TP school to schedule a Reflective Practice meeting
focusing on the test construction and marking scheme activities undertaken this week. DO
Participate in the Reflective Practice meeting Wh t
ill th
il d
h
th
fi i h th t
t? Y3 TP Week 11 - Summary of the Task Activity 11.1: Mentor and Student Teachers plan together a lesson for each day for Student
Teachers to practise test administration, scoring and recording the results of summative
assessment Activity 11.2: Student Teachers teach the summative assessment lesson individually each day and
the Mentor observes Competencies: (i) Use of summative assessment to support independent study skills for effective
learning: Competency 8(a-h) (see Annex 1) Y3 TP Week 11 - Summary of the Task When you have completed these three Y3 Week 11 activities with your Mentor, you will have: When you have completed these three Y3 Week 11 activities with your Mentor, you will have: • planned together, a lesson for each day, to practise test administration, scoring and recording
the results of summative assessment; the results of summative assessment; • taught each day, the summative assessment lesson individually using appropriate subject
knowledge and the Mentor observes; g
• reflected, together with your Mentor, on the summative assessment activities Year 3 TP Activity 11.1: Plan a lesson for each day to practise test administration,
scoring and recording the results of summative assessment Year 3 TP Activity 11.1: Plan a lesson for each day to practise test administration,
scoring and recording the results of summative assessment DO
Participate in the Reflective Practice meeting Come to the Reflective Practice meeting ready to present and discuss your reflections on the
summative assessment activities this week – test construction and marking scheme preparation. During the meeting the discussion should address these important questions:
• How appropriate were the summative test items and the marking scheme? • How well did the pupils perform in the summative assessment? • How did the girls preform in the tests compared to the boys? • What does the pupils’ performance tell you about their understanding of the topic? REFLECT TOGETHER
Complete the following Self-Assessment Questionnaire and record your score in your TP Journal:
STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE:
Answer the following questions based on the summative assessment activities in Week 10. Teaching skill/competence
Score
a) How prepared was I for the summative assessment activities? b) How well did my test questions match the topics the pupils have studied? c) Were my summative assessment questions suitable for testing all pupils? d) Did my marking scheme work well? Score: needs improvement = 1; satisfactory = 2; good performance = 3 REFLECT TOGETHER
Complete the following Self-Assessment Questionnaire and record your score in your TP Journal:
STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE:
Answer the following questions based on the summative assessment activities in Week 10. Teaching skill/competence
Score
a) How prepared was I for the summative assessment activities? b) How well did my test questions match the topics the pupils have studied? c) Were my summative assessment questions suitable for testing all pupils? d) Did my marking scheme work well? Score: needs improvement = 1; satisfactory = 2; good performance = 3 STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE:
Answer the following questions based on the summative assessment activities in Week 10. Teaching skill/competence
Score
a) How prepared was I for the summative assessment activities? b) How well did my test questions match the topics the pupils have studied? c) Were my summative assessment questions suitable for testing all pupils? d) Did my marking scheme work well? DO
Participate in the Reflective Practice meeting Score: needs improvement = 1; satisfactory = 2; good performance = 3 Transforming Teacher Education and Learning 99 Transforming Teacher Education and Learning
www.t-tel.org 3 Year 1 - 3: Teaching Practice Y3 TP Topic 11: Administer the test, score and record test results
Student Teachers teach specific periods individually
Purpose: In this Topic you will practise administering the test, scoring and recording test results
Activity 11.1: Mentor and Student Teachers plan together a lesson for each day for Student
Teachers to practise test administration, scoring and recording the results of summative
assessment
Activity 11.2: Student Teachers teach the summative assessment lesson individually each day and
the Mentor observes
Activity 11.3: Mentor and Student Teachers reflect together on summative assessment activities
Competencies: (i) Use of summative assessment to support independent study skills for effective
learning: Competency 8(a-h) (see Annex 1)
When you have completed these three Y3 Week 11 activities with your Mentor, you will have:
• planned together, a lesson for each day, to practise test administration, scoring and recording
the results of summative assessment;
• taught each day, the summative assessment lesson individually using appropriate subject
knowledge and the Mentor observes;
• reflected, together with your Mentor, on the summative assessment activities
Year 3 TP Activity 11.1: Plan a lesson for each day to practise test administration,
scoring and recording the results of summative assessment
PLAN TOGETHER
Last week, you practised construction of tests and preparation of marking schemes for summative
assessment. This week, in collaboration with your Mentor, you will plan a lesson for each day to
practise the use of summative assessment including administering the test, scoring and recording
the result. First, you must agree with your Mentor the English, maths or science topics you will teach
and test this week. Then construct the tests and prepare the marking schemes as you did last week. In planning how to administer the tests consider the following points:
• How will the pupils be arranged for the test? • How long will the test take? • What instructions will you give the pupils? • How will you make the pupils understand the instructions if they cannot read accurately? • Will the test be written on the board or printed? • How will the class be managed if the test is to be written on the board? DO DO
Prepare the Week 11 lesson plans including how to administer, score and record test results
Plan the Week 11 lessons taking advice from your Mentor on how to administer, score and record
test results. You should follow the system that is used in the school and is familiar to the pupils. As you prepare the tests and plan how to administer, score and record the results, remember
that the summative assessment tests are important. They form the end of year record of each
pupil’s progress and achievement. REFLECT TOGETHER REFLECT TOGETHER
Discuss and reflect together with your TP partner, checking on the lessons each of you will be
teaching. Compare notes on how you plan to administer, score and record the results of the
summative assessment tests. You should be consistent in your approach to this task. Discuss and reflect together with your TP partner, checking on the lessons each of you will be
teaching. Compare notes on how you plan to administer, score and record the results of the
summative assessment tests. You should be consistent in your approach to this task. You may want to trial the plans for administering, scoring and recording the test results before the
lesson. Ask your TP partner and/or Mentor to help you with this. You may want to trial the plans for administering, scoring and recording the test results before the
lesson. Ask your TP partner and/or Mentor to help you with this. Reflect back on the summative tests you used in last week’s lessons. Did you encounter any
problems with administering the tests, scoring or recording the test results? If so, how can you
overcome those problems in this week’s teaching practice lessons? PLAN TOGETHER Last week, you practised construction of tests and preparation of marking schemes for summative
assessment. This week, in collaboration with your Mentor, you will plan a lesson for each day to
practise the use of summative assessment including administering the test, scoring and recording
the result. First, you must agree with your Mentor the English, maths or science topics you will teach
and test this week. Then construct the tests and prepare the marking schemes as you did last week. In planning how to administer the tests consider the following points: • How will the pupils be arranged for the test? • How long will the test take? • What instructions will you give the pupils? • How will you make the pupils understand the instructions if they cannot read accurately? • Will the test be written on the board or printed? • How will the class be managed if the test is to be written on the board? • What will the pupils do when they finish the test? • How long will the test take? When planning how to administer the test be gender sensitive so that the girls especially wil When planning how to administer the test be gender sensitive so that the girls especially will
participate in the exercise actively without feeling intimidated. In planning how to score the tests, think about these points: will you score the test papers during
or after the lesson? For some tests, can the pupils mark each other’s test answers and calculate the
score? How would you organise this? In planning how to score the tests, think about these points: will you score the test papers during
or after the lesson? For some tests, can the pupils mark each other’s test answers and calculate the
score? How would you organise this? Student Teacher Handbook for Teaching Pratice 100 In planning how you will record the results, bear the following points in mind: In planning how you will record the results, bear the following points in mind: YEAR 3 - OFF-CAMPUS TP • How does the class teacher record test results? • How does the school gather all the test results from each class? • How are the test results used? • Will you announce the scores to the whole class or post the results on the wall, or give
individual pupils their score? PLAN TOGETHER • Will the test score be a numerical score or a grade score? How can you be sure that you
have marked the pupils’ test fairly? Discuss with your Mentor to ensure that your approach to the summative assessment task is
consistent with classroom practice and the requirements of the TP school, and is familiar to
the pupils. REFLECT TOGETHER After each lesson which incorporates summative assessment, your Mentor will guide you to make
a critical evaluation of your own performance. Your TP partner should also be involved in the
discussion. As you are both teaching the same class it is important that you are using a consistent
approach to the summative assessment activities and there may be some common points for both
of you to follow up. After each lesson which incorporates summative assessment, your Mentor will guide you to make
a critical evaluation of your own performance. Your TP partner should also be involved in the
discussion. As you are both teaching the same class it is important that you are using a consistent
approach to the summative assessment activities and there may be some common points for both
of you to follow up. Y3 TP Activity 11.3: Reflect together with your Mentor on the summative assessment
activities PLAN TOGETHER You and your Mentor will have planned together the approaches you will use when administering
the summative assessment test, scoring and recording of the results. As a final preparation before
you teach the lesson and administer the summative assessment test each day, ensure that you have
everything ready that you will need as the teacher and everything that the pupils will need for the
test. Think about how to ensure that the test is an enjoyable activity for the pupils and not a
stressful experience, as stress can adversely affect individual pupils’ performance. Bear in mind
that the test will be administered and scored in a way that is fair to all pupils, girls and boys alike. 101 Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 3 Year 1 - 3: Teaching Practice DO
Teach the lesson incorporating summative test administration, scoring and recording the result
Teach the specific lesson for the Mentor to observe. Teach the lesson and administer the summative
assessment test according to the prepared lesson plan. The Mentor will observe and record some
points for feedback specifically in relation to your administration of the summative assessment
activity, the scoring and recording of results. PLAN TOGETHER Arrange with the Mentors and Student Teachers in your TP school, a convenient time and place
for this week’s Reflective Practice meeting. You should also invite any other officials including the
Link Tutor, TPC, GEO and CS to attend if they are visiting the TP school at the time of the Reflective
Practice meeting. The focus of the meeting will be the effectiveness of the summative assessment
activities all Student Teachers have engaged in during Week 11. Prepare for the Reflective Practice
meeting by reviewing how effectively you feel you administered the summative assessment
activities. In particular think about any advice relating to summative assessment that you would like
to receive from the Mentors during the meeting. Arrange with the Mentors and Student Teachers in your TP school, a convenient time and place
for this week’s Reflective Practice meeting. You should also invite any other officials including the
Link Tutor, TPC, GEO and CS to attend if they are visiting the TP school at the time of the Reflective
Practice meeting. The focus of the meeting will be the effectiveness of the summative assessment
activities all Student Teachers have engaged in during Week 11. Prepare for the Reflective Practice
meeting by reviewing how effectively you feel you administered the summative assessment
activities. In particular think about any advice relating to summative assessment that you would like
to receive from the Mentors during the meeting. DO • How effectively were the summative assessment tasks administered, scored and results
recorded? • What further advice is needed for the Student Teachers to improve their practice in future? Record in your TP Journal any advice that is given by the Lead Mentor and Mentors, or any
other visiting officials, during the Reflective Practice meeting that will help you to improve
in future. REFLECT TOGETHER
Complete the following Self-Assessment Questionnaire and record your score in your TP Journal:
STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE:
Answer the following questions based on the summative assessment activities in Week 11. Teaching skill/competence
Score
a) How prepared was I for the summative assessment activities? b) How well did I administer the summative assessment activities? c) Was my scoring system fair for all pupils? d) Did my system for recording results work well? Score: needs improvement = 1; satisfactory = 2; good performance = 3 Student Teacher Handbook for Teaching Pratice 102 YEAR 3 - OFF-CAMPUS TP Y3 TP Topic 12: Interpret test results - Student Teachers individually teach specific period
Purpose: In this Topic you will practise interpreting test results in summative assessment
Activity 12.1: Mentor and Student Teachers plan together a lesson for each day for the Student
Teachers to practise interpreting test results of summative assessment
Activity 12.2: Student Teachers teach the summative assessment lesson individually each day and
the Mentor observes
Activity 12.3: Mentor and Student Teachers reflect together on the summative assessment test
results and their interpretation
Competencies: (i) Use of strategies to assess pupils using summative assessment and interpret
the test results: Competency 8(h) (see Annex 1) Activity 12.2: Student Teachers teach the summative assessment lesson individually each day and
the Mentor observes Activity 12.3: Mentor and Student Teachers reflect together on the summative assessment test
results and their interpretation Competencies: (i) Use of strategies to assess pupils using summative assessment and interpret
the test results: Competency 8(h) (see Annex 1) When you have completed these three Y3 Week 12 activities you will have: • planned together with your Mentor a lesson for each day to help you to practise interpreting
test results of summative assessment; • planned together with your Mentor a lesson for each day to help you to practise interpreting
test results of summative assessment; • taught the summative assessment lesson individually using appropriate subject knowledge a
the Mentor observes; • reflected together with your Mentor on the summative assessment activities. Y3 TP Activity 12.1: Plan a lesson for each day to practise the use of summative assess-
ment and interpretation of test results Y3 TP Activity 12.1: Plan a lesson for each day to practise the use of summative assess-
ment and interpretation of test results Y3 TP Week 12 - Summary of the Task When you have completed these three Y3 Week 12 activities you will have: REFLECT TOGETHER You will need to seek the guidance of your Mentor when you prepare the test questions and the
marking scheme, as you did last week. Check with the Mentor that the summative assessment
activities are appropriate for the pupils and the subject content you will be teaching. Review your
and your TP partner’s summative assessment activities to ensure they are consistent and will not
be too burdensome for the pupils. In particular, share your strategies for interpreting the results. For example, interpretation of individual pupil performance by grading, class performance, teacher
performance, comparison of girls and boys results, etc. DO Prepare lesson plans including summative assessment activities to interpret the test results
At this stage, you will have to prepare the lesson plans you have discussed with your Mentor. Develop the test questions and the marking scheme, bearing in mind how you intend to interpret
the summative assessment test results. Y3 TP Activity 12.2: Teach the summative assessment lesson individually each day and
interpret the summative assessment results as the Mentor observes Y3 TP Activity 12.2: Teach the summative assessment lesson individually each day and
interpret the summative assessment results as the Mentor observes PLAN TOGETHER Interpretation of summative assessment
results - examples:
• For pupils and parents
• For next years’ teachers in the same
school or next years’ teachers in the
next school
• For District Office and National level
reporting
Information from summative assessment
results - examples:
• Individual performance
• Class performance
• School performance
• Performance of girls and boys
• Position in the class
• Remedial teaching needed
• Teacher performance In your final week of Y3 teaching practice,
you will conclude the practice of summative
assessment skills by interpreting the test
results (see Text Box examples). You will still
need to practise the skills learned during
Weeks 10 and 11 TP – test construction,
marking scheme design, administering the
test, scoring and recording the results. The
final task of the Student Teacher in the
process of conducting summative assessment
is the interpretation of the results, especially
in this instance when the summative
assessment is conducted at the end of the
school year. Check with your Mentor on how the
summative assessment test results are used. For example, are the summative assessment
test results: (a) included in an end-of-year
school report to pupils and parents? (b)
collated and submitted to the district
education office? (c) passed on to the class
teacher who will teach the pupils next year? Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 103 www.t-tel.org 3 Year 1 - 3: Teaching Practice Year 1 - 3: Teaching Practice Discuss with your TP partner what you recall from your college studies about how test results are
interpreted. Review your DBE notes on summative assessment. Under the supervision of your Mentor, plan a lesson each day to practise using summative
assessment and interpretation of results. You will need to agree with your Mentor the English,
maths or science topics you will teach this week and the types of test you will construct. Discuss how
the assessment results should be interpreted depending on the purpose. For example, a reading
comprehension test might be included in pupils’ end-of-year grade or to inform the parents that
the pupil needs to be encouraged to practise reading at home. The class scores in a numeracy test
may be used to report on improved learning achievement in basic numeracy or they may be used to
compare the performance of girls and boys. PLAN TOGETHER After you have taught the lesson, administered the summative assessment activity, marked,
scored and recorded the results you will have to focus on interpretation of the test results. Plan together with your Mentor and TP partner how you will present your interpretation of the
summative assessment results. After you have taught the lesson, administered the summative assessment activity, marked,
scored and recorded the results you will have to focus on interpretation of the test results. Plan together with your Mentor and TP partner how you will present your interpretation of the
summative assessment results. DO
Teach the lesson, conduct and interpret the summative assessment results
Teach the lesson individually according to the lesson plan each day. Administer the summative
assessment activity, mark, score and record the results. Interpret and present the results of the
summative assessment according to the lesson plan agreed with the Mentor. The Mentor will
observe the lesson, focusing specifically on the interpretation of results from the summative
assessment activity. REFLECT TOGETHER REFLECT TOGETHER
After each lesson, record your interpretation of the summative assessment results in your
TP Journal. Review, with your Mentor and TP partner, your interpretation of the summative
assessment results. Consider how the interpretation of the summative assessment results provides
information that can influence your teaching in future. After each lesson, record your interpretation of the summative assessment results in your
TP Journal. Review, with your Mentor and TP partner, your interpretation of the summative
assessment results. Consider how the interpretation of the summative assessment results provides
information that can influence your teaching in future. Student Teacher Handbook for Teaching Pratice 104 Y3 TP Activity 12.3: Mentor and Student Teachers reflect together on the
interpretation of summative assessment results PLAN TOGETHER In this final week of Y3 TP, you will arrange the last Reflective Practice meeting at a time convenient
to the Lead Mentor, all Mentors and Student Teachers in your TP school. The topics for discussion
in this final meeting will be “How can we as teachers make best use of our interpretation of
summative assessment results? How can we check that they are accurate and useful to the teacher
and the pupil? How does the interpretation inform teaching and learning?” Participate in the final Reflective Practice meeting You should come to the final Reflective Practice meeting with your TP Journal entries on
interpretation of the summative assessment activities. In the discussion, try to relate your
interpretations to each of the Reflective Practice meeting topics: You should come to the final Reflective Practice meeting with your TP Journal entries on
interpretation of the summative assessment activities. In the discussion, try to relate your
interpretations to each of the Reflective Practice meeting topics: • How can we as teachers make the best use of our interpretation of summative assessment
results? • How can we check that they are accurate and useful to the teacher and the pupil? • How does the interpretation of summative assessment results inform teaching and learnin STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE: STUDENT TEACHER SELF-ASSESSMENT QUESTIONNAIRE:
Answer the following questions based on the summative assessment activities in Week 12. Teaching skill/competence
Score
a) Did my strategy for interpretation of the summative assessment results work well? b) Was I able to identify differences in learning achievement between girls and boys? c) How was my overall performance throughout the Y3 teaching practice? d) How well did I develop my teaching skills from beginning to end of Y3 TP? Score: needs improvement = 1; satisfactory = 2; good performance = 3 You will need to take your completed TP Journal with you when you attend the Y3 Post-TP week in
your college next week. Check that it is up-to-date and all entries are well presented as this forms
part of your final assessment. It will also provide you with a comprehensive record of your teaching
practice experience and information on which to reflect as a beginner teacher next year. 105 Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning www.t-tel.org 3 Year 1 - 3: Teaching Practice Y3 Post-TP Topic: Post-Off-Campus Activities (college-based) led by the TPC with Tutors and
Student Teachers Post-TP Activity 2: Reflection and discussion between Student Teachers and Tutors to review
the Off-Campus TP tasks Y3 Post-TP Activity 1: Review of Teacher Competencies for Year 3 and Student Teacher
performance Y3 Post-TP Activity 1: Review of Teacher Competencies for Year 3 and Student Teacher
performance Y3 Post-TP Activities - Summary of the Task When you have completed these two Y3 TP Post-Off-Campus TP activities with your TPC and Tutor,
you will have: • reviewed the Teacher Competencies for Year 3 and Student Teacher performance;
• reflected discussed and reviewed the Off Campus TP tasks with your TPC and Tutor • reviewed the Teacher Competencies for Year 3 and Student Teacher performance;
• reflected, discussed and reviewed the Off-Campus TP tasks with your TPC and Tutor. • reflected, discussed and reviewed the Off-Campus TP tasks with your TPC and Tuto PLAN TOGETHER PLAN TOGETHER
In this final teaching practice, you have worked continuously for 12 weeks with guidance and
support from your Mentors. During this week, you will review your teaching practice achievements
including the Teacher Competencies you practised. This review will be led by the Tutors and TPC,
with all Student Teachers in attendance. At this review meeting, each Student Teacher will talk
about their TP experiences in relation to achievement of the Teacher Competencies. In this final teaching practice, you have worked continuously for 12 weeks with guidance and
support from your Mentors. During this week, you will review your teaching practice achievements
including the Teacher Competencies you practised. This review will be led by the Tutors and TPC,
with all Student Teachers in attendance. At this review meeting, each Student Teacher will talk
about their TP experiences in relation to achievement of the Teacher Competencies. Your Link Tutor and the TPC will organise the Post-TP review meeting using the THINK-PAIR-SHARE
approach. For the THINK activity, begin by reviewing, together with your TP partner, the Teacher
Competencies listed in Annex 1. Identify in which week of the Y3 TP you practised each competency. Refer to your TP Journal for each week and check how well you achieved each of the competencies. Be ready to present a specific example of your best competency achievement and an example of a
competency you found more difficult to achieve, from your TP Journal records. Identify any specific
points about Teacher Competencies you might want to discuss in the review meeting. DO DO
You should have your TP Journal with you for the meeting, and you should have your
self-assessment results ready to share with the TP group. There should be opportunity for you to
comment on your TP experiences with your TP group. ou should have your TP Journal with you for the meeting, and you should have your
lf-assessment results ready to share with the TP group. There should be opportunity for you t
mment on your TP experiences with your TP group. PLAN TOGETHER In preparation for the final review meeting, re-read your TP Journal entries. In particular, re-read
each of the self-assessment questionnaires you completed in Week 6 to Week 12 – but you must not
change any of your scores. If you completed the self-assessment questionnaires honestly you will
have some questions with a score of 1, some questions with a score of 2 and some questions with
a score of 3. Collate your scores for each week into one summary table and analyse your results. Compare your results with your TP partner’s results. DO In the PAIR stage, work with your TP group, that is, all the Student Teachers from your TP school. Present the competencies that each of you rated as your best achievement and the competencies
that you rated as more difficult to achieve. In the PAIR stage, work with your TP group, that is, all the Student Teachers from your TP school. Present the competencies that each of you rated as your best achievement and the competencies
that you rated as more difficult to achieve. In the SHARE stage each TP group should share with the whole group to find out whether there are
common patterns to the responses from all Y3 Student Teachers. Your responses may be collated
by your TP group members onto a flip chart, according to the competencies you feel were most
strongly and less strongly achieved. In the larger group, led by the Link Tutors and TPC, you will have the opportunity to share your
experiences and any specific points about Teacher Competencies you might want to discuss in the
review meeting. Student Teacher Handbook for Teaching Pratice 106 REFLECT TOGETHER
Reflect with your TP group on the competencies you all feel most confident about and
competencies you all feel you need to practise more to improve. Your Tutor will gather from all
the Student Teachers the strategies and ideas for improving those weaker competencies. YEAR 3 - OFF-CAMPUS TP Y3 Post-TP Activity 2: Reflection and discussion between Student Teachers and Tutors
to review the Off-Campus TP tasks Y3 Post-TP Activity 2: Reflection and discussion between Student Teachers an
to review the Off-Campus TP tasks Y3 Post-TP Activity 2: Reflection and discussion between Student Teachers and Tutor
to review the Off-Campus TP tasks PLAN TOGETHER
In this final Y3 TP activity, you will have the opportunity to reflect and discuss with your Tutors
your Off-Campus TP tasks. You will be able to review how you performed individually during the
teaching practice. This review process will be led by the TPC and Link Tutors with all Student
Teachers in attendance. Student Teacher Handbook for Teaching Pratice REFLECT TOGETHER REFLECT TOGETHER
In a final wrap-up meeting, reflect with your TP groups and your Tutor on a general overview of
the Teaching Practice experience. Your Tutor will share ideas on the successes and challenges
experienced during the Y3 TP and they will gather ideas from you to find out how to improve the
TP arrangements in future years. In a final wrap-up meeting, reflect with your TP groups and your Tutor on a general overview of
the Teaching Practice experience. Your Tutor will share ideas on the successes and challenges
experienced during the Y3 TP and they will gather ideas from you to find out how to improve the
TP arrangements in future years. The discussion should be open and everyone should feel free to express their views. This final
reflection may include suggestions on a range of issues including Student Teachers’ assessment,
effectiveness of the Mentoring arrangements, accommodation and safety of Student Teachers,
especially female Student Teachers, during teaching practice, and teaching practice as a preparation
for becoming a qualified teacher. Your TPC and Tutors will keep a careful note of the discussion points raised in this final meeting, for
the College record and future action. 107 Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning Year 1 - 3: Teaching Practice Year 1 - 3: Teaching Practice Year 1 - 3: Teaching Practice 108 Student Teacher Handbook for Teaching Pratice Annex 1. Teacher Competencies Does
everybody agree or disagree? Is there an alternative answer?)
e) Uses strategies to increase participation during questioning
(e.g. calls on pupils randomly, doesn’t require pupils to raise
their hands, gives praise for effort)
f) Other:
6. The teacher promotes and
manages whole class discussion
a) Uses brainstorming (asks for pupils’ views/ideas on a topic)
b) Writes pupils’ views/ideas down
c) Organises/summarises pupils’ views/ideas
d) Builds on what is said
e) Encourages pupils to build on what is said
f) Amount of time pupils talk = amount of time the teacher
talks/lectures
g) Other:
7. The teacher uses strategies to
organise and execute group or
pair work
(Be sympathetic to large class
sizes when marking)
a) Demonstrates strategies for group formation (e.g. assigns
groups/pairs by who is sharing a desk, through ability,
randomly)
b) Demonstrates strategies for group management (e.g. assigning roles, randomly selecting somebody to report back
to class)
c) Has pupils discuss a topic with each other in groups or pairs
d) Uses Think-Pair-Share (pupils think individually, discuss with a
partner and then share views/ideas with the class)
e) Has groups/pairs complete the same activity/task
f) Has groups/pairs complete different activities/tasks
g) Other:
8. The teacher uses strategies to
assess pupil understanding
a) Uses questions to see whether pupils understand
b) Uses peer-assessment (pupils discuss/mark each other’s
work)
c) Uses pupil self-assessment
d) Uses pupil presentations to see whether they understand
e) Assesses through individual work assignment/homework
f) Has pupils vote on different answers to see if they
understand
g) Uses traffic lights (red-yellow-green cards for groups to
indicate how they are dealing with a task)
h) Other: e.g. end of term examinations, end of year
examinations
9. The teacher gives constructive
feedback on pupil’s answers, work
or effort
a) Gives written feedback on work
b) Gives verbal feedback to the class or individuals (You would
benefit from looking at …. This area needs improvement…
You could do this to strengthen…)
c) Corrects wrong answers kindly
d) Gives praise for correct answers/good effort
e) Other: Teacher Competencies
Examples of Actions or Strategies
d) Asks follow-up questions to build on student responses (How
did you come to this answer? Why do you think this? Does
everybody agree or disagree? Is there an alternative answer?)
e) Uses strategies to increase participation during questioning
(e.g. Annex 1. Teacher Competencies calls on pupils randomly, doesn’t require pupils to raise
their hands, gives praise for effort)
f) Other:
6. The teacher promotes and
manages whole class discussion
a) Uses brainstorming (asks for pupils’ views/ideas on a topic)
b) Writes pupils’ views/ideas down
c) Organises/summarises pupils’ views/ideas
d) Builds on what is said
e) Encourages pupils to build on what is said
f) Amount of time pupils talk = amount of time the teacher
talks/lectures
g) Other:
7. The teacher uses strategies to
organise and execute group or
pair work
(Be sympathetic to large class
sizes when marking)
a) Demonstrates strategies for group formation (e.g. assigns
groups/pairs by who is sharing a desk, through ability,
randomly)
b) Demonstrates strategies for group management (e.g. assigning roles, randomly selecting somebody to report back
to class)
c) Has pupils discuss a topic with each other in groups or pairs
d) Uses Think-Pair-Share (pupils think individually, discuss with a
partner and then share views/ideas with the class)
e) Has groups/pairs complete the same activity/task
f) Has groups/pairs complete different activities/tasks
g) Other:
8. The teacher uses strategies to
assess pupil understanding
a) Uses questions to see whether pupils understand
b) Uses peer-assessment (pupils discuss/mark each other’s
work)
c) Uses pupil self-assessment
d) Uses pupil presentations to see whether they understand
e) Assesses through individual work assignment/homework
f) Has pupils vote on different answers to see if they
understand
g) Uses traffic lights (red-yellow-green cards for groups to
indicate how they are dealing with a task)
h) Other: e.g. end of term examinations, end of year
examinations
9. The teacher gives constructive
feedback on pupil’s answers, work
or effort
a) Gives written feedback on work
b) Gives verbal feedback to the class or individuals (You would
benefit from looking at …. This area needs improvement…
You could do this to strengthen…)
c) Corrects wrong answers kindly
d) Gives praise for correct answers/good effort
e) Other: Annex 1. Teacher Competencies Abstract from Teacher Competencies Matrix in the Handbook on Mentorship and Supervision of
Teacher Trainees on School Attachment (GES 2002). Teacher Trainees on School Attachment (GES 2002). Teacher Competencies
Examples of Actions or Strategies
1. The teacher uses strategies to
open the lesson
a) Asks pupils what they remember from last class
b) Gives a summary of what pupils have learned
c) Ask pupils questions about the topic
d) Gives a clear introduction to the topic of the lesson
e) Shares learning objectives with pupils
f) Other:
2. The teacher uses strategies to
provide clear explanations for
new concepts, knowledge or skills
a) Gives an explanation
b) Gives examples
c) Gives a definition(s)
d) Gives a demonstration
e) Gives instructions
f) Follows the process ‘I do’, ‘We do’, ‘You do’
g) Other:
3. The teacher uses different
TLMs to facilitate learning
a) Uses white board/chalk board effectively
b) Real-life objects/hand-made objects
c) Pictures/posters/charts
d) Newspapers/magazine articles
e) Textbooks
f) Reference/supplementary books
g) Radio or other electronic audio/visual device
h) Teacher has pupils bring resources to the lesson
i) Other:
4. The teacher uses different
interactive methods/activities to
facilitate learning
a) Question and answer (where pupils also ask questions
of teacher)
b) Whole class dialogue (e.g. discussion, debate,
brainstorming)
c) Small group work (e.g. discussion, debate, brainstorming)
d) Class assignment
e) Student presentation
f) Role-play
g) Games
h) Story telling
i) Songs, poems or rhymes
j) Other:
5. The teacher asks pupils a range
of questions during the lesson
a) Asks questions that require remembering (What do you
remember about…? How do you define….? What is….?)
b) Asks questions that require analysing and evaluating (How
can you classify…? Why do you think…? Can you compare
with ….? What are the advantages/disadvantages of …? What is the most important…? Is there a better solution
than…? Can you suggest an alternative? Can you predict the
outcome if…?)
c) Handles student responses well (sufficient wait time,
doesn’t immediately correct wrong answers, asks follow
up questions) ANNEX c) Handles student responses well (sufficient wait time,
doesn’t immediately correct wrong answers, asks follow
up questions) Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 109 Year 1 - 3: Teaching Practice Year 1 3: Teaching Practice
Teacher Competencies
Examples of Actions or Strategies
d) Asks follow-up questions to build on student responses (How
did you come to this answer? Why do you think this? Examples of Actions or Strategies 9. The teacher gives constructive
feedback on pupil’s answers, work
or effort 110 Student Teacher Handbook for Teaching Pratice Teacher Competencies
Examples of Actions or Strategies
10. The teacher uses techniques to
address mixed abilities
a) Uses peer teaching
b) Gives individual coaching/support
c) Has pupils give each other support in mixed ability groups
d) Gives more/different activities to keep excellent pupils
challenged
e) Other:
11. The teacher applies all
teaching methods equally to boys
and girls
a) Gives equal chance to girls and boys to ask/answer questions
b) Assigns leadership roles equally to girls and boys in lesson
activities
c) Makes sure girls and boys participate equally in activities
d) Allows sufficient time for girls and boys who may be shy or
afraid to speak out
e) Checks if both girls and boys equally understand the lesson
f) Provides constructive feedback to both girls and boys in class
g) Other:
12. The teacher uses strategies to
challenge traditional gender roles
and norms
a) Uses learning materials that do not portray traditional
gender roles (e.g. women cooking/cleaning and men
working)
b) Points out and discusses traditional gender roles that appear
in materials (e.g. discusses how gender roles are
problematic)
c) Actively uses examples (in exercises or activities) that
challenge or reverse traditional gender roles (e.g. show men
cooking)
d) Makes girls and boys feel comfortable/confident to not
follow traditional gender roles (e.g. boys cooking, girls being
doctors)
e) Other:
13. The teacher uses strategies to
effectively manage a class
(particularly a large class)
a) Uses strategies to get the class ready for learning which do
not require shouting, threats or violence
b) Makes the lesson interesting to draw the attention of almost
all pupils in class
c) Is able to divide and share resources amongst all the pupils
d) Establishes clear rules and expectations for pupil conduct
e) Other:
14. The teacher has clearly paid
attention to the seating
arrangements in the classroom
a) Pupils who need more support (physical/learning disabilities)
are sitting at the front
b) Girls and boys are equally mixed throughout the classroom
c) Quiet and more vocal pupils are equally mixed throughout
the classroom (quiet pupils are not all sitting at the back)
d) Other
15. Student Teacher Handbook for Teaching Pratice Examples of Actions or Strategies The teacher uses strategies to
close the lesson
a) Reviews core concepts/skills from the lesson
b) Summarises what has been taught
c) Asks if pupils have any more questions
d) Other ANNEX Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 111 Year 1 - 3: Teaching Practice Teacher Competencies
Examples of Actions or Strategies
16. The teacher has a clear,
high-quality lesson plan or
activity plan for parts of the
lesson
a) Lesson objectives were completed during the observed
lesson
b) Lesson plan demonstrates the use of appropriate TLM
c) Lesson plan demonstrated the use of interactive methods
d) Time was allocated appropriately for each part of the lesson
e) Lesson plan demonstrated a logical development for the
lesson
f) Other: 112 Student Teacher Handbook for Teaching Pratice Annex 2. Lesson Plan Format
Week Ending:
Class:
Subject:
Average Age:
Reference:
No. on Roll:
DAY/DURATION
TOPIC/SUB-TOPIC/ASPECT
OBJECTIVES/RPK
TEACHER/LEARNER
ACTIVITY
TEACHING/LEARNING
MATERIALS
CORE POINTS
EVALUATION/REMARKS Annex 2. Lesson Plan Format ANNEX Transforming Teacher Education and Learning
www.t-tel.org 113 Year 1 - 3: Teaching Practice Annex 3. Lesson Observation Format Annex 3. Lesson Observation Format
This lesson observation format may be used by you, as a Student Teacher
Student Teacher’s Name:
TP School:
Class:
Subject:
Topic:
Topic
Observable evidence
Remarks
Lesson Sequence - start of
the lesson
Lesson Sequence -
development of the lesson
Lesson Sequence - end of the
lesson
Learner participation –
pupils activities
Learning activities for
different pupil abilities
Use of teaching and learning
resources
Teacher’s use of praise and
encouragement
Teacher communication –
teacher talk
Teacher communication –
pupil talk
Classroom Management –
use of instructional time
Management of pupil
behaviour
Individual, group and
class activities Student Teacher Handbook for Teaching Pratice 114 Topic
Observable evidence
Remarks
Subject knowledge –
appropriate for pupils’ level
Informal assessment of
pupils’ understanding
Formal assessment of pupils’
understanding
Feedback to pupils on
learning achievement ANNEX 115 Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning Year 1 - 3: Teaching Practice Annex 4. Assessment Procedures Teacher states summaries/
core points for all activities
which relate to main skills/
concepts
Teacher states some
summaries/core points
teaching/learning activities
which relate to main skills/
concepts
Teacher states summaries/
core points but are not related
to main skills/concepts
Teaching/learning activities
are stated in lesson plan
sequentially but are not
logical. Teaching/learning activities
stated in lesson plan are
adequate but are not
sequenced. Teaching/learning activities
stated in lesson plan but are
inadequate. Teacher states appropriate
TLMs and when and how they
will be used in the lesson. Teacher states TLMs and when
and how they will be used in
the lesson. Teacher states TLMs to be
used in advanced preparation
but does not indicate when
they will be used in the lesson. Teacher demonstrates subject
knowledge, indicates a variety
of techniques to ensure active
student participation, and
provides sample questions and
Teacher demonstrates subject
knowledge, and indicates a
variety of techniques to
ensure active student
participation. Teacher demonstrates subject
knowledge, but does not
indicate techniques to ensure
active student participation. NAME OF STUDENT
LEVEL
REG NO. PROGRAMME
SCHOOL OF PRACTICE
FORM/CLASS
SUBJECT
DATE
TIME
LESSON TOPIC NAME OF STUDENT
LEVEL
REG NO. PROGRAMME
SCHOOL OF PRACTICE
FORM/CLASS
SUBJECT
DATE
TIME
LESSON TOPIC ANNEX 3 - Good
2 - Satisfactory
1 - Poor
Teacher states specific
relevant and measurable
objectives but they are not
achievable within the duration
of the lesson. Teacher states objectives
that are relevant but not
measurable. Teacher states lesson
objectives that are relevant
but not specific. Teacher states summaries/
core points for all activities
which relate to main skills/
concepts
Teacher states some
summaries/core points
teaching/learning activities
which relate to main skills/
concepts
Teacher states summaries/
core points but are not related
to main skills/concepts
Teaching/learning activities
are stated in lesson plan
sequentially but are not
logical. Teaching/learning activities
stated in lesson plan are
adequate but are not
sequenced. Teaching/learning activities
stated in lesson plan but are
inadequate. Teacher states appropriate
TLMs and when and how they
will be used in the lesson. Teacher states TLMs and when
and how they will be used in
the lesson. Teacher states TLMs to be
used in advanced preparation
but does not indicate when
they will be used in the lesson. Teacher demonstrates subject
knowledge, indicates a variety
of techniques to ensure active
student participation, and
provides sample questions and
answers. Annex 4. Assessment Procedures The assessment of DBE student teaching practice will be based on the following scoring profile: The assessment of DBE student teaching practice will be based on the following scoring profile: The assessment of DBE student teaching practice will be based on the following scoring profile:
Year 1 Observation TP 15%
Year 2 On-Campus TP 25%
Year 3 Off Campus TP 60% Teaching Practice Assessment Form A has been developed by University of Cape Coast and
approved for all CoEs to be used as the standard Assessment Tool by CoE Tutors. Item and Score
5- Outstanding
4 – Very Good
Objectives And Core Points In Lesson Plan
1. Objectives
Teacher states specific,
relevant, measurable and
achievable objectives which
are linked to classroom
activities. Teacher states specific
relevant, measurable and
achievable objectives. 2. Summarises Core Points
Teacher states summaries/
core points for all activities
which relate to and clarify
main skills/concepts and are
also related to lesson
objectives. Teacher states summaries/
core points for all activities
which relate to and clarify
main skills/concepts
3. Teaching and Learning
Activities
Teacher provides varied
teaching/learning activities
that are logical, timely and
direct student learning. Teaching/learning activities
are sequenced, logical and
appropriate, time indicated. 4. TLMs
Teacher states appropriate
and varied TLMs and indicates
when in suitable stages of the
lesson to use them. Teacher states appropriate
and varied TLMs and indicates
when and how they will be
used in the lesson. 5. Subject and Pedagogical
knowledge
Teacher’s subject
knowledge is logical; is linked
to objectives(s); provides
answers to sample questions;
provides activities to engage
students; uses vocabulary /
technical terms. Teacher shows logical subject
knowledge linked to
objectives, provides answers
to sample questions &
activities to engage students. 116 Student Teacher Handbook for Teaching Pratice NAME OF STUDENT
LEVEL
REG NO. PROGRAMME
SCHOOL OF PRACTICE
FORM/CLASS
SUBJECT
DATE
TIME
LESSON TOPIC
3 - Good
2 - Satisfactory
1 - Poor
Teacher states specific
relevant and measurable
objectives but they are not
achievable within the duration
of the lesson. Teacher states objectives
that are relevant but not
measurable. Teacher states lesson
objectives that are relevant
but not specific. Annex 4. Assessment Procedures Teacher demonstrates subject
knowledge, and indicates a
variety of techniques to
ensure active student
participation. Teacher demonstrates subject
knowledge, but does not
indicate techniques to ensure
active student participation. 3 - Good Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning
www.t-tel.org 117 Year 1 - 3: Teaching Practice Item and Score
5- Outstanding
4 – Very Good
Objectives And Core Points In Lesson Plan
6. Introduction to the Lesson
Teacher introduces the lesson,
reviews students’ RPK, links it
up with topic and stimulates
student interest. Teacher
shares lesson objectives with
students. Teacher introduces the lesson,
reviews students’ RPK, links it
up with topic and stimulates
student interest. 7. Presentation - Teaching and
Learning activities
Teacher organises teaching
and learning activities
sequentially, logically, uses
varied pedagogical skills,
maintains balance between
teacher and student activities. Teaching and learning
activities are organized,
sequentially and logical. Teacher uses varied
pedagogical skills. 8. Pace of lesson and audibility
of voice
Pace is appropriate for the
entire lesson and all students
can hear the teacher clearly. Teacher does not shout. Pace is appropriate some of
the time and voice is audible
from the back of the class
throughout the lesson. Teacher does not shout. 9. Questioning and feedback
Teacher asks a well-balanced
mixture of factual, probing,
high order & divergent
questions: distributes
questions fairly, facilitates
independent & co-operative
learning. Stimulates critical
thinking in students. Teacher asks a mixture of
factual and high order
questions: distributes
questions fairly, facilitates
independent & co-operative
learning. Encourages students’
questions and answers. 10. Use of chalkboard
Teacher manages chalkboard
effectively, date, subject and
topic are written on the board:
all core points are on the
board; writing is legible; gives
students time to write down
core points into their books:
cleans board at the end of
lesson. Teacher manages chalkboard
effectively, date, subject and
topic are written on the board:
all core points are on the
board; writing is legible; gives
students time to write down
core points into their books
11. Use of Teaching Learning
Resources. TLR/TLMs
Teacher uses appropriate
TLRs, links them to students’
previous knowledge and
lesson objectives at key
stage/s of the lesson. Teacher uses appropriate
TLRs, links them to students’
previous knowledge and
lesson objectives. Teacher introduces the lesson,
reviews students’ RPK, links it
up with topic and stimulates
student interest. Teacher
shares lesson objectives with
students. Annex 4. Assessment Procedures Teacher introduces the lesson,
reviews students’ RPK, links it
up with topic and stimulates
student interest. Teacher manages chalkboard
effectively, date, subject and
topic are written on the board:
all core points are on the
board; writing is legible; gives
students time to write down
core points into their books Teacher uses appropriate
TLRs, links them to students’
previous knowledge and
lesson objectives at key
stage/s of the lesson. Teacher uses appropriate
TLRs, links them to students’
previous knowledge and
lesson objectives. 118 Student Teacher Handbook for Teaching Pratice 3 - Good Teacher introduces the lesson,
reviews students’ RPK, and
links it up with topic. Teacher introduces the lesson,
reviews students’ Relevant
Previous Knowledge RPK,
Teacher merely introduces the
lesson. Teaching and learning
activities are organized,
sequentially and logical. Teaching and learning
activities are organized,
sequential. Teaching and learning
activities are organized. Pace is appropriate some of
the time and voice is audible
from the back of the class
throughout the lesson
Pace is appropriate some of
the time but voice is not
audible from the back of the
class throughout the lesson
Pace of lesson is not
appropriate. Teacher speaks
too fast/too slow/too loud/
too low
Teacher asks a mixture of
factual and high order
questions: distributes
questions fairly, facilitates
independent learning. Accommodates students’
questions and answers. Teacher asks mostly
factual questions, he/she is
receptive to student
questions and answers and
acknowledges students’
answers. Teacher asks only factual
questions, he/she is not
receptive to student questions
and answers
Teacher manages chalkboard
effectively, date, subject and
topic are written on the board:
all core points are on the
board; writing is legible. Teacher manages chalkboard
effectively, date, subject and
topic are written on the board:
all core points are on the
board; writing not is legible
Teacher writes haphazardly on
the board or hardly writes on
the board at all. Teacher uses appropriate
TLRs, links them to students’
previous knowledge. Teacher uses appropriate
TLRs, but does not link them
to students’ previous
knowledge
Teacher does not have TLRs or
does not use TLRs brought to
class. ANNEX Teacher manages chalkboard
effectively, date, subject and
topic are written on the board:
all core points are on the
board; writing is legible. Teacher uses correct
grammatical expressions;
corrects students’ language
errors; uses effective verbal
and nonverbal communication;
avoids language mannerisms. Teacher engages all students
in lesson; facilitates
problem-solving among
students; monitors progress
and provides feedback. Teacher uses correct
grammatical expressions;
corrects students’ language
errors; occasionally exhibits
language mannerisms. Teacher engages some
students in lesson; does not
monitor progress effectively.
Feedback to students is
minimal. Teacher uses incorrect
grammatical expressions and
often exhibits some language
mannerisms. Annex 4. Assessment Procedures Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning
www.t-tel.org 119 Year 1 - 3: Teaching Practice Item and Score
5- Outstanding
4 – Very Good
Objectives And Core Points In Lesson Plan
12. Communication/Use of
language
Teacher uses correct
grammatical expressions;
corrects students’ language
errors; encourages students
to pay attention to their use
of language; uses effective
verbal and nonverbal
communication; avoids
language mannerisms. Teacher uses correct
grammatical expressions;
corrects students’ language
errors; uses effective verbal
and nonverbal communication;
avoids language mannerisms. 13. Student Participation
Teacher engages all students
in lesson; facilitates
problem-solving among
students; encourages
co-operative learning and
peer-tutoring; monitors
progress and provides
feedback. Teacher engages all students
in lesson; facilitates
problem-solving among
students; encourages
co-operative learning;
monitors progress and
provides feedback. 14. Assessment of student
learning
Teacher uses variety of
assessment techniques to
determine understanding;
assessments are appropriate
and timely; he/she encourages
students’ self-assessment and
application of learning. Teacher uses variety of
assessment techniques to
check understanding;
encourages students’
self-assessment and
application of learning. 15. Mastery of Subject matter
Teacher exhibits command
over subject matter, gives
precise information;
exhibits confidence, cites lots
of examples; relates content
to students’ experience,
fosters critical thinking. Teacher has command over
subject matter; exudes
confidence gives accurate
information, cites lots of
examples; relates content to
students’ experience. 16. Classroom Management
Teacher establishes a
purposeful learning
environment, interacts with
students, uses student’s ideas,
encourages co-operative
learning and monitors student
learning activities. Teacher establishes a
purposeful learning
environment, interacts with
students, uses students’ ideas,
and monitors student learning
activities. 4 – Very Good Teacher uses correct
grammatical expressions;
corrects students’ language
errors; encourages students
to pay attention to their use
of language; uses effective
verbal and nonverbal
communication; avoids
language mannerisms. Teacher uses correct
grammatical expressions;
corrects students’ language
errors; uses effective verbal
and nonverbal communication;
avoids language mannerisms. Teacher has command over
subject matter; exudes
confidence gives accurate
information, cites lots of
examples; relates content to
students’ experience. Teacher establishes a
purposeful learning
environment, interacts with
students, uses students’ ideas,
and monitors student learning
activities. 120 Student Teacher Handbook for Teaching Pratice 3 - Good 2 - Satisfactory 1 - Poor Teacher uses correct
grammatical expressions;
corrects students’ language
errors; occasionally exhibits
language mannerisms. Annex 4. Assessment Procedures Teacher uses correct
grammatical expressions; but
does not correct students’
grammatically incorrect
expressions; exhibits some
language mannerisms
Teacher uses incorrect
grammatical expressions and
often exhibits some language
mannerisms. Teacher engages all students
in lesson; facilitates
problem-solving among
students; monitors progress
and provides feedback. Teacher engages some
students in lesson; does not
monitor progress effectively. Feedback to students is
minimal. Students participation in the
lesson is low; there is virtually
no monitoring or feedback. Teacher uses a variety of
assessment techniques to
check understanding;
encourages students’
self-assessment of
understanding linked to lesson
objectives. Teacher uses only one
assessment method or tool
(e.g oral questioning, written
exercise, one- minute test, etc)
to check understanding. They
are linked to objectives. Teacher uses only one
assessment method or tool
(e.g oral questioning, written
exercise, one- minutes test,
etc) to check understanding. They are not linked to
objectives. Teacher has command over
subject matter; exudes
confidence gives accurate
information, cites lots of
examples; gives/elicits correct
answers to/from students. Teacher has command of
subject matter, gives relevant
information, cites some
examples. Some aspects of lesson are
not relevant; teacher exhibits
lack of confidence. Teacher establishes a
purposeful learning
environment, and monitors
student learning activities. Teacher establishes a learning
environment and interactions
with students are purposeful. Teacher establishes a learning
environment but interactions
with students are not directed
towards learning. Teacher uses incorrect
grammatical expressions and
often exhibits some language
mannerisms. Teacher uses correct
grammatical expressions;
corrects students’ language
errors; occasionally exhibits
language mannerisms. Teacher uses correct
grammatical expressions; but
does not correct students’
grammatically incorrect
expressions; exhibits some
language mannerisms ANNEX Teacher engages all students
in lesson; facilitates
problem-solving among
students; monitors progress
and provides feedback. Teacher has command over
subject matter; exudes
confidence gives accurate
information, cites lots of
examples; gives/elicits correct
answers to/from students. Teacher establishes a
purposeful learning
environment, and monitors
student learning activities. Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 121 Year 1 - 3: Teaching Practice Item and Score
5- Outstanding
4 – Very Good
Objectives And Core Points In Lesson Plan
17. Class Control
Teacher establishes clear
parameters for student
conduct, develops
appropriate strategies for
preventing problems, deals
with misbehaviour promptly. He/she is fair, firm but
friendly. Teacher establishes cl
parameters for studen
conduct, develops
appropriate strategie
preventing problems,
with misbehaviour pro
18. Annex 4. Assessment Procedures Closure
Teacher draws attention to
end of the lesson; uses
questions & answers,
summaries, practices to
clarify main points along
lesson objectives; gives &
marks written exercises to
evaluate learning; assigns
activity for next lesson. Teacher uses question
answers, summaries,
practices to clarify ma
points along lesson
objectives, gives & ma
written exercises to e
learning. 19. Teacher Behaviour
Teacher shows enthusiasm in
teaching, maintains students’
attention throughout the
lesson, is creative and
innovative, exhibits decorum
in speech and behaviour and
is smartly dressed. Teacher shows enthus
in teaching, maintains
students’ attention in
lesson, model polite
classroom behaviour a
creative and innovativ
20. Record keeping and
attitude to teaching
Teacher keeps records of
lessons in a bound
notebook for reference; ac-
cepts constructive feedback
to improve teaching; reflects
on teaching; shows positive
attitude towards teaching. Teacher keeps records
lessons in a bound
notebook for referenc
cepts constructive fee
to improve teaching; r
on teaching remarks. Item and Score
5- Outstanding
4 – Very Good
Objectives And Core Points In Lesson Plan
17. Class Control
Teacher establishes clear
parameters for student
conduct, develops
appropriate strategies for
preventing problems, deals
with misbehaviour promptly. He/she is fair, firm but
friendly. Teacher establishes clear
parameters for student
conduct, develops
appropriate strategies for
preventing problems, deals
with misbehaviour promptly. 18. Closure
Teacher draws attention to
end of the lesson; uses
questions & answers,
summaries, practices to
clarify main points along
lesson objectives; gives &
marks written exercises to
evaluate learning; assigns
activity for next lesson. Teacher uses questions &
answers, summaries,
practices to clarify main
points along lesson
objectives, gives & marks
written exercises to evaluate
learning. 19. Teacher Behaviour
Teacher shows enthusiasm in
teaching, maintains students’
attention throughout the
lesson, is creative and
innovative, exhibits decorum
in speech and behaviour and
is smartly dressed. Teacher shows enthusiasm
in teaching, maintains
students’ attention in the
lesson, model polite
classroom behaviour and is
creative and innovative. 20. Record keeping and
attitude to teaching
Teacher keeps records of
lessons in a bound
notebook for reference; ac-
cepts constructive feedback
to improve teaching; reflects
on teaching; shows positive
attitude towards teaching. Teacher keeps records of
lessons in a bound
notebook for reference; ac-
cepts constructive feedback
to improve teaching; reflects
on teaching remarks. 4 – Very Good Teacher establishes clear
parameters for student
conduct, develops
appropriate strategies for
preventing problems, deals
with misbehaviour promptly. Annex 4. Assessment Procedures Teacher establishes clear
parameters for student
conduct, develops
appropriate strategies for
preventing problems, deals
with misbehaviour promptly. Teacher establishes clear
parameters for student
conduct, develops
appropriate strategies for
preventing problems, deals
with misbehaviour promptly. He/she is fair, firm but
friendly. Teacher uses questions &
answers, summaries,
practices to clarify main
points along lesson
objectives, gives & marks
written exercises to evaluate
learning. Teacher draws attention to
end of the lesson; uses
questions & answers,
summaries, practices to
clarify main points along
lesson objectives; gives &
marks written exercises to
evaluate learning; assigns
activity for next lesson. Teacher shows enthusiasm
in teaching, maintains
students’ attention in the
lesson, model polite
classroom behaviour and is
creative and innovative. Teacher keeps records of
lessons in a bound
notebook for reference; ac-
cepts constructive feedback
to improve teaching; reflects
on teaching remarks. TOTAL SCORE GRADE:
Name of Supervisor
Signature
OVERALL COMMENTS: Student Teacher Handbook for Teaching Pratice Student Teacher Handbook for Teaching Pratice 122 3 - Good 2 - Satisfactory Teacher establishes clear
parameters for student
conduct, develops
appropriate strategies for
preventing problems, deals
with misbehaviour. Teacher establishes clear
parameters for student
conduct, develops
appropriate strategies for
preventing problems. Teacher establishes clear
parameters for student
conduct. Teacher uses questions &
answers, summaries,
practices to clarify main
points along lesson
objectives. Teacher uses questions &
answers, summaries and
practices to end lesson. The bell announces the end
of the lesson; teacher
finishes lesson abruptly/
teacher finishes lesson well
ahead of time. Teacher shows enthusiasm
in teaching, focuses student
attention on the lesson and
models polite classroom
behaviour. Teacher shows evidence of
enthusiasm in teaching and
focuses student attention on
the lesson. Teacher shows evidence of
enthusiasm in teaching but
exhibits distractive
mannerisms. Teacher keeps records of
lessons in a bound
notebook for reference;
accepts constructive
feedback to improve
teaching. Teacher keeps records of
lessons on sheets of paper. Teacher has no record of
previous lessons, current
lesson plan is on some sheets
of paper. Teacher establishes clear
parameters for student
conduct, develops
appropriate strategies for
preventing problems, deals
with misbehaviour. Teacher establishes clear
parameters for student
conduct, develops
appropriate strategies for
preventing problems. Teacher establishes clear
parameters for student
conduct. Teacher uses questions &
answers, summaries,
practices to clarify main
points along lesson
objectives. Teacher uses questions &
answers, summaries and
practices to end lesson. Teacher shows enthusiasm
in teaching, focuses student
attention on the lesson and
models polite classroom
behaviour. Annex 4. Assessment Procedures The bell announces the end
of the lesson; teacher
finishes lesson abruptly/
teacher finishes lesson well
ahead of time. Teacher shows enthusiasm
in teaching, focuses student
attention on the lesson and
models polite classroom
behaviour. Teacher shows evidence of
enthusiasm in teaching and
focuses student attention on
the lesson. Teacher shows evidence of
enthusiasm in teaching but
exhibits distractive
mannerisms. Teacher keeps records of
lessons in a bound
notebook for reference;
accepts constructive
feedback to improve
teaching. Teacher keeps records of
lessons on sheets of paper. Teacher has no record of
previous lessons, current
lesson plan is on some sheets
of paper. Teacher establishes clear
parameters for student
conduct. Teacher establishes clear
parameters for student
conduct, develops
appropriate strategies for
preventing problems, deals
with misbehaviour. Teacher establishes clear
parameters for student
conduct, develops
appropriate strategies for
preventing problems. ANNEX Teacher uses questions &
answers, summaries,
practices to clarify main
points along lesson
objectives. Teacher uses questions &
answers, summaries and
practices to end lesson. Teacher keeps records of
lessons in a bound
notebook for reference;
accepts constructive
feedback to improve
teaching. 123 Transforming Teacher Education and Learning Transforming Teacher Education and Learning
www.t-tel.org Year 1 - 3: Teaching Practice Annex 5. Guidelines for Protection Strategies
for Student Teachers on Teaching Practice The Gender Responsive competencies that are described in the table below provide a
comprehensive set of guidelines or indicators for protection of Student Teachers, particularly
female Student Teachers, during their teaching practice. The Gender Responsive competencies
highlight the systems and procedures that should be in place in all TP schools to safeguard Student
Teachers. These Gender Responsive competencies are included as a core component of the T-TEL
Project Monitoring and Evaluation Scorecard. Gender Responsive
competencies for Mentors
Example actions or strategies:
1. The Mentor makes sure
that their school sexual
harassment policy has been
developed and implemented
a) The school has received and adopted the National Teaching
Council’s Sexual Harassment Policy. If the policy has not yet been
circulated, the Lead Mentor should help the school complete the
following actions:
i. The school’s staff develops an agreed definition of what
constitutes sexual harassment (e.g. sexual bullying, suggestion,
touching and favours. Coming from staff/community towards
other teachers, Student Teachers and pupils)
ii. The school staff agree on a transparent reporting system for
teachers, Student Teachers or pupils experiencing sexual harass-
ment (e.g. victim can go to a designated female staff member,
problem then taken to HT/SMC, then circuit supervisor, etc.)
iii. The school staff nominates an appropriate, trained staff member
to act as the first point for reporting and to act as a counsellor for
those experiencing sexual harassment
iv. The school staff develops a reprimand system for those guilty of
sexual harassment (e.g. develop appropriate
punishments for staff, HT, community members, pupils, etc.)
v. The HT disseminates the sexual harassment definition, reporting
system and reprimand system to all teachers,
Student Teachers, pupils and community members
b) The Mentor ensures that all Student Teachers are aware of the
school’s sexual harassment policy and reporting system
c) The Mentor monitors female Student Teachers’ relationships with
staff/community members to make sure they are free from sexual
harassment
2. Annex 5. Guidelines for Protection Strategies
for Student Teachers on Teaching Practice The Mentor helps to secure
gender responsive
accommodation for female
Student Teachers
a) The Mentor meets with their Student Teacher before
teaching practice begins to help them find safe
accommodation
b) The Mentor ensures that their Student Teacher does not have to
live with a teacher, Mentor or member of staff
c) The Mentor ensures that if their Student Teacher lives in a com-
munity member’s house, the community member has been vetted
and is aware of the school sexual harassment policy
d) The Mentor ensures that their Student Teacher’s accommoda-
tion is close to the school and is safe at night (adequate lighting,
security guards/watchmen), particularly for females
e) If possible, the Mentor tries to ensure that pairs of female
Student Teachers find accommodation together a) The school has received and adopted the National Teaching
Council’s Sexual Harassment Policy. If the policy has not yet been
circulated, the Lead Mentor should help the school complete the
following actions: a) The school has received and adopted the National Teaching
Council’s Sexual Harassment Policy. If the policy has not yet been
circulated, the Lead Mentor should help the school complete the
following actions: 1. The Mentor makes sure
that their school sexual
harassment policy has been
developed and implemented i. The school’s staff develops an agreed definition of what
constitutes sexual harassment (e.g. sexual bullying, suggestion,
touching and favours. Coming from staff/community towards
other teachers, Student Teachers and pupils)f ii. The school staff agree on a transparent reporting system for
teachers, Student Teachers or pupils experiencing sexual harass-
ment (e.g. victim can go to a designated female staff member,
problem then taken to HT/SMC, then circuit supervisor, etc.) iii. The school staff nominates an appropriate, trained staff member
to act as the first point for reporting and to act as a counsellor for
those experiencing sexual harassment iv. The school staff develops a reprimand system for those guilty of
sexual harassment (e.g. develop appropriate
punishments for staff, HT, community members, pupils, etc.)i c) The Mentor monitors female Student Teachers’ relationships with
staff/community members to make sure they are free from sexual
harassment 2. The Mentor helps to secure
gender responsive
accommodation for female
Student Teachers 124 Student Teacher Handbook for Teaching Pratice Gender Responsive
competencies for Mentors
Example actions or strategies:
3. Annex 5. Guidelines for Protection Strategies
for Student Teachers on Teaching Practice The Mentor ensures gender
responsive treatment of
Student Teachers at the
school
a) The Mentor and staff’s daily behaviour does not reinforce
traditional gender roles (e.g. only female Student Teachers run
errands or clean up)
b) The Mentor ensures that female and male Student Teachers are
treated equally by staff, community and pupils
c) The Mentor monitors female Student Teachers’ relationships
with staff/community to make sure they are free from bullying,
intimidation and any form of abuse
d) The Mentor ensures that female and male Student Teachers have
equal access to school resources and teaching/learning materials
e) The Mentor encourages and supports female Student Teachers in
teaching subjects like maths and science
f) The Mentor encourages female Student Teachers to take
leadership roles during teaching practice
g) The Mentor supports and makes allowances for female Student
Teachers who have to balance family responsibilities (such as
marriage, pregnancy, child-care)
4. The Mentor provides
gender responsive support to
Student Teacher
well-being
a) The Mentor builds confidence in Student Teachers, especially
females
b) The Mentor monitors female Student Teachers’ day-to-day
well-being
c) The Mentor takes action to address female Student Teachers’
problems/needs
d) The Mentor makes sure that female Student Teachers feel
comfortable and secure
e) The Mentor ensures that female teacher toilets have water and
hygiene bins and that female Student Teachers are allowed to use
these
5. The Mentor
demonstrates gender
responsive classroom
practices while co-teaching
with Student Teachers
a) The Mentor shows Student Teachers how to give equal chances to
girls and boys to ask/answer questions
b) The Mentor shows Student Teachers how to assign leadership
roles equally to girls and boys in lesson activities
c) The Mentor shows Student Teachers how to make sure girls and
boys participate equally in activities
d) The Mentor shows Student Teachers how to be patient with girls
and boys who may be shy or afraid to speak out
e) The Mentor shows Student Teachers how to check if both girls
and boys equally understand the lesson
f) The Mentor shows Student Teachers how to provide constructive
verbal feedback to both girls and boys in class
6. The Mentor demonstrates
classroom strategies to
challenge traditional gender
roles
a) The Mentor shows Student Teachers how to use teaching
materials that do not show or reinforce traditional gender roles
(e.g. Student Teacher Handbook for Teaching Pratice Annex 5. Guidelines for Protection Strategies
for Student Teachers on Teaching Practice women cooking/cleaning and men in professional roles)
b) The Mentor shows Student Teachers how to point out traditional
gender roles that appear in books and discuss how traditional
gender roles can limit what girls think they can achieve in their
education and their lives Example actions or strategies: 3. The Mentor ensures gender
responsive treatment of
Student Teachers at the
school 3. The Mentor ensures gender
responsive treatment of
Student Teachers at the
school ANNEX Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning 125 Year 1 - 3: Teaching Practice Gender Responsive
competencies for Mentors
Example actions or strategies:
c) The Mentor shows Student Teachers how to use examples (in
exercises/activities) that challenge or reverse traditional gender
roles (e.g. like showing men/boys cleaning the house)
d) The Mentor shows Student Teachers how to make girls and boys
feel confident to challenge traditional gender roles in general
(e.g. encourage boys to cook and girls to be doctors)
e) The Mentor shows Student Teachers how to support/encourage
girls in studying and achieving in subjects like maths and science
7. The Lead Mentor
contributes to improving the
school’s gender responsive
Mentoring practices
a) The Lead Mentor checks that there is an equal number of female
and male Mentors at the school (if there is an imbalance, the
Mentor advocates for the gender balance of Mentors)
b) The Lead Mentor completes the Gender Responsive Scorecard
after every teaching practice and reflects on how they can
improve next time
c) The Lead Mentor has all Student Teachers anonymously appraise
their Mentors using the Gender Responsive Scorecard at the end
of their teaching practice
d) The Lead Mentor collects and looks at all the Student Teachers’
appraisals of their Mentors and identifies areas that need
improvement
e) The Lead Mentor facilitates discussion with other Mentors to
choose/prioritise follow-up actions for areas that need
improvement
f) The Lead Mentor advocates/budgets for gender responsive
training for all staff and Mentors
g) The Lead Mentor advocates/budgets for gender responsive
infrastructure improvements g) The Lead Mentor advocates/budgets for gender responsive
infrastructure improvements 126 Student Teacher Handbook for Teaching Pratice Annex 6. Example Page from a TP Journal ANNEX 127 Transforming Teacher Education and Learning
www.t-tel.org Transforming Teacher Education and Learning
|
https://openalex.org/W4390703365
|
https://ejournal.lumbungpare.org/index.php/maras/article/download/108/91
|
Indonesian
| null |
PERAMALAN TUNGGAKAN TAGIHAN PELANGGAN MENGGUNAKAN MODEL AUTOREGRESSIVE FRACTIONALLY INTEGRATED MOVING AVERAGE (ARFIMA)
|
Maras
| 2,023
|
cc-by-sa
| 5,295
|
PERAMALAN TUNGGAKAN TAGIHAN PELANGGAN
MENGGUNAKAN MODEL AUTOREGRESSIVE
FRACTIONALLY INTEGRATED MOVING AVERAGE
(ARFIMA) Tri Wulanda Fitri1*, Gumgum Darmawan2, Sri Winarni3, Yuyun Hidayat4,
Resa Septiani Pontoh5
1,2,3,4,5Universitas Padjadjaran
triwulandafitri@gmail.com* Tri Wulanda Fitri1*, Gumgum Darmawan2, Sri Winarni3, Yuyun Hidayat4,
Resa Septiani Pontoh5
1,2,3,4,5Universitas Padjadjaran
triwulandafitri@gmail.com* Abstrak
:
Kenaikan
jumlah
pelanggan
pada
perusahaan X mengakibatkan kenaikan yang sama
pada besar tunggakan tagihan pelanggan. Data
tunggakan tersebut cenderung memiliki pola tren
naik dan memiliki fluktiasi yang besar, akibatnya
perusahaan memerlukan adanya peramalan guna
perencanaan
dan
acuan
dalam
pengambilan
keputusan serta evaluasi kebijakan yang tepat untuk
mencegah peningkatan jumlah tunggakan tagihan di
masa mendatang dan mengurangi risiko kerugian. Metode peramalan yang akan digunakan pada
penelitian ini adalah Autoregressive Fractionally
Integrated Moving Average (ARFIMA). Metode
ARFIMA dipilih karena data memiliki sifat long
memory, dilihat dari lag pada plot ACF data yang
menurun secara hiperbolik menuju nilai nol. Artinya
observasi yang terpisah jauh masih memiliki perilaku
berkelanjutan atau ketergantungan yang kuat. Ciri
lain dari metode ARFIMA adalah nilai differencing-
nya. Berbeda dengan metode ARIMA yang memiliki
parameter d berupa bilangan asli, metode ARFIMA
memiliki parameter d berupa bilangan pecahan. Pada
penelitian digunakan differencing sebesar 0,697
dengan model ARFIMA (1, d, 0) untuk melakukan
peramalan 6 periode kedepan. Diperoleh nilai MAPE
untuk peramalan tersebut sebesar 9,28%. e-ISSN: 2987-811X
MARAS: Jurnal Penelitian Multidisplin
https://ejournal.lumbungpare.org/index.php/maras
Vol. 1 No. 3 Desember 2023
Page: 614-625
Article History:
Received: 27-12-2023
Accepted: 30-12-2023 e-ISSN: 2987-811X
MARAS: Jurnal Penelitian Multidisplin
https://ejournal.lumbungpare.org/index.php/maras
Vol. 1 No. 3 Desember 2023
Page: 614-625 Article History:
Received: 27-12-2023
Accepted: 30-12-2023 Kata Kunci : ARFIMA, Long Memory, Differencing,
Tunggakan Tagihan Kata Kunci : ARFIMA, Long Memory, Differencing,
Tunggakan Tagihan Kata Kunci : ARFIMA, Long Memory, Differencing,
Tunggakan Tagihan PENDAHULUAN Perusahaan X adalah perusahaan yang bergerak di bidang jasa layanan
teknologi informasi komunikasi (TIK) dan jaringan telekomunikasi di Indonesia. Pada
penjalanan bisnisnya, perusahaan X memiliki unit yang bergerak khusus untuk
melayani para pelanggan bisnis dan korporat yang mana pelanggan unit ini mengalami
peningkatan yang signifikan seiring berjalannya waktu. Tercatat bahwa jumlah | 614 Fitri et al. 10.60126/maras.v1i3.108 pelanggan unit tersebut pada Juli 2021 adalah sebanyak 493 pelanggan, dan pada Juli
2022 sebanyak 577 pelanggan. Hal ini menunjukkan adanya pertumbuhan jumlah
pelanggan sebanyak 84 pelanggan atau tumbuh 17,04% dibanding periode yang sama
tahun lalu. pelanggan unit tersebut pada Juli 2021 adalah sebanyak 493 pelanggan, dan pada Juli
2022 sebanyak 577 pelanggan. Hal ini menunjukkan adanya pertumbuhan jumlah
pelanggan sebanyak 84 pelanggan atau tumbuh 17,04% dibanding periode yang sama
tahun lalu. Pertumbuhan jumlah pelanggan tidak hanya berdampak positif bagi
perusahaan. Di sisi lain, pertumbuhan jumlah pelanggan juga memberikan masalah
baru bagi perusahaan, terutama dalam hal keuangan. Salah satu masalah yang
ditimbulkan oleh bertumbuhnya jumlah pelanggan adalah tunggakan tagihan
pelanggan yang cenderung meningkat seiring berjalannya waktu. Kenaikan pada
tunggakan ini dapat dilihat dari grafik data bulanan Tunggakan Tagihan periode
Januari 2018 sampai Desember 2023 berikut. Gambar 1. Grafik Tunggakan Tagihan Pelanggan Gambar 1. Grafik Tunggakan Tagihan Pelanggan Grafik data tunggakan di atas terlihat memiliki pola trend naik dan fluktuasi
yang sangat besar. Data tersebut juga diduga memiliki sifat long memory yang artinya
data deret waktu memiliki perilaku berkelanjutan atau ketergantungan yang kuat
antara data saat ini dan pengamatan yang terjadi jauh di masa lalu. Hal tersebut
diduga karena banyaknya tunggakan yang terjadi pada observasi terakhir disebabkan
oleh kurangnya tindakan dari internal perusahaan dalam menanggulangi isu
tunggakan pelanggan yang sudah berlangsung cukup lama yang mengakibatkan
tunggakan pelanggan semakin naik seiring berjalannya waktu. Hal selanjutnya yang menjadi alasan bahwa data penelitian ini memilki sifat
long memory terletak pada plot ACF-nya. Diketahui bahwa apabila nilai autokorelasi
pada plot ACF menurun secara hiperbolik lambat menuju nilai nol, maka data
diidentifikasi sebagai data deret waktu long memory (Wei, 2006). Berikut adalah plot
ACF data Tunggakan Tagihan Pelanggan. Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| 615
Gambar 2. Plot ACF Data Tunggakan Pelanggan Gambar 2. Plot ACF Data Tunggakan Pelanggan Gambar 2. Plot ACF Data Tunggakan Pelanggan Gambar 2. Data Penelitian Data yang digunakan berupa data tunggakan tagihan jasa pelanggan
perusahaan X periode Januari 2018 sampai dengan Desember 2023. Data tersebut akan
dibagi menjadi data pelatihan dan data uji. Data pelatihan digunakan dalam
pembentukan model dan data uji digunakan untuk memeriksa keakuratan model yang
dibangun. Data yang digunakan dalam penelitian ini berjumlah 72 observasi. Sebanyak
66 data digunakan sebagai data pelatihan yaitu observasi periode Januari 2018 sampai
dengan Juni 2023, dan 6 observasi lainnya akan digunakan sebagai data uji. Metode Analisis ARFIMA PENDAHULUAN Plot ACF Data Tunggakan Pelanggan Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| 615 Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| 615 Fitri et al. 10.60126/maras.v1i3.108 Berdasarkan Gambar 2 di atas dapat dilihat bahwa lag pada plot ACF tersebut
sudah turun secara lambat menuju nilai nol. Hal ini membenarkan dugaan bahwa data
deret waktu pada penelitian ini memliki sifat long memory. Berdasarkan penjelasan di atas, penulis tertarik untuk membahas model dan
peramalan pada data long memory. Diketahui metode yang paling tepat untuk
pemodelan dan peramalan data long memory adalah model ARFIMA (Autoregressive
Fractionally Integrated Moving Average), sehingga penulis akan menggunakan metode
analisis ARFIMA pada penelitian ini. Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| 616 1. Identifikasi Long Memory Tahapan pertama untuk mengetahui apakah sebuah data deret waktu memiliki
ciri long memory adalah dengan melihat plot ACF data tersebut. Jika nilai
autokorelasi pada plot data ACF menurun secara hiperbolik menuju nilai nol, maka
data tersebut dapat dianggap sebagai data long memory. Kemudian untuk
memastikan data bersifat long memory secara statistik dilakukan pengujian
menggunakan statistik uji Hurst (Hurst, 1951) dengan langkah-langkah sebagai
berikut: a. Menentukan rata-rata, adjusted mean, dan simpangan baku dari data deret
waktu dengan persamaan sebagai berikut: g
𝑌̅ = 1
𝑇∑𝑌𝑡
𝑇
𝑖=1
(7)
𝑌𝑡
𝑎𝑑𝑗= 𝑌𝑡−𝑌̅ (8)
𝑆𝑡= √1
𝑇∑(𝑌𝑡
𝑎𝑑𝑗)
2
𝑇
𝑖=1
(9) dengan 𝑡= 1, 2, … , 𝑇 b. Menentukan deviasi kumulatif dan rentang dari deviasi kumulatif. 𝑇 b. Menentukan deviasi kumulatif dan rentang dari deviasi kumulatif. 𝑇 𝑌𝑡
∗= ∑𝑌𝑡
𝑎𝑑𝑗
𝑇
𝑖=1
(10)
𝑗, … , 𝑌𝑡
𝑎𝑑𝑗) −𝑀𝑖𝑛(𝑌1
𝑎𝑑𝑗, 𝑌2
𝑎𝑑𝑗, … , 𝑌𝑡
𝑎𝑑𝑗) (11) 𝑌𝑡
∗= ∑𝑌𝑡
𝑎𝑑𝑗
𝑇
𝑖=1
(10)
𝑗
𝑗
𝑗
𝑗
𝑗 𝑌𝑡
∗= ∑𝑌𝑡
𝑎𝑑𝑗
𝑇
𝑖=1
(10)
𝑗
𝑑𝑗
𝑑 𝑌𝑡
∗= ∑𝑌𝑡
𝑎𝑑𝑗
𝑖=1
(10)
𝑅𝑡= 𝑀𝑎𝑥(𝑌1
𝑎𝑑𝑗, 𝑌2
𝑎𝑑𝑗, … , 𝑌𝑡
𝑎𝑑𝑗) −𝑀𝑖𝑛(𝑌1
𝑎𝑑𝑗, 𝑌2
𝑎𝑑𝑗, … , 𝑌𝑡
𝑎𝑑𝑗) (11)
dengan 𝑡= 1, 2, … , 𝑇 𝑡
∑
𝑡
𝑖=1
(
)
𝑅𝑡= 𝑀𝑎𝑥(𝑌1
𝑎𝑑𝑗, 𝑌2
𝑎𝑑𝑗, … , 𝑌𝑡
𝑎𝑑𝑗) −𝑀𝑖𝑛(𝑌1
𝑎𝑑𝑗, 𝑌2
𝑎𝑑𝑗, … , 𝑌𝑡
𝑎𝑑𝑗) (11)
dengan 𝑡= 1, 2, … , 𝑇 𝑖=1
𝑅𝑡= 𝑀𝑎𝑥(𝑌1
𝑎𝑑𝑗, 𝑌2
𝑎𝑑𝑗, … , 𝑌𝑡
𝑎𝑑𝑗) −𝑀𝑖𝑛(𝑌1
𝑎𝑑𝑗, 𝑌2
𝑎𝑑𝑗, … , 𝑌𝑡
𝑎𝑑𝑗) (11)
1, 2, … , 𝑇 g
, ,
,
c. Menentukan nilai eksponensial Hurst (H) melalui statistik R/S dari data deret
waktu. g
c. Menentukan nilai eksponensial Hurst (H) melalui statistik R/S dari data deret
waktu. c. Menentukan nilai eksponensial Hurst (H) melalui statistik R/S dari data deret
waktu. | 616 Fitri et al. 10.60126/maras.v1i3.108 𝐻=
𝑙𝑜𝑔(𝑅
𝑆)
𝑡
log (𝑡) (12) Kriteria uji statistik Hurst: Kriteria uji statistik Hurst: j
- 0 < 𝐻 < 0,5 menunjukkan sifat short memory - 0 < 𝐻 < 0,5 menunjukkan sifat short memory - 0,5 < 𝐻 < 1 menunjukkan sifat long memory - 0,5 < 𝐻 < 1 menunjukkan sifat long memory 2. Estimasi Parameter d dengan Metode GPH
Estimasi GPH adalah salah satu teknik pengujian paling umum digunakan
untuk fraksi integrasi d. 1. Identifikasi Long Memory Kelebihan dari metode Geweke dan Porter-Hudak adalah
parameter d dapat langsung diestimasi tanpa terlebih dahulu mengetahui nilai orde
AR (p) dan MA (q). Parameter d dapat ditaksir melalui persamaan (Geweke dan
Porter-Hudak, 1983): ,
𝑑̂ =
∑
(𝑥𝑗−𝑥̅)(𝑦𝑗−𝑦̅)
𝑚
𝑗=1
∑
(𝑥𝑗−𝑥̅)2
𝑚
𝑗=1
, 𝑚= [𝑇
1 2
⁄ ] (13)
dengan,
𝑥𝑗= 𝑙𝑛(
1
4𝑠𝑖𝑛2 (λ𝑗
2
⁄ )
) , λ𝑗= 2𝜋𝑗
𝑇 𝑑̂ =
∑
(𝑥𝑗−𝑥̅)(𝑦𝑗−𝑦̅)
𝑚
𝑗=1
∑
(𝑥𝑗−𝑥̅)2
𝑚
𝑗=1
, 𝑚= [𝑇
1 2
⁄ ] (13) g
𝑥𝑗= 𝑙𝑛(
1
4𝑠𝑖𝑛2 (λ𝑗
2
⁄ )
) , λ𝑗= 2𝜋𝑗
𝑇 dimana, 𝑇= banyaknya pengamatan, 𝑗= 1, 2, … , 𝑚
1
𝑇1 dimana, 𝑇= banyaknya pengamatan, 𝑗= 1, 2, … , 𝑚
1
𝑇1 𝑦𝑗= ln 𝐼(λ𝑗), 𝐼(λ𝑗) = 1
2𝜋{𝛾0 + 2 ∑
𝛾𝑡cos (𝑡 λ𝑗)
𝑇−1
𝑡=1
} , λ𝑗∈(−𝜋, 𝜋) Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| 617 ntegrated Moving Average (ARFIMA) eramalan Tunggakan Tagihan Pelanggan Men
(
) 3. Pemodelan Proses pemodelan pada metode ARFIMA hampir sama dengan pemodelan pada
model ARIMA yaitu, dengan mengidentifikasi orde AR(p) dan MA(q) pada plot ACF
(autocorrelation function) dan PACF (partial autocorrelation function) untuk data
yang sudah stasioner terhadap varians dan rata-rata. Langkah selanjutnya adalah
melihat plot ACF dan PACF dari correlogram cut off di lag berapa. Dari plot ACF
dan PACF tersebut bisa diidentifikasi beberapa kemungkinan model yang cocok
untuk dijadikan model. Proses pemodelan pada metode ARFIMA hampir sama dengan pemodelan pada
model ARIMA yaitu, dengan mengidentifikasi orde AR(p) dan MA(q) pada plot ACF
(autocorrelation function) dan PACF (partial autocorrelation function) untuk data
yang sudah stasioner terhadap varians dan rata-rata. Langkah selanjutnya adalah
melihat plot ACF dan PACF dari correlogram cut off di lag berapa. Dari plot ACF
dan PACF tersebut bisa diidentifikasi beberapa kemungkinan model yang cocok
untuk dijadikan model. 4. Uji Signifikansi Parameter
Uji signifikansi parameter dilakukan untuk melihat apakah model yang
didapatkan dari proses sebelumnya cukup baik atau tidak. Berikut hipotesis yang
digunakan dalam uji signifikansi parameter: 4. Uji Signifikansi Parameter
Uji signifikansi parameter dilakukan untuk melihat apakah model yang
didapatkan dari proses sebelumnya cukup baik atau tidak. Berikut hipotesis yang
digunakan dalam uji signifikansi parameter: Hipotesis:
𝐻0 ∶𝜃= 0 (parameter tidak menjelaskan variabel respon)
𝐻1 ∶𝜃≠0 (parameter menjelaskan variabel respon) Statistik Uji:
̂ Statistik Uji: 𝑡ℎ𝑖𝑡𝑢𝑛𝑔=
𝜃̂
𝑆𝐸(𝜃̂) (14) dengan,
𝜃̂ : nilai estimasi parameter 𝑆𝐸(𝜃̂) : standar error dari nilai estimasi parameter (𝜃̂) Kriteria Uji: Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| 617 Fitri et al. 10.60126/maras.v1i3.108 Tolak 𝐻0 jika nilai |𝑡ℎ𝑖𝑡𝑢𝑛𝑔| > 𝑡𝑎
2,(𝑇−𝑝), dengan nilai n sebagai banyaknya observasi
dan p adalah jumlah parameter yang ditaksir. Tolak 𝐻0 jika nilai |𝑡ℎ𝑖𝑡𝑢𝑛𝑔| > 𝑡𝑎
2,(𝑇−𝑝), dengan nilai n sebagai banyaknya observasi
dan p adalah jumlah parameter yang ditaksir. Uji Di
ik 5. Uji Diagnostik
Tahap ini menguji apakah residual data memenuhi asumsi white noise, yaitu
apakah residual data bersifat acak dan terdistribusi normal. Dalam pengujian
diagnostik dilakukan prosedur sebagai berikut: 5. Uji Diagnostik
Tahap ini menguji apakah residual data memenuhi asumsi white noise, yaitu
apakah residual data bersifat acak dan terdistribusi normal. Dalam pengujian
diagnostik dilakukan prosedur sebagai berikut: a. Asumsi nilai residual berdistribusi normal
Pengujian diagnostik model yang pertama adalah melakukan uji normalitas
untuk melihat kenormalan dari residual data. Untuk menguji normalitas residual
dapat digunakan salah satunya adalah dengan menggunakan uji Kolmogorov-
Smirnov. a. Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| 618 3. Pemodelan Asumsi nilai residual berdistribusi normal
Pengujian diagnostik model yang pertama adalah melakukan uji normalitas
untuk melihat kenormalan dari residual data. Untuk menguji normalitas residual
dapat digunakan salah satunya adalah dengan menggunakan uji Kolmogorov-
Smirnov. Hipotesis:
𝐻0 ∶𝑎𝑡 ~ 𝑁(0, 𝜎𝑎𝑡
2 ) (residual berdistribusi normal)
𝐻1 ∶𝑎𝑡≠𝑁(0, 𝜎𝑎𝑡
2 ) (residual tidak berdistribusi normal) Statistik Uji :
𝐷= 𝑚𝑎𝑥|𝐹0(𝑎𝑡) −𝑆(𝑎𝑡)| (15)
dengan,
𝐹0(𝑎𝑡) ∶ fungsi peluang kumulatif dari distribusi normal
𝑆(𝑎𝑡) ∶ fungsi peluang kumulatif data 𝐷= 𝑚𝑎𝑥|𝐹0(𝑎𝑡) −𝑆(𝑎𝑡)| (15) dengan,
𝐹0(𝑎𝑡) ∶ fungsi peluang kumulatif dari distribusi normal
𝑆(𝑎𝑡) ∶ fungsi peluang kumulatif data g
,
𝐹0(𝑎𝑡) ∶ fungsi peluang kumulatif dari distribusi normal
𝑆(𝑎) ∶f
i
l
k
l tif d t 𝑆(𝑎𝑡) ∶ fungsi peluang kumulatif data Kriteria Uji:
𝐻0 ditolak jika nilai maksimum |𝐹0(𝑎𝑡) −𝑆(𝑎𝑡)| > nilai pada tabel Kolmogorov-
Smirnov, sebaliknya apabila nilai maksimum |𝐹0(𝑎𝑡) −𝑆(𝑎𝑡)| < nilai pada tabel
Kolmogorov-Smirnov maka 𝐻0 diterima. Cara lain adalah dengan melihat nilai
p-value dari analisis uji Kolmogorov-Smirnov yaitu, tolak 𝐻0 apabila p-value <
α. b. Asumsi nilai residual bersifat acak Pengujian diagnostik model yang kedua dilakukan adalah menguji keacakan
(korelasi) terhadap residual data dengan menggunakan statistik uji Box – Pierce
dan Ljung – Box. Pengujian diagnostik model yang kedua dilakukan adalah menguji keacakan
(korelasi) terhadap residual data dengan menggunakan statistik uji Box – Pierce
dan Ljung – Box. Hi
t
i Hipotesis:
𝐻0 ∶ 𝜌1 = 𝜌2 = ⋯= 𝜌𝑘= 0 (residual bersifat acak (uncorrelated))
𝐻1 ∶ minimal terdapat satu nilai 𝜌𝑘≠0 (terdapat korelasi pada residual) Hipotesis:
𝐻0 ∶ 𝜌1 = 𝜌2 = ⋯= 𝜌𝑘= 0 (residual bersifat acak (uncorrelated))
𝐻1 ∶ minimal terdapat satu nilai 𝜌𝑘≠0 (terdapat korelasi pada resid Hipotesis
𝐻0 ∶ 𝜌1 = 𝜌2 = ⋯= 𝜌𝑘= 0 (residual bersifat acak (uncorrelated))
𝐻1 ∶ minimal terdapat satu nilai 𝜌𝑘≠0 (terdapat korelasi pada residual)
St ti tik Uji: Statistik Uji: 𝑄= 𝑛(𝑛+ 2) ∑
𝑟𝑘
2
𝑛−𝑘
𝑚
𝑘=1
(16) 𝑄= 𝑛(𝑛+ 2) ∑
𝑟𝑘
2
𝑛−𝑘
𝑚
𝑘=1
(16) dimana 𝑟𝑘
2 adalah autokorelasi residual lag-k, dan m adalah lag maksimum (lag
maksimum ditentukan sembarang, tetapi untuk lag yang cukup besar). Autokorelasi residual : Autokorelasi residual : 𝑟𝑘
2 =
∑
𝑒𝑡𝑒𝑡−𝑘
𝑛
𝑡=𝑘+1
∑
𝑒𝑡2
𝑛
𝑡=1
(17) Kriteria Uji:
𝑄≤𝑋(𝑎),(𝑘−𝑝−𝑞)
2
𝐻0 diterima,
𝑄> 𝑋(𝑎),(𝑘−𝑝−𝑞)
2
𝐻0 ditolak. Kriteria Uji:
𝑄≤𝑋(𝑎),(𝑘−𝑝−𝑞)
2
𝐻0 diterima, 𝑄≤𝑋(𝑎),(𝑘−𝑝−𝑞)
2
𝐻0 diterima, 𝑄> 𝑋(𝑎),(𝑘−𝑝−𝑞)
2
𝐻0 ditolak. Integrated Moving Average (ARFIMA) g
𝐴𝐼𝐶= 𝑛 𝑙𝑛𝜎̂𝑎
2 + 2𝑀 (18) Nilai Mean Absolute Percentage Error (MAPE) digunakan untuk mengukur
keakuratan nilai model yang diestimasi dan dinyatakan dalam bentuk rata-rata
persentase absolute kesalahan. MAPE juga menunjukkan seberapa besar kesalahan
prediksi ketika membandingkan nilai sebenarnya dari data dengan hasil prediksi. Model berkinerja sangat baik ketika MAPE kurang dari 10% dan berkinerja baik
ketika MAPE antara 10% dan 20% (Lewis, 1982). Berikut persamaan MAPE: 𝑀𝐴𝑃𝐸=
∑
|𝑦𝑡−𝑦𝑡̂ |
𝑦𝑡
𝑛
𝑡=1
𝑛
× 100% (19) dengan,
𝑦𝑡 : nilai aktual waktu t
𝑦𝑡̂ : nilai peramalan 𝑦𝑡
𝑛∶ jumlah data historis 𝑦𝑡̂ : nilai peramalan 𝑦𝑡 𝑦𝑡̂ : nilai peramalan 𝑦𝑡 𝑛∶ jumlah data historis Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| 619 Fitri et al. Fitri et al. 10.60126/maras.v1i3.108 Model yang telah memenuhi asumsi akan dilihat nilai Akaike Information
Criterion (AIC). Model dengan nilai AIC terkecil yang akan menjadi model terbaik
untuk peramalan. Berikut persamaan untuk menghitung nilai AIC (Wei, 2006): g
𝐴𝐼𝐶= 𝑛 𝑙𝑛𝜎̂𝑎
2 + 2𝑀 (18)
( 3. Pemodelan ( ),(
𝑝𝑞)
6. Pemilihan Model Terbaik ( ),(
𝑝𝑞)
6. Pemilihan Model Terbaik | 618 HASIL DAN PEMBAHASAN 1. Identifikasi Long Memory
Tahapan pertama untuk mengidentifikasi data deret waktu long memory adalah
dengan melihat plot ACF data. Plot ACF data penelitian ini dapat dilihat pada
Gambar 2. Pada gambar tersebut terlihat bahwa data deret waktu telah memiliki
sifat long memory dengan ciri lag pada plot ACF-nya yang turun secara lambat
menuju nilai nol (Wei, 2006). Langkah selanjutnya untuk mengidentifiksi sifat long memory pada data deret
waktu adalah dengan uji statistik Hurst. Didapatkan hasil uji Hurst menggunakan
bantuan software R untuk data ini yaitu sebesar 0,766 yang mana nilai tersebut
sudah memenuhi kriteria 0,5 < H < 1 yang menunjukkan bahwa suatu data deret
waktu mengandung sifat long memory. g
y
Tahapan pertama untuk mengidentifikasi data deret waktu long memory adalah
dengan melihat plot ACF data. Plot ACF data penelitian ini dapat dilihat pada
Gambar 2. Pada gambar tersebut terlihat bahwa data deret waktu telah memiliki
sifat long memory dengan ciri lag pada plot ACF-nya yang turun secara lambat
menuju nilai nol (Wei, 2006). Langkah selanjutnya untuk mengidentifiksi sifat long memory pada data deret
waktu adalah dengan uji statistik Hurst. Didapatkan hasil uji Hurst menggunakan
bantuan software R untuk data ini yaitu sebesar 0,766 yang mana nilai tersebut
sudah memenuhi kriteria 0,5 < H < 1 yang menunjukkan bahwa suatu data deret
waktu mengandung sifat long memory. Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| 619
2. Uji Stasioneritas
a. Stasioneritas Varians
Pemeriksaan stasioneritas varians pada data dapat dilihat dari nilai lambda. Apabila nilai lambda mendekati 1 maka, data sudah stasioner dalam varians. Data yang belum stasioner dalam varians harus melewati tahapan tranformasi
Box-Cox terlebih dahulu dengan rumus 𝑌𝑡
λ. Berikut adalah nilai estimasi lambda
sebelum dan sesudah transformasi. Tabel 1. Estimasi Nilai λ
Transformasi 𝑌𝑡
λ
Sebelum Transformasi
Sesudah Transformasi
Estimasi Nilai λ
0,21
1,19
Keterangan
Belum Stasioner
Sudah Stasioner
Dari tabel di atas dapat dilihat bahwa data sudah stasioner dalam varians setelah
dilakukannya 1 kali transformasi dengan nilai akhir lambda 1,194. b. Stasioneritas Rata-rata
Stasioneritas rata-rata pada data dapat dilihat melalui pengujian ADF
(Augmented Dikcey Fuller). Jika nilai p-value > 𝛼 = 5% maka, data tidak stasioner Integrated Moving Average (ARFIMA) a. Stasioneritas Varians Pemeriksaan stasioneritas varians pada data dapat dilihat dari nilai lambda. Apabila nilai lambda mendekati 1 maka, data sudah stasioner dalam varians. Data yang belum stasioner dalam varians harus melewati tahapan tranformasi
Box-Cox terlebih dahulu dengan rumus 𝑌𝑡
λ. Berikut adalah nilai estimasi lambda
sebelum dan sesudah transformasi. Tabel 1. Estimasi Nilai λ
Transformasi 𝑌𝑡
λ
Sebelum Transformasi
Sesudah Transformasi
Estimasi Nilai λ
0,21
1,19
Keterangan
Belum Stasioner
Sudah Stasioner
Dari tabel di atas dapat dilihat bahwa data sudah stasioner dalam varians setelah
dilakukannya 1 kali transformasi dengan nilai akhir lambda 1,194. b. Stasioneritas Rata-rata Stasioneritas rata-rata pada data dapat dilihat melalui pengujian ADF
(Augmented Dikcey Fuller). Jika nilai p-value > 𝛼 = 5% maka, data tidak stasioner | 619 Fitri et al. 10.60126/maras.v1i3.108 dalam rata-rata, sebaliknya nilai p-value < 𝛼 = 5% menyatakan data deret waktu
memiliki rata-rata yang stasioner. Didapatkan hasil analisis p-value dengan
menggunakan bantuan software R adalah sebesar 0,37 yang artinya data belum
stasioner dalam rata-rata. Apabila rata-rata data belum stasioner maka,
diperlukan adanya proses differencing. Pada data long memory proses differencing
dilakukan dengan parameter bernilai pecahan. Hal ini differencing yang
dilakukan dengan nilai pecahan membantu menghilangkan komponen tren atau
pola sekuensial dari data dengan cara yang lebih lembut daripada differencing
dengan bilangan bulat (J. Pan et al, 2008). Metode yang sering digunakan untuk memperoleh parameter d untuk data long
memory adalah dengan metode Geweke Poter-Hudak (GPH). Berikut hasil
pengujian menggunakan bantuan software R: Tabel 2. Hasil Differencing
p-value
awal
𝛼
Keterangan Differencing
p-value setelah
differencing
Keterangan
0,37
0,05
Belum
Stasioner
0,69
0,01
Sudah
Stasioner
Dari tabel di atas dapat dilihat bahwa data telah stasioner dalam rata-rata setelah
dilakukan differencing sebesar 0,69. d
tifik
i M d l Tabel 2. Hasil Differencing
p-value
awal
𝛼
Keterangan Differencing
p-value setelah
differencing
Keterangan
0,37
0,05
Belum
Stasioner
0,69
0,01
Sudah
Stasioner
Dari tabel di atas dapat dilihat bahwa data telah stasioner dalam rata-rata setelah
dilakukan differencing sebesar 0,69. 0,37
0,05
Stasioner
0,69
0,01
Stasioner
Dari tabel di atas dapat dilihat bahwa data telah stasioner dalam rata-rata setelah
dilakukan differencing sebesar 0,69. Dari tabel di atas dapat dilihat bahwa data telah stasioner dalam rata-rata setelah
dilakukan differencing sebesar 0,69. 3. Identifikasi Model
Langkah selanjutnya adalah menentukan orde AR(p) dan MA(q) untuk
mengidentifikasi kemungkinan model peramalan terbaik dengan melihat plot ACF
dan PACF data deret waktu setelah stasioner dalam varians dan rata-rata. Berikut
disajikan plot ACF dan PACF data setelah stasioner. Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| 621 a. Stasioneritas Varians j
p
Gambar 3. Plot ACF Setelah Differencing
Gambar 4. Plot PACF Setelah Differencing Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| 620
Gambar 3. Plot ACF Setelah Differencing
Gambar 4. Plot PACF Setelah Differencing mbar 3. Plot ACF Setelah Differencing Gambar 4. Plot PACF Setelah Differencing Gambar 4. Plot PACF Setelah Differencing Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| | 620 Fitri et al. 10.60126/maras.v1i3.108 Dapat dilihat cut off pada plot ACF dan PACF di atas sama-sama terjadi setelah
lag pertama, dengan parameter d sebesar 0,69 sehingga estimasi model ARFIMA
yang akan digunakan adalah sebagai berikut: Tabel 3. Model ARFIMA
Model ARFIMA
ARFIMA (1,d,0)
ARFIMA (1,d,1)
ARFIMA (0,d,1) Tabel 3. Model ARFIMA Tabel 3. Model ARFIMA
Model ARFIMA
ARFIMA (1,d,0)
ARFIMA (1,d,1)
ARFIMA (0,d,1) Tabel 3. Model ARFIMA
Model ARFIMA
ARFIMA (1,d,0)
ARFIMA (1,d,1)
ARFIMA (0,d,1) ARFIMA (0,d,1) Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA) 4. Uji Signifikasi Parameter 4. Uji Signifikasi Parameter Estimasi model yang telah didapatkan perlu dilakukan uji signifikansi untuk
melihat apakah parameter berpengaruh secara signifikan terhadap model. Kriteria
uji untuk parameter yang berpengaruh signifikan terhadap model adalah ketika
nilai |𝑡ℎ𝑖𝑡𝑢𝑛𝑔| > 𝑡𝑎
2,(𝑇−𝑝). Berikut hasil analisis signifikansi parameter pada model-
model estimasi sebelumnya: model estimasi sebelumnya: Tabel 4. Parameter Model ARFIMA (1,d,0)
ϕ1
Estimasi Parameter
0,96
Standard Error
0,03
|𝑡ℎ𝑖𝑡𝑢𝑛𝑔|
27,61
𝑡𝑎
2,(𝑇−𝑝)
2,00
Keterangan
Signifikan
Tabel 5. Parameter Model ARFIMA (1,d,1)
ϕ1
𝜃1
Estimasi Parameter
0,96
0,37
Standard Error
0,04
0,25
|𝑡ℎ𝑖𝑡𝑢𝑛𝑔|
23,47
1,45
𝑡𝑎
2,(𝑇−𝑝)
2,00
2,00
Keterangan
Signifikan
Tidak Signifikan Berdasarkan tabel di atas, dapat dilihat bahwa kedua model estimasi
memperoleh nilai p-value > 𝛼 = 5% artinya asumsi nilai residual bersifat normal pada
kedua model estmasi terpenuhi. 5. Uji Diagnostik a. Uji Normalitas Residual
Uji normalitas pada nilai sisa bertujuan untuk melihat apakah residual
berdistribusi normal atau tidak. Pengujian dilakukan menggunakan uji
Kolmogorov-Smirnov
dengan
bantuan
software
R. Berikut
ditampilkan
rangkuman output pengujian normalitas residual menggunakan uji Kolmogorov-
Smirnov pada software R: p
Tabel 6. Hasil Analisis Uji Normalitas Residual
Model ARFIMA
(𝑑= 0,69)
p-value
Keterangan
(1,d,0)
0,07
𝐻0 diterima
(0,d,1)
0,06
𝐻0 diterima 10.60126/maras.v1i3.108 Fitri et al. b. Uji Non-autokorelasi Residual
Pengujian non-autokorelasi pada nilai sisa menggunakan statistik uji Box-Ljung. Berikut rangkuman output uji non-autokorelasi residual menggunakan statistik
uji Box-Ljung menggunakan software R:
Tabel 7. Hasil Analisis Uji Non-Autokorelasi Residual
Model ARFIMA
(𝑑= 0,69)
p-value
Keterangan
(1,d,0)
0,75
𝐻0 diterima
(0,d,1)
0,02
𝐻0 ditolak
Berdasarkan tabel di atas dapat dilihat bahwa model estimasi yang memenuhi
asumsi non-autokorelasi hanyalah model ARFIMA (1,d,0), artinya model tersebut
iliki
id
l d
b
if
k
l
d b. Uji Non-autokorelasi Residual
Pengujian non-autokorelasi pada nilai sisa menggunakan statistik uji Box-Ljung. Berikut rangkuman output uji non-autokorelasi residual menggunakan statistik
uji Box-Ljung menggunakan software R: Pengujian non-autokorelasi pada nilai sisa menggunakan statistik uji Box-Ljung. Berikut rangkuman output uji non-autokorelasi residual menggunakan statistik
uji Box-Ljung menggunakan software R: g
p
j
gg
uji Box-Ljung menggunakan software R: g
p
j
uji Box-Ljung menggunakan software R: Tabel 7. Hasil Analisis Uji Non-Autokorelasi Residual Tabel 7. Hasil Analisis Uji Non-Autokorelasi Residual
Model ARFIMA
(𝑑= 0,69)
p-value
Keterangan
(1,d,0)
0,75
𝐻0 diterima
(0,d,1)
0,02
𝐻0 ditolak Berdasarkan tabel di atas dapat dilihat bahwa model estimasi yang memenuhi
asumsi non-autokorelasi hanyalah model ARFIMA (1,d,0), artinya model tersebut
memiliki residual data yang bersifat acak atau uncorrelated. UCAPAN TERIMA KASIH UCAPAN TERIMA KASIH CAPAN TERIMA KASIH Penulis mengucapkan terima kasih kepada dosen-dosen Prodi Statistika
Universitas Padjadjaran
D
G
D
S Si
M Si lis mengucapkan terima kasih kepada dosen-dosen Prodi Statistika
Padjadjaran Penulis mengucapkan terima kasih kepada dosen-dosen Prodi Statistika
Universitas Padjadjaran Penulis mengucapkan terima kasih kepada dosen-dosen Prodi
Universitas Padjadjaran Universitas Padjadjaran 1. Dr. Gumgum Darmawan, S.Si., M.Si. 1. Dr. Gumgum Darmawan, S.Si., M.Si. 2. Sri Winarni, S.Si., M.Si. 3. Prof. Yuyun Hidayat, MS., Ph.D. 3. Prof. Yuyun Hidayat, MS., Ph.D. 3. Prof. Yuyun Hidayat, MS., Ph.D. 4. Resa Septiani Pontoh, S.Si., M.A.B., M.Stat.Sci. 4. Resa Septiani Pontoh, S.Si., M.A.B., M.Stat.Sci. 4. Resa Septiani Pontoh, S.Si., M.A.B., M.Stat.Sci. Atas bimbingan dan telaah keilmiahan yang dilakukan guna meningkatkan
kualitas artikel ilmiah ini. Semoga ilmu yang diberikan dapat bermanfaat bagi bidang
keilmuan terkait. 6. Model Terbaik Dari analisis sebelumnya, diketahui bahwa hanya terdapat 1 model yang
signifikan dan memenuhi asumsi residual yaitu model ARFIMA (1,d,0) dengan
parameter 𝑑= 0,69. Selanjutnya akan dilihat nilai AIC dan MAPE dari model
tersebut. Tabel 8. Nilai AIC dan MAPE Model Terpilih
Model ARFIMA
(𝑑= 0,69)
AIC
MAPE (%)
(1,d,0)
2256,88
9,28 Nilai MAPE didapat dari data testing sebanyak 6 observasi yang diambil dari
periode Juli 2023 sampai Desember 2023. Sebelumnya telah dilakukan peramalan
dan pemodelan pada data training periode Januari 2018 sampai Juni 2023
menggunakan model ARFIMA (1,d,0) dengan 𝑑= 0,69. Berikut adalah tabel analisis
nilai MAPE untuk data testing pada penelitian ini. Tabel 9. Analisis MAPE Model Terpilih
Periode
Data Aktual
Forecast
Persentase (%)
Juli 2023
2464640158
2604807195
5,68
Agustus 2023
2745750660
2605284602
5,11
September 2023
2825728957
2601503245
7,93
Oktober 2023
2904822571
2595024804
10,66
November 2023
2983229008
2586610332
13,29
Desember 2023
2961108837
2576716156
12,98
MAPE
9,28 ,
Model ARFIMA (1; 𝑑= 0,69; 0) sudah dapat dikategorikan baik karena memiliki
nilai persentase MAPE sebesar 9,28% yang mana MAPE dengan persentase antara
0% - 20% menunjukkan bahwa model memiliki akurasi peramalan yang baik (Meng
Dun et al, 2020). Apabila model ARFIMA (1; 𝑑= 0,69; 0) dimasukkan ke dalam
persamaan (1) dan (6) maka, dapat dituliskan sebagai berikut:
ϕ𝑝(𝐵)(1 −𝐵)𝑑𝑌𝑡= 𝜃𝑞(𝐵)𝑎𝑡
0,961(𝐵)(1 −𝐵)0,69𝑌𝑡= 𝜃𝑞(𝐵)𝑎𝑡
(1 −𝐵)0,69(1 −0,96)𝑌𝑡= 0 (20)
dengan, Model ARFIMA (1; 𝑑= 0,69; 0) sudah dapat dikategorikan baik karena memiliki
nilai persentase MAPE sebesar 9,28% yang mana MAPE dengan persentase antara
0% - 20% menunjukkan bahwa model memiliki akurasi peramalan yang baik (Meng
Dun et al, 2020). Apabila model ARFIMA (1; 𝑑= 0,69; 0) dimasukkan ke dalam
persamaan (1) dan (6) maka, dapat dituliskan sebagai berikut:
ϕ𝑝(𝐵)(1 −𝐵)𝑑𝑌𝑡= 𝜃𝑞(𝐵)𝑎𝑡
0,961(𝐵)(1 −𝐵)0,69𝑌𝑡= 𝜃𝑞(𝐵)𝑎𝑡
(1 −𝐵)0,69(1 −0,96)𝑌𝑡= 0 (20)
dengan, Model ARFIMA (1; 𝑑= 0,69; 0) sudah dapat dikategorikan baik karena memiliki
nilai persentase MAPE sebesar 9,28% yang mana MAPE dengan persentase antara
0% - 20% menunjukkan bahwa model memiliki akurasi peramalan yang baik (Meng
Dun et al, 2020). Apabila model ARFIMA (1; 𝑑= 0,69; 0) dimasukkan ke dalam
persamaan (1) dan (6) maka, dapat dituliskan sebagai berikut:
ϕ𝑝(𝐵)(1 −𝐵)𝑑𝑌𝑡= 𝜃𝑞(𝐵)𝑎𝑡 ϕ𝑝( )(
)
𝑡
𝑞( ) 𝑡
0,961(𝐵)(1 −𝐵)0,69𝑌𝑡= 𝜃𝑞(𝐵)𝑎𝑡
(1 −𝐵)0,69(1 −0,96)𝑌𝑡= 0 (20)
dengan, Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| 6 | 622 10.60126/maras.v1i3.108 Fitri et al. 6. Model Terbaik (1 −𝐵)𝑑= 1 −𝑑𝐵−1
2 (1 −𝑑)𝑑𝐵2 −1
6 (1 −𝑑)(2 −𝑑)𝑑𝐵3 + ⋯
(1 −𝐵)0,69 = 1 −(0,69)(𝐵) −1
2 (1 −0,69)(0,69)𝐵2 −1
6 (1 −0,69)(2 −0,69)(0,69)𝐵3 + ⋯
(1 −𝐵)0,69 = 1 −0,69𝐵−0,10𝐵2 −0,04𝐵3 + ⋯ 2
6
(1 −𝐵)0,69 = 1 −(0,69)(𝐵) −1
2 (1 −0,69)(0,69)𝐵2 −1
6 (1 −0,69)(2 −0,69)(
(1 −𝐵)0,69 = 1 −0,69𝐵−0,10𝐵2 −0,04𝐵3 + ⋯ sehingga model menjadi, gg
j
,
(1 −0,69𝐵−0,10𝐵2 −0,04𝐵3 + ⋯)(1 −0,96)𝑌𝑡= 0
(1 −0,69𝐵−0,10𝐵2 −0,04𝐵3 + ⋯)(0,03)𝑌𝑡= 0
(0,03𝑌𝑡−0,02𝐵𝑌𝑡−0,0031𝐵2𝑌𝑡−0,0013𝐵3𝑌𝑡+ ⋯) = 0
dengan 𝐵𝑌𝑡= 𝑌𝑡−1, maka:
(0,0301𝑌𝑡−0,021𝑌𝑡−1 −0,00317𝑌𝑡−2 −0,001378𝑌𝑡−3 + ⋯) = 0
diperoleh persamaan ARFIMA (1; 𝑑= 0,69; 0) sebagai berikut:
𝑌𝑡= 0,021𝑌𝑡−1 + 0,00317𝑌𝑡−2 + 0,001378𝑌𝑡−3 −⋯
0,0301
(21)
P
l gg
j
,
(1 −0,69𝐵−0,10𝐵2 −0,04𝐵3 + ⋯)(1 −0,96)𝑌𝑡= 0
(1 −0,69𝐵−0,10𝐵2 −0,04𝐵3 + ⋯)(0,03)𝑌𝑡= 0
(0,03𝑌𝑡−0,02𝐵𝑌𝑡−0,0031𝐵2𝑌𝑡−0,0013𝐵3𝑌𝑡+ ⋯) = 0
d
k dengan 𝐵𝑌𝑡= 𝑌𝑡−1, maka:
(0,0301𝑌𝑡−0,021𝑌𝑡−1 −0,00317𝑌𝑡−2 −0,001378𝑌𝑡−3 + ⋯) = 0
diperoleh persamaan ARFIMA (1; 𝑑= 0,69; 0) sebagai berikut:
𝑌𝑡= 0,021𝑌𝑡−1 + 0,00317𝑌𝑡−2 + 0,001378𝑌𝑡−3 −⋯
0,0301
(21) 7. Peramalan Peramalan data tunggakan akan dilakukan untuk 6 observasi kedepan yaitu
untuk tunggakan periode bulan Januari 2024 sampai Juni 2024. Peramalan
dilakukan dengan model yang telah terpilih sebelumnya yaitu, model ARFIMA
(1; 𝑑= 0,69; 0) dan MAPE sebesar 9,28% menggunakan persamaan : gg
= 0,021𝑌𝑡−1 + 0,00317𝑌𝑡−2 + 0,001378𝑌𝑡−3 −⋯ 𝑌𝑡= 0,021𝑌𝑡−1 + 0,00317𝑌𝑡−2 + 0,001378𝑌𝑡−3
0,0301 Berikut hasil peramalannya : Tabel 10. Hasil Peramalan ARFIMA (1; 𝑑= 0,69; 0)
Periode
Peramalan Tunggakan
Januari 2024
Rp2.961.997.399,00
Februari 2024
Rp2.960.660.303,00
Maret 2024
Rp2.957.341.131,00
April 2024
Rp2.952.430.036,00
Mei 2024
Rp2.946.227.012,00
Juni 2024
Rp2.938.956.800,00 Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA)
| 623 0,0301 Model dipilih karena bersifat signifikan dan telah memenuhi asumsi residual
normalitas dan non-autokorelasi. Model terpilih juga sudah memiliki akurasi
peramalan yang cukup baik dengan MAPE sebesar 9,28%. Saran yang dapat diberikan untuk penelitian selanjutnya adalah dalam
mengestimasi parameter d dapat menggunakan metode lainnya seperti SPR, GPHTr,
GPHTa, dan MGPH. Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA) KESIMPULAN DAN SARAN Penelitian ini membahas pemodelan dan peramalan menggunakan ARFIMA
untuk data tunggakan tagihan pelanggan observasi bulan Januari 2018 sampai
Desember 2023 dengan peramalan untuk periode Januari 2024 sampai Juni 2024. 10.60126/maras.v1i3.108 Fitri et al. Model terbaik yang digunakan untuk penilitian ini adalah model ARFIMA (1; 𝑑=
0,69; 0) dengan persamaan: Model terbaik yang digunakan untuk penilitian ini adalah model ARFIMA (1; 𝑑=
0,69; 0) dengan persamaan: = 0,021𝑌𝑡−1 + 0,00317𝑌𝑡−2 + 0,001378𝑌𝑡−3 −⋯ 𝑌𝑡= 0,021𝑌𝑡−1 + 0,00317𝑌𝑡−2 + 0,001378𝑌𝑡−3 −⋯
0,0301 DAFTAR PUSTAKA [1]
Agustiana, Ferdian., dkk. (2016). Perbandingan Penaksiran Parameter Pembeda
(d) ARFIMA Nonstasioner GPH dengan Modifikasi GPH (Geweke and Porter-
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Baillie, R., & Chung, S. (2002). Modeling and forecasting from trend-stationary
long memory models with applications to climatology. International Journal of
Forecasting, 18, 215-226. https://doi.org/10.1016/S0169-2070(01)00154-6. [4]
Breidert, Christoph. (2006). Estimation of Willingness-to-Pay. Wiesbaden: Gabler
Edition Wissenschaft. [5]
Caraka, Rezzy Eko, dkk. (2016). Model Long Memory Dalam Prediksi Suhu. Prosiding 2016 Anomali Cuaca dan Iklim Sebagai Peluang dan Tantangan, 222-
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Darmawan, Gumgum. (2009). Perbandingan Metode Peramalan ARIMA dan
ARFIMA pada Data Long Memory. Jurnal Unisba, 9(2), 109-113. [7]
Dun, M., Xu, Z., Chen, Y., & Wu, L. (2020). Short-Term Air Quality Prediction
Based on Fractional Grey Linear Regression and Support Vector Machine. Mathematical
Problems
in
Engineering,
2020,
1-13. https://doi.org/10.1155/2020/8914501. [8]
Geweke, J dan Porter-Hudak, S. 1983. The Estimation and Application of Long
Memory Time Series Models. Journal of Time Series Analysis. 4(4), 221-238. [9]
Haba, A., dkk. (2021). Prediksi Jumlah Tunggakan Tagihan Rekening Air
Menggunakan Metode Least Square. Jurnal Nasional Cosphi, 5(2), 31-35. [10] Hartini, Dwi. (2016). Penerapan Model ARFIMA (Autoregressive Fractionally
Integrated Moving Average) dalam Prakiraan Data Suku Bunga PUAB (Pasar
Uang Antar Bank). [11] Hurst, HE. (1951). The Problem of Long Term Storage in Reservoirs: An
Experimental Study. International Association of Scientific Hydrology, Bullein. 116: 770-799. Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA) | 624 10.60126/maras.v1i3.108 Fitri et al. [12] Hutabarat, Magdaniar. (2017). Pemodelan Hubungan Antara IHSG, Nilai Tukar
Dolar Amerika Serikat Terhadap Rupiah (Kurs) dan Inflasi dengan Vector Error
Correction Model (VECM). [13] Lewis,
C. (1982). Industrial
and
Business
Forecasting
Methods. London:Butterworth. [14] Montgomery, Douglas C., C. L. Jennings., M. Kulahci. (2008). Introduction to
Time Series Analysis and Forecasting. New Jersey: John Wiley & Sons, Inc. [15] Palma, Wilfredo. (2007). Long-Memory Time Series. New Jersey: John Wiley &
Sons, Inc. [16] Panjaitan, Helmi, A. Prahutama, dan Sudarno. (2018). Peramalan Jumlah
Penumpang Kereta Api Menggunakan Metode ARIMA, Intervensi, dan ARFIMA. Jurnal Gaussian, 7(1), 96-109. [17] Pan, J., & Chen, S. (2008). Monitoring long‐memory air quality data using
ARFIMA model. Environmetrics, 19. https://doi.org/10.1002/ENV.882. [18] Paridi. (2018). Peramalan Tunggakan Tagihan Pelanggan Menggunakan Model Autoregressive Fractionally
Integrated Moving Average (ARFIMA) [24] Wei, William W.S. (2006). Time Series Analysis: Univariate and Multivariate
Methods, 2nd edition. New York: Pearson Education, Inc. DAFTAR PUSTAKA Perbandingan Metode ARIMA (Box Jenkins) dan ARFIMA dalam
Peramalan Jumlah Kasus Demam Berdarah Dengue di Rumah Sakit Hasan
Sadikin Bandung. Jurnal Pendidikan Mandala, 3(3), 338-348. [19] Petriella, Yanita. (2022). Tagihan Indihome Naik Mulai April 2022, Imbas PPN 11
Persen. Diakses pada Agustus 2022, dari https://ekonomi.bisnis.com/. [20] Prasetya, B. D., F. S. Pamungkas, I. Kharisudin. (2020). Pemodelan dan
Peramalan Data Saham dengan Analisis Time Series menggunakan Python. PRISMA. Prosiding Seminar Nasional Matematika 3, 714-718. [21] Pudiastuti, V. E., F. Hasun, M. Dellaroswati. (2015). Evaluasi Besaran Tarif
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Methods, 2nd edition. New York: Pearson Education, Inc. | 625
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https://europepmc.org/articles/pmc3334645?pdf=render
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English
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The fine specificity of IgM anti-citrullinated protein antibodies (ACPA) is different from that of IgG ACPA
|
Arthritis research & therapy
| 2,011
|
cc-by
| 5,801
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© 2011 Suwannalai 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 Introduction: The antigen recognition pattern of immunoglobulin M (IgM) could, when directed against protein
antigens, provide an indication of the antigenic moieties triggering new B cells. The half-life of IgM is short and
memory B cells against T-cell-dependent protein antigens typically produce IgG and not IgM antibodies. In this
study, we analyzed whether a difference exists between the fine specificity of IgM versus IgG anti-citrullinated
protein antibodies (ACPAs). Methods: We determined the fine specificity of IgM and IgG ACPAs in 113 ACPA-positive rheumatoid arthritis
patients with IgM cyclic citrullinated peptide 2 (CCP2) levels above 100 AU/ml. Fine specificity was assessed by
performing ELISA using citrullinated peptides derived from vimentin, fibrinogen-b, fibrinogen-a and a-enolase, as
well as citrullinated proteins fibrinogen and myelin basic protein. The arginine counterparts were used as controls. Results: Recognition of defined citrullinated antigens by IgM ACPA was confined to samples that also displayed
recognition by IgG ACPA. However, the IgM ACPA response displayed a more restricted antigen recognition profile
than IgG ACPA (OR = 0.26, P < 0.0001). Conclusion: Our data show that several defined citrullinated antigens are recognized only by IgG ACPA, whereas
others are also recognized by IgM ACPA. These observations suggest that not all citrullinated antigens are able to
activate new B cells despite concurrent recognition by IgG ACPA. evolution of such a response is typically characterized by
a first wave of IgM antibodies after the first antigen con-
tact, quickly followed by the presence of IgG. After
repeated antigen exposure, the IgG responses are further
boosted while the IgM peak declines. The latter observa-
tion is explained by the presence of Ig-switched, affinity
matured, memory B cell that are formed in the presence
of CD4+ T cells. These T cells provide the helper activ-
ity required for affinity maturation, isotype switching
and memory cell formation. When such T-cell help can-
not be provided, as in the case of hyper-IgM syndrome,
IgG, IgA and IgE antibody levels are absent or severely
reduced [7]. The presence of IgG, IgA and IgE ACPAs
[5,8], therefore, provides another line of evidence for the
T-cell-dependent nature of ACPA responses. The fine specificity of IgM anti-citrullinated
protein antibodies (ACPA) is different from that
of IgG ACPA Parawee Suwannalai1, Annemiek Willemze1, Linda van Toorn1, Gerrie Stoeken-Rijsbergen1, Nivine Levarht1,
Jan Wouter Drijfhout2, Tom W J Huizinga1, Rene E M Toes1 and Leendert A Trouw1* * Correspondence: L.A.Trouw@lumc.nl
1Department of Rheumatology, Leiden University Medical Center (LUMC), PO
Box 9600, NL-2300 RC Leiden, The Netherlands
Full list of author information is available at the end of the article Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Stability of the anti-citrullinated protein antibody I
response Samples from 18 patients were used to analyze the specific
reactivity of IgM against CCP2, Fib-a and Fib-b over time. We used serum samples obtained at baseline and at 1, 2
and 5 years of follow-up. Samples were tested by ELISA as
described above. Samples were considered positive when
they displayed an absorbance value higher than the cut-off
and a difference of at least 0.1 absorbance units when
comparing the reactivity against the Cit-containing peptide
and its arginine-containing control peptide. Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Page 2 of 7 arginine-containing peptides (10 μg/ml) were incubated
on streptavidin-coated plates. After being washed, sera
were incubated at 1:25 dilution at 37°C for 1 hour, bound
antibodies were detected using rabbit anti-human IgM
(Dako Denmark A/S, Glostrup, Denmark), followed by
washing with horseradish peroxidase-labeled goat anti-
rabbit IgG (Dako Denmark A/S). 2,2’-azino-bis(3-ethyl-
benzothiazoline-6-sulfonic acid) was used as a substrate,
and absorbance was determined at 415 nm. In each ELISA
plate, we included on the Cit-containing peptide eight
representative gout controls and used these to calculate
the cut-off for positivity [8]. These eight gout controls
represent a set of fifty controls as established before [8]
and were used on each plate to minimize plate-to-plate
variation. This was defined as the mean ± 2 SD of the
absorbance on the Cit-containing peptide. In addition, we
verified that the difference in absorbance between wells
coated with the Cit- and the arginine-containing peptide
was at least 0.1 as previously described [8,12]. Therefore,
sera that fulfilled both criteria were considered positive. described, in contrast to T-cell-independent B-cell
responses against, for example, repetitive sugar residues
on bacteria [9,10]. For these reasons, it is most conceiva-
ble that the presence of IgM ACPA suggests that activa-
tion of recently recruited naïve B cells recognize
citrullinated antigens because the half-life of circulating
IgM is short. In this study, we hypothesized that there
might be certain antigens which drive the ACPA IgM
response in RA. We therefore sought to determine
whether there is a difference in the fine specificity of IgG
and IgM ACPA. Statistical analysis
ff
b Demographic data (age, gender, disease duration and
radiographic damage) of ACPA IgG-positive RA patients
who had ACPA IgM ≥100 AU/ml were not different
from patients displaying ACPA IgM levels < 100 AU/ml. The peptides used in this study are linear citrulline (Cit)-
containing peptides, which are known ACPA IgG epi-
topes in RA, as well as their arginine counterparts [12]. Specifically, we used vimentin (Vim) 59 to 74: VYAT-
CitSSAVCitLCitSSVP; fibrinogen-a (Fib-a) 27 to 43:
FLAEGGGVCitGPRVVERH; Fib-b 36 to 52: NEEGFFSA-
CitGHRPLDKK; and a-enolase 5 to 20: KIHACitEIFDS-
CitGNPTV. In addition, we tested citrullinated protein
antigens (Fib and myelin basic protein (MBP)) and all of
their arginine counterparts. As described previously, the
presence of anti-Cit-MBP antibodies on ELISA is clini-
cally equivalent to the original anti-Sa (Cit-Vim) on
Western blot assays [13]. Differences between groups were analyzed using the
Mann-Whitney U test or analysis of variance. The associa-
tions between ACPA IgM and ACPA IgG positivity were
evaluated by using 2 × 2 tables for the estimation of ORs
and 95% CIs. The correlation between ACPA IgM and
ACPA IgG responses were determined using Spearman’s
rank-correlation coefficients. The c2 test was used to eval-
uate differences in the frequency distribution of ACPA
IgG and IgM. All data were analyzed with GraphPad
Prism version 4.0 software (GraphPad Software, San
Diego, CA, USA) or SPSS for Windows release 16.0 soft-
ware (SPSS Inc, Chicago, IL, USA). In all tests, P < 0.05
was considered significant. Fine specificity of anticitrullinated protein antibody
immunoglobulin M We determined the fine specificity of ACPA IgM and IgG
in 113 serum samples of anti-cyclic citrullinated peptide
(CCP2) IgG and IgM double-positive RA patients collected
from the Leiden Early Arthritis Clinic (EAC), an inception
cohort of recent-onset arthritis that was initiated at the
Department of Rheumatology at Leiden University Medi-
cal Center in 1993 [11]. We selected those patients who
had a relatively high titer of IgM CCP2 (levels ≥100 AU/
ml) to ensure that differences between IgG and IgM reac-
tivity could not be explained by differences in the sensitiv-
ity associated with the detection of IgG or IgM antibodies
by ELISA. For the determination of ACPA status and
ACPA levels of IgM anti-CCP2, we used a commercial
immunoassay kit (Euro-Diagnostica, Malmö, Sweden)
with minor modifications for the detection of IgM. The
collection and use of patient samples was approved by the
local medical ethics committee and in compliance with
the Declaration of Helsinki. All patients provided their
written informed consent. Introduction Anti-citrullinated protein antibodies (ACPA) may be
involved in the disease pathogenesis of rheumatoid arthri-
tis (RA). ACPA can be found early in the disease course
[1], even before disease onset [2], and the presence of
ACPA at the time of diagnosis can predict disease course
[3]. Moreover, ACPA can contribute to disease pathogen-
esis by activating immune cells [4,5] and the complement
system [6]. The ACPA response likely represents a T-cell-depen-
dent B-cell response, given the protein nature of the
antigen recognized and the strong association with the
human leukocyte antigen shared epitope alleles. The * Correspondence: L.A.Trouw@lumc.nl
1Department of Rheumatology, Leiden University Medical Center (LUMC), PO
Box 9600, NL-2300 RC Leiden, The Netherlands
Full list of author information is available at the end of the article To the best of our knowledge, IgM-producing memory
B cells against T-cell-dependent antigens have not been Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Page 2 of 7 Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Page 3 of 7 ACPA response [8]. If IgM is the result of triggering of
naïve B cells, then the fine specificity of ACPA IgM
might conceivably differ from ACPA IgG, as IgM will
mainly be directed against the antigens that have
recently been detected by B cells [14]. Therefore, we
determined the ACPA IgM and IgG responses against a
set of citrullinated peptides from Fib, enolase and Vim,
as well as the responses against two citrullinated pro-
teins (Figures 1A and 1B). We next determined the recognition profile of IgG
ACPA. Although not all patient sera recognized all citrulli-
nated epitopes, the IgG ACPA epitope recognition pattern
was clearly much broader than the epitope recognition
profile of IgM ACPA (Figures 1A and 2). In fact, when all
ACPA reactivities were analyzed as a group, the chance of
having an IgM-positive response was four times lower
compared to IgG (OR = 0.26, P < 0.0001). For those reac-
tivities where, next to IgG ACPA, IgM ACPA responses
also were apparent, a correlation between the titers of IgG
and IgM ACPAs was observed between the positive sam-
ples (data not shown). Our data show that some but not all citrullinated epi-
topes are recognized by IgM ACPA (Table 1 and Figure
1B). These data indicate a restricted epitope recognition
profile by IgM ACPA, as some epitopes were not recog-
nized by IgM in all analyzed patient sera. When reactivity patterns were directly compared at an
individual level, we observed that IgM ACPAs against When reactivity patterns were directly compared at an
individual level, we observed that IgM ACPAs against A
B
0
1
2
3
4
***
***
***
***
***
***
***
CCP2
Vim
Fib-D
Fib-E
D-eno
FIB
MBP
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
ACPA IgG Abs 415 nm
0
1
2
3
4
***
***
***
***
CCP2
Vim
Fib-D
Fib-E
D-eno
FIB
MBP
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
ACPA IgM Abs 415 nm
Figure 1 The fine specificity of immunoglobulin G (IgG) and IgM anticitrullinated protein antibodies (ACPAs). The absorbance (Abs) at
415 nm of ACPA IgG (A) and IgM (B) ACPA fine specificity. ***P < 0.001. Anti-citrullinated protein antibody fine specificity is
different from IgG fine specificity It has been shown that IgM ACPA can be detected in
the sera of RA patients with established disease, suggest-
ing an ongoing recruitment of new B cells into the Fine specificity assays of ACPA IgG were performed
essentially as described before [12], with minor modifica-
tions for ACPA IgM [8]. In brief, for IgM-, Cit- and Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Data for gout controls from three ELISA plates are depicted. a-eno =
a-enolase; Arg = arginine; CCP2 = cyclic citrullinated peptide 2; Cit = citrulline; Fib = fibrinogen; MBP = myelin basic protein; Vim = vimentin. A
0
1
2
3
4
***
***
***
***
***
***
***
CCP2
Vim
Fib-D
Fib-E
D-eno
FIB
MBP
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
ACPA IgG Abs 415 nm B
0
1
2
3
4
***
***
***
***
CCP2
Vim
Fib-D
Fib-E
D-eno
FIB
MBP
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
Cit
Arg
Gout
ACPA IgM Abs 415 nm
Figure 1 The fine specificity of immunoglobulin G (IgG) and IgM anticitrullinated protein antibodies (ACPAs). The absorbance (Abs) at B Figure 1 The fine specificity of immunoglobulin G (IgG) and IgM anticitrullinated protein antibodies (ACPAs). The absorbance (Abs) at
415 nm of ACPA IgG (A) and IgM (B) ACPA fine specificity. ***P < 0.001. Data for gout controls from three ELISA plates are depicted. a-eno =
a-enolase; Arg = arginine; CCP2 = cyclic citrullinated peptide 2; Cit = citrulline; Fib = fibrinogen; MBP = myelin basic protein; Vim = vimentin. Figure 1 The fine specificity of immunoglobulin G (IgG) and IgM anticitrullinated protein antibodies (ACPAs). The absorbance (Abs) at
415 nm of ACPA IgG (A) and IgM (B) ACPA fine specificity. ***P < 0.001. Data for gout controls from three ELISA plates are depicted. a-eno =
a-enolase; Arg = arginine; CCP2 = cyclic citrullinated peptide 2; Cit = citrulline; Fib = fibrinogen; MBP = myelin basic protein; Vim = vimentin. Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Suwannalai et al. Discussion In this study, we observed that ACPA IgM responses are
different from ACPA IgG responses, as they display a
more restricted antigen recognition pattern. These data
are intriguing, as they indicate that the regulation of the
IgM ACPA response differs from the regulation of B cells
producing IgG-directed against citrullinated antigens. Although the reason for this difference is not known, we
think it is most conceivable that these findings are
explained by a limited recruitment of new B cells into the
ACPA response that is driven by some, but not other,
citrullinated antigens. Given the short half-life of circulat-
ing IgM and the lack of memory B cells producing IgM Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Page 4 of 7 Table 1 Specific positivity of anticitrullinated protein
antibody immunoglobulin G and immunoglobulin M in
113 rheumatoid arthritis patients directed against a set
of defined specificities
Epitope
IgG (%)
IgM (%)
CCP2
100
100
Vim
56
7
Fib-a
33
4
Fib-b
94
40
a-enolase
55
8
Fib
53
40
MBP
63
40
CCP2 = cyclic citrullinated peptide 2; Fib = fibrinogen; IgG = immunoglobulin
G; IgM = immunoglobulin M; MBP = myelin basic protein; Vim = vimentin. Table 1 Specific positivity of anticitrullinated protein
antibody immunoglobulin G and immunoglobulin M in
113 rheumatoid arthritis patients directed against a set
of defined specificities peptides over time. The reactivity of ACPA IgM against
CCP2, Fib-a and Fib-b at baseline and at 1, 2 and 5 years
of follow-up was analyzed in sera of 18 patients. We
observed that the ACPA IgM levels against these peptides
appeared rather stable (Figures 3A to 3C). Some patients
whose sera reacted with CCP2, Fib-a and Fib-b at base-
line may over time become IgM-negative (Figure 3D). Importantly, none of the patients tested became IgM-ser-
opositive against either Fib-a or Fib-b peptides. These
data indicate that the observations made using the base-
line samples (Figures 1 and 2) would also be made at any
of the follow-up time points. Collectively, these data
therefore indicate that the ACPA IgM response is nar-
rower than the ACPA IgG response, an observation not
applicable only to the baseline results but also during fol-
low-up. CCP2 = cyclic citrullinated peptide 2; Fib = fibrinogen; IgG = immunoglobulin
G; IgM = immunoglobulin M; MBP = myelin basic protein; Vim = vimentin. CCP2 = cyclic citrullinated peptide 2; Fib = fibrinogen; IgG = immunoglobulin
G; IgM = immunoglobulin M; MBP = myelin basic protein; Vim = vimentin. some citrullinated epitopes, but not others, were pre-
sent. However, in all cases, IgM positivity was paralleled
by the presence of IgG recognizing the same epitopes. In contrast, patients can display certain IgG ACPA reac-
tivities in the absence of IgM reactivities against the
same antigen (Figure 2). Together, these data indicate
that IgM ACPAs display a more restricted antigen
recognition profiles as compared to IgG ACPAs. Stability of the anti-citrullinated protein antibody I
response To address the question whether the observed differences
between IgG and IgM reactivity are limited to baseline
samples only or whether they are also present at later fol-
low-up, we analyzed ACPA IgM reactivity against three CCP
vim
Fib-Į
IgG
Fib-ȕ
Į-eno
Fib
MBP
CCP
vim
Fib-Į
IgM
Fib-ȕ
Į-eno
Fib
MBP
Figure 2 Positivity of immunoglobulin G (IgG) and IgM anticitrullinated protein antibodies (ACPAs). The citrullinated epitopes recognized
by IgG and IgM ACPAs in each individual. The gray area represents positive reactivity, and the white area represents lack of reactivity. a-eno =
a-enolase; CCP = cyclic citrullinated peptide; Fib = fibrinogen; MBP = myelin basic protein; Vim = vimentin CCP
vim
Fib-Į
IgG
Fib-ȕ
Į-eno
Fib
MBP
CCP
vim
Fib-Į
IgM
Fib-ȕ
Į-eno
Fib
MBP
Figure 2 Positivity of immunoglobulin G (IgG) and IgM anticitrullinated protein antibodies (ACPAs). The citrullinated epitopes recognized
by IgG and IgM ACPAs in each individual. The gray area represents positive reactivity, and the white area represents lack of reactivity. a-eno =
a-enolase; CCP = cyclic citrullinated peptide; Fib = fibrinogen; MBP = myelin basic protein; Vim = vimentin. Figure 2 Positivity of immunoglobulin G (IgG) and IgM anticitrullinated protein antibodies (ACPAs). The citrullinated epitopes recognized
by IgG and IgM ACPAs in each individual. The gray area represents positive reactivity, and the white area represents lack of reactivity. a-eno =
a-enolase; CCP = cyclic citrullinated peptide; Fib = fibrinogen; MBP = myelin basic protein; Vim = vimentin. Figure 2 Positivity of immunoglobulin G (IgG) and IgM anticitrullinated protein antibodies (ACPAs). The citrullinated epitopes recognized
by IgG and IgM ACPAs in each individual. The gray area represents positive reactivity, and the white area represents lack of reactivity. a-eno =
a-enolase; CCP = cyclic citrullinated peptide; Fib = fibrinogen; MBP = myelin basic protein; Vim = vimentin. Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
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response Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Page 5 of 7 IgM
tested pos BL neg FU pos FU
CCP2
18
18
3
0
Fib-D
18
7
3
0
Fib-E
18
17
6
0
A
B
C
D
CCP
BL
1
2
5
0
1
2
3
years
Abs 415 nm (cit - arg)
Fib D
BL
1
2
5
0
1
2
3
years
Abs 415 nm (cit - arg)
Fib E
BL
1
2
5
0
1
2
3
years
Abs 415 nm (cit - arg)
Figure 3 Stability of the immunoglobulin M anticitrullinated protein antibody response. Sera taken from 18 patients at baseline (BL) and
at 1, 2 and 5 years of follow-up were analyzed by ELISA for reactivity against cyclic citrullinated peptide 2 (CCP2), fibrinogen (Fib)-a and Fib-b. (A) through (C) Graphs displaying the immunoglobulin M (IgM) reactivity to the indicated peptides over time. The absorbance of citrulline (cit)
reactivity minus absorbance of the arginine (arg) reactivity is shown. (D) Table showing the number of positive patients at baseline (BL) as well
as the conversion toward negative or positive during one of the follow-up (FU) time points analyzed. A
B
C
D
CCP
BL
1
2
5
0
1
2
3
years
Abs 415 nm (cit - arg)
Fib D
BL
1
2
5
0
1
2
3
years
Abs 415 nm (cit - arg) B A C
D
years
Fib E
BL
1
2
5
0
1
2
3
years
Abs 415 nm (cit - arg) C D Figure 3 Stability of the immunoglobulin M anticitrullinated protein antibody response. Sera taken from 18 patients at baseline (BL) and
at 1, 2 and 5 years of follow-up were analyzed by ELISA for reactivity against cyclic citrullinated peptide 2 (CCP2), fibrinogen (Fib)-a and Fib-b. (A) through (C) Graphs displaying the immunoglobulin M (IgM) reactivity to the indicated peptides over time. The absorbance of citrulline (cit)
reactivity minus absorbance of the arginine (arg) reactivity is shown. (D) Table showing the number of positive patients at baseline (BL) as well
as the conversion toward negative or positive during one of the follow-up (FU) time points analyzed. who had ACPA IgM < 100 AU/ml and observed no
differences. against protein antigens, we think that the IgM ACPAs
detected in this study are produced by new B cells that are
recruited into the ACPA response against certain citrulli-
nated antigens. References 1. van Venrooij WJ, Hazes JM, Visser H: Anticitrullinated protein/peptide
antibody and its role in the diagnosis and prognosis of early
rheumatoid arthritis. Neth J Med 2002, 60:383-388. ,
2. Nielen MM, van der Horst AR, van Schaardenburg D, van der Horst-
Bruinsma IE, van de Stadt RJ, Aarden L, Dijkmans BA, Hamann D:
Antibodies to citrullinated human fibrinogen (ACF) have diagnostic and
prognostic value in early arthritis. Ann Rheum Dis 2005, 64:1199-1204. 3. Lindqvist E, Eberhardt K, Bendtzen K, Heinegård D, Saxne T: Prognostic
laboratory markers of joint damage in rheumatoid arthritis. Ann Rheum
Dis 2005, 64:196-201. ,
2. Nielen MM, van der Horst AR, van Schaardenburg D, van der Horst-
Bruinsma IE, van de Stadt RJ, Aarden L, Dijkmans BA, Hamann D:
Antibodies to citrullinated human fibrinogen (ACF) have diagnostic and
prognostic value in early arthritis. Ann Rheum Dis 2005, 64:1199-1204. 3. Lindqvist E, Eberhardt K, Bendtzen K, Heinegård D, Saxne T: Prognostic
laboratory markers of joint damage in rheumatoid arthritis. Ann Rheum
Dis 2005, 64:196-201. 2. Nielen MM, van der Horst AR, van Schaardenburg D, van der Horst-
Bruinsma IE, van de Stadt RJ, Aarden L, Dijkmans BA, Hamann D:
Antibodies to citrullinated human fibrinogen (ACF) have diagnostic and
prognostic value in early arthritis. Ann Rheum Dis 2005, 64:1199-1204. prognostic value in early arthritis. Ann Rheum Dis 2005, 64:1199 1204. 3. Lindqvist E, Eberhardt K, Bendtzen K, Heinegård D, Saxne T: Prognostic
laboratory markers of joint damage in rheumatoid arthritis. Ann Rheum
Dis 2005, 64:196-201. 4. Clavel C, Nogueira L, Laurent L, Iobagiu C, Vincent C, Sebbag M, Serre G:
Induction of macrophage secretion of tumor necrosis factor α through
Fcγ receptor IIa engagement by rheumatoid arthritis-specific
autoantibodies to citrullinated proteins complexed with fibrinogen. Arthritis Rheum 2008, 58:678-688. Abbreviations ACPA: anticitrullinated protein antibody; Arg: arginine; AU: arbitrary unit; BL:
baseline; Cit: citrulline; CCP2: cyclic citrullinated peptide 2; EAC: Early Arthritis
Clinic; ELISA: enzyme-linked immunosorbent assay; Fib: fibrinogen; FU:
follow-up; Ig: immunoglobulin; IgM: immunoglobin M LUMC: Leiden
University Medical Center; MBP: myelin basic protein; RA: rheumatoid
arthritis; RF: rheumatoid factor. Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Cyanogen bromide-activated-Sepharose beads (Sigma-
Aldrich, St Louis, MO, USA), there was no reduction of
ACPA IgM levels. Moreover, after mixing sera which were
highly positive for RF IgM but negative for ACPA IgG and
ACPA IgM with sera which were RF IgM- and ACPA
IgM-negative but ACPA IgG-positive, ACPA IgM could
not be detected [8]. Collectively, these observations sup-
port the notion that true IgM ACPA and not IgM RF
bound to ACPA IgG were detected by the methods
employed. Author details
1 1Department of Rheumatology, Leiden University Medical Center (LUMC), PO
Box 9600, NL-2300 RC Leiden, The Netherlands. 2Department of
Immunohematology and Blood Tranfusion, PO Box 9600, NL-2300 RC Leiden
University Medical Center (LUMC), Leiden, The Netherlands. 1Department of Rheumatology, Leiden University Medical Center (LUMC), PO
Box 9600, NL-2300 RC Leiden, The Netherlands. 2Department of
Immunohematology and Blood Tranfusion, PO Box 9600, NL-2300 RC Leiden
University Medical Center (LUMC), Leiden, The Netherlands. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 5 July 2011 Revised: 14 October 2011
Accepted: 30 November 2011 Published: 30 November 2011 Received: 5 July 2011 Revised: 14 October 2011
Accepted: 30 November 2011 Published: 30 November 2011 Acknowledgements
f PS received a grant from Ramathibodi Hospital, Mahidol University, Bangkok,
Thailand. AW is supported by the Dutch Organization for Scientific Research
(AGIKO grant). REMT is a recipient of a NWO-VICI grant, and LAT is a
recipient of a NWO-VENI grant. This study was supported by the European
Union (Sixth Framework Programme integrated project Autocure and
Seventh Framework Programme integrated project Masterswitch 223404) as
well as IMI-BTCure. This study was also supported by national funding from
The Netherlands Genomics Initiative (NGI) as part of The Netherlands
Proteomics Center (NPC) and the Center for Medical Systems Biology
(CMSB). Although it is tempting to speculate, these studies
should not be taken as an argument for the involvement
of the antigens analyzed here in the recruitment of new B
cells into the ACPA response. Peptides are unlikely to
reflect correctly the three-dimensional structure of citrulli-
nated proteins that form the epitope for antibodies. More-
over, ACPA IgG is cross-reactive to multiple citrullinated
antigens [15,16], and therefore recognition of a citrulli-
nated antigen by ACPA IgG does not indicate that this
antigen is necessarily involved in the induction of B-cell
responses. Nonetheless, our data do show that the IgM
ACPA response is significantly more restricted than that
of the IgG ACPA present in the same patient. Authors’ contributions PS, TWJH, REMT and LAT conceived the study and participated in its design
and edited the manuscript. PS and LAT drafted and prepared the
manuscript and performed the statistical analyses. AW assisted with
manuscript preparation. PS, LVT, GSR and NL performed blood analyses. JWD
provided peptides essential for the assays. All authors contributed to the
interpretation of the study findings and approved the final manuscript. g
How IgM can be formed in the presence of an active
IgG response against the same antigen is not clear. In
other situations, as exemplified by the prophylactic
administration of anti-Rhesus D antigen antibodies to
pregnant women carrying a Rhesus D-positive child, the
presence of IgG against a certain antigen will prevent the
induction of a novel IgM response. The mechanism
behind this protective measure is thought to be mediated
by either the capture and clearance of circulating Rhesus
D antigen and/or by IgG-Rhesus D immune complexes
that inactivate new Rhesus D-reactive B cells through
FcgRIIB, the inhibitory Fcg IIB receptor [17]. Clearly, IgG
ACPAs do not inhibit the activation of IgM-positive,
citrullinated antigen-reactive B cells. The reason for this
finding is not known but could possibly be explained by
the low avidity of the ACPA [18], conceivably resulting in
“nonstable” immune complexes unable to trigger
FcgRIIB. 8.
Verpoort KN, Jol-van der Zijde CM, Papendrecht-van der Voort EA, Ioan-
Facsinay A, Drijfhout JW, van Tol MJ, Breedveld FC, Huizinga TW, Toes RE: Stability of the anti-citrullinated protein antibody I
response Even in the case where ACPA IgM-produ-
cing memory B cells exist, it is still interesting that such
cells are present only against certain citrullinated antigens. Since IgM rheumatoid factor (RF) could also be a con-
founding factor, we compared RF levels between the
patients who displayed ACPA IgM ≥100 AU/ml with
those who had ACPA IgM < 100 AU/ml and observed
that in RA patients with ACPA IgM ≥100 AU/ml, the
levels of RF IgM were higher than in RA patients who had
ACPA IgM < 100 AU/ml (RF IgM 50 AU/ml (25 to 118)
and 28 AU/ml (12 to 60), respectively). To exclude the
possibility that our findings were influenced by IgM RF,
we analyzed ACPA IgM specificities in relation to IgM RF. We observed that IgM RF-positive samples can be nega-
tive for IgM ACPA reactivities and that, in the absence of
IgM RF, IgM ACPA reactivities can be detected readily
(data not shown). RF-positive samples that have IgG reac-
tivity against all fine specificity epitopes may have IgM
ACPA against only some antigens and not others, con-
firming that our assay did not merely detect IgM RF. Pre-
viously, we addressed this issue experimentally [8]. When
RFs were depleted from RF IgM-positive, ACPA IgM-posi-
tive and ACPA IgG-positive sera using IgG-coated For this study, we selected patients who are double-posi-
tive for IgG and IgM CCP2 reactivity. To exclude the pos-
sibility that differences between IgG and IgM may be
explained by differences in the sensitivity associated with
the detection of IgG or IgM antibodies by ELISA, we
selected only patients with an IgM level of at least 100
AU/ml. Indeed, in the setup we used, we observed that,
although the IgG responses against the different citrulli-
nated antigens were rather similar in absorbance units,
IgM reactivity was detected only against some antigens
but not others. This observation was made at baseline but
appears also to be present at follow-up. To ensure that we
did not introduce additional bias into our data, we com-
pared the demographic data (age, gender, disease duration
and radiographic damage) of ACPA IgG-positive RA
patients who had ACPA IgM ≥100 AU/ml with those Page 6 of 7 Page 6 of 7 Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
http://arthritis-research.com/content/13/6/R195 Isotype distribution of anti-cyclic citrullinated peptide antibodies in
undifferentiated arthritis and rheumatoid arthritis reflects an ongoing
immune response. Arthritis Rheum 2006, 54:3799-3808. Isotype distribution of anti-cyclic citrullinated peptide antibodies in
undifferentiated arthritis and rheumatoid arthritis reflects an ongoing
immune response. Arthritis Rheum 2006, 54:3799-3808. 9. Rajewsky K: Clonal selection and learning in the antibody system. Nature
1996, 381:751-758. 9. Rajewsky K: Clonal selection and learning in the antibody system. Nature
1996, 381:751-758. 9. Rajewsky K: Clonal selection and learning in the antibody system. Nature
1996, 381:751-758. 10. Racine R, McLaughlin M, Jones DD, Wittmer ST, MacNamara KC,
Woodland DL, Winslow GM: IgM production by bone marrow
plasmablasts contributes to long-term protection against intracellular
bacterial infection. J Immunol 2011, 186:1011-1021. 10. Racine R, McLaughlin M, Jones DD, Wittmer ST, MacNamara KC,
Woodland DL, Winslow GM: IgM production by bone marrow
plasmablasts contributes to long-term protection against intracellular
bacterial infection. J Immunol 2011, 186:1011-1021. 11. van Aken J, van Dongen H, le Cessie S, Allaart CF, Breedveld FC,
Huizinga TW: Comparison of long term outcome of patients with
rheumatoid arthritis presenting with undifferentiated arthritis or with
rheumatoid arthritis: an observational cohort study. Ann Rheum Dis 2006,
65:20-25. 12. Verpoort KN, Cheung K, Ioan-Facsinay A, van der Helm-van Mil AH, de
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http://arthritis-research.com/content/13/6/R195 doi:10.1186/ar3524
Cite this article as: Suwannalai et al.: The fine specificity of IgM anti-
citrullinated protein antibodies (ACPA) is different from that of IgG
ACPA. Arthritis Research & Therapy 2011 13:R195. Suwannalai et al. Arthritis Research & Therapy 2011, 13:R195
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antibodies against recall antigens. Ann Rheum Dis 2011, 70:373-379. Submit your next manuscript to BioMed Central
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Detecting circular RNA from high-throughput sequence data with de Bruijn graph
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METHODOLOGY Open Access © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Detecting circular RNA from
high-throughput sequence data with de
Bruijn graph Xin Li*
and Yufeng Wu From 14th International Symposium on Bioinformatics Research and Applications (ISBRA’18)
Beijing, China. 8–11 June 2018 Abstract Background: Circular RNA is a type of non-coding RNA, which has a circular structure. Many circular RNAs are stable
and contain exons, but are not translated into proteins. Circular RNA has important functions in gene regulation and
plays an important role in some human diseases. Several biological methods, such as RNase R treatment, have been
developed to identify circular RNA. Multiple bioinformatics tools have also been developed for circular RNA detection
with high-throughput sequence data. Results: In this paper, we present circDBG, a new method for circular RNA detection with de Bruijn graph. We
conduct various experiments to evaluate the performance of CircDBG based on both simulated and real data. Our
results show that CircDBG finds more reliable circRNA with low bias, has more efficiency in running time, and performs
better in balancing accuracy and sensitivity than existing methods. As a byproduct, we also introduce a new method
to classify circular RNAs based on reads alignment. Finally, we report a potential chimeric circular RNA that is found by
CircDBG based on real sequence data. CircDBG can be downloaded from https://github.com/lxwgcool/CircDBG. Conclusions: We develop a new method called CircDBG for circular RNA detection, which is based on de Bruijn
graph. We conduct extensive experiments and demonstrate CircDBG outperforms existing tools, especially in saving
running time, reducing bias and improving capability of balancing accuracy and sensitivity. We also introduce a new
method to classify circular RNAs and report a potential case of chimeric circular RNA. Keywords: Circular RNA, High-throughput sequencing, Genomics, RNA-Seq, de Bruijn graph biological functions in some human diseases have started
to be recognized [1–3]. Many of these circRNAs are sta-
ble and contain exons, but they are not translated into
proteins [4]. *Correspondence: xin.li@uconn.edu
Department of Computer Science and Engineering, University of Connecticut,
Storrs 06269, CT, USA Li and Wu BMC Genomics 2019, 21(Suppl 1):749
https://doi.org/10.1186/s12864-019-6154-7 Li and Wu BMC Genomics 2019, 21(Suppl 1):749
https://doi.org/10.1186/s12864-019-6154-7 Background Circular RNA, the RNA in a circular form through a
usually 5’ to 3’ phosphodiester bond, is a type of non-
coding RNA [1]. Circular RNA (or circRNA) is recently
recognized as a new class of functional molecule [1]. CircRNA consists no 5’ or 3’ free terminus, as illustrated
in Fig. 1, which makes it much more stable in the cells
than linear RNA [1]. CircRNAs were originally thought
as the byproduct from the process of mis-splicing, and
considered to be of low abundance. Recently, however,
the importance of circRNAs in gene regulation and their There are two types of experimental methods currently
that can be used to identify circular RNA [5]. First, since
circRNAs lack a poly(A) tail [6], they can be retained in
rRNA-depleted libraries by using expected depletion pro-
file to assess results. Second, circRNA can be enriched in
libraries treated with RNase R to digest linear RNA and
make it easier to detect lowly expressed circRNA. With
the high-throughput sequencing technologies, multiple
bioinformatics tools have been developed recently for cir-
cRNAs detection from RNA sequence reads. Some of Li and Wu BMC Genomics 2019, 21(Suppl 1):749 Page 2 of 11 Fig. 1 Ei: exons, Li: linear splicing junction (for linear RNA). Ci: circular splicing junction (for circRNA). Two sets of isoforms of linear RNA and circRNA
are shown on left and right sides. 3‘ in circular splicing junction is donor, while 5‘ is acceptor. All linear isoforms are sensitive to RNase R. Circular
isoforms show no significant decrease in abundance after RNase R treatment Fig. 1 Ei: exons, Li: linear splicing junction (for linear RNA). Ci: circular splicing junction (for circRNA). Two sets of isoforms of linear RNA and circRNA
are shown on left and right sides. 3‘ in circular splicing junction is donor, while 5‘ is acceptor. All linear isoforms are sensitive to RNase R. Circular
isoforms show no significant decrease in abundance after RNase R treatment repetitive k-mers. Moreover, CircMarker becomes slow
for large data. them require gene annotation while others do not. Those
methods could be divided into two categories: (a) reads-
mapping-based methods, such as CIRI [7]/CIRI2 [8],
CIRCExplorer [9], Find-circ [10] and CircRNAFinder [11],
and (b) k-mer-based methods, such as CircMarker [12]. In this paper, we present a new method named CircDBG
for circular RNA detection with de Bruijn graph. High-level approach The key idea of CircDBG is creating a de Bruijn graph
based on k-mers from the boundary parts of exons in
annotated genome. As shown in Fig. 2, we take advan-
tage of this graph to find the relationship between k-mer
of reads and the potential donor/acceptor exon by track-
ing the path in the graph for circular RNA detection. Since the path provides a stronger signal for calling the
two exons involved in the back splicing than individual
k-mers, CircDBG can filter out more false positives than
CircMarker. This is especially true when there are dupli-
cate k-mers in exons and/or there are errors in the reads. To make CircDBG more efficient, we also develop various
techniques. Recently, we developed a k-mer-based tool called
CircMarker [12], which uses an efficient k-mer table
for circular RNA detection. Compared with the reads-
mapping-based method, CircMarker has two major
advantages. First, CircMarker looks for the circRNA-
related reads for detection and does not depend on any
third party mapping tool. Thus CircMarker is much faster
than reads-mapping-based methods, especially for small
data. Second, since the minimum comparison unit for
CircMarker is a k-mer rather than reads, it can tolerate
more errors and find more circular RNAs. However,
CircMarker still has some issues. A key issue for Circ-
Marker is the potential loss of information. CircMarker
considers k-mers individually. That is, CircMarker does
not consider the order of k-mers from either reads or
exons, and this may lead to false positives when there are Background Different
from the normal de Bruijn graph, CircDBG uses it in a
specialized way designed to call circular RNA, which is
the first algorithm using de Bruijn graph for circular RNA
detection. Through experiments based on simulated and
real data, we demonstrate that this new method finds
more reliable circRNA with low bias, runs faster and has
better performance in balancing accuracy and sensitivity
than existing methods. Reads-mapping-based methods first map the RNA-seq
reads onto a reference. For this purpose, CIRI uses BWA
[13], while bowtie [14] and Tophat (TopHat-fusion) [15]
are used by Find-circ and CIRCExplorer respectively. Since BWA and bowtie do not require annotations, all
RNA reads need to be mapped to the entire reference
genome. As CIRCExplorer, CircRNAFinder only performs
reads mapping by using STAR in the range of annotated
genomes as provided by annotation file. Those mapping
methods have two major issues. First, reads-mapping-
based tools are often computationally inefficient because
mapping all reads can be slow, yet we note that many
RNA-seq reads are irrelevant to circRNA detection. Sec-
ond, these tools may miss circRNA in some cases due to
errors in reads mapping [12]. Finally, we introduce a new method of classifying circu-
lar RNAs based on reads alignment and report a potential
chimeric circular RNA that is found by CircDBG based on
real sequence data. CircDBG Our new CircDBG method contains three parts: (a) build-
ing de Bruijn graph, (b) finding potential donor/acceptor Page 3 of 11 Li and Wu BMC Genomics 2019, 21(Suppl 1):749 Fig. 2 Five exons are in the genome (top left). Back splicing occurs from exon 4 to exon 2, which generates a circular RNA (top right). The
bidirectional de Bruijn graph (bottom left) is built from the k-mers from each exon in the genome, where each exon is represented by the path with
the same color in graph. The dotted line represents a RNA reads which supports the presence of circRNA case: the starting part of the read overlaps
with the ending part of exon 4, and the ending part of the read overlaps with the starting part of exon 2 Fig. 2 Five exons are in the genome (top left). Back splicing occurs from exon 4 to exon 2, which generates a circular RNA (top right). The
bidirectional de Bruijn graph (bottom left) is built from the k-mers from each exon in the genome, where each exon is represented by the path with
the same color in graph. The dotted line represents a RNA reads which supports the presence of circRNA case: the starting part of the read overlaps
with the ending part of exon 4, and the ending part of the read overlaps with the starting part of exon 2 cannot cover the whole exon, especially when the exon is
very long, we only use k-mers near the boundaries of an
exon when building the graph. The length of extraction is
identified as: sites, and (c) detecting circular RNA. First, a memory-
efficient de Bruijn graph is created, which records rel-
evant information of annotated genome. Second, we
filter out circRNA-related reads and find the potential
donor/accepter exon. Finally, we compare k-mers from
reads with the graph for circular RNA detection. Some
parameters should be determined before running Cir-
cDBG, such as the length of k-mer. The maximum length
of k-mers in CircDBG is 16, and we set 15 as its default
value for all data analysis reported in this paper. Lseq = Lreads −k −5 This means we require that there are at least 6 contin-
uous k-mers should come from the other side of circular
splicing junction. CircDBG In another word, we require at least the
length of k + 5 in reads to come from the other site of cir-
cular RNA. If the length of an eligible exon is shorter than
2 × Lseq, the whole exon is used to build graph. Build de Bruijn graph
d c The graph is constructed by these collected k-mers: exon
1/exon 3 are represented by 2 separate green/blue paths in the graph, and exon 2 is represented by one red path Finally, since the last node doesn’t have the next neigh-
bor and the first node doesn’t have the previous neighbor,
we set the value of this kind of next and previous neighbor
node as 0 with tag “U”. These two special nodes indicate
the ending and beginning of the path. in one exon, multiple groups of exon information are asso-
ciated with each node. Each exon information contains the
node’s position and multiple links. As same as the strategy
of CircMarker, we use 5 values to represent the node posi-
tion: one tag and four indexes (including chromosome,
gene, transcript, and exon). Since the maximum index
value (e.g. the number of exons in one transcript) is not
large, we use light weight data structure to store those
additional messages, such as exon index and transcrip-
tion index, in each node to save the RAM[12]. The tag
contains 4 different values: S/E and H/T, which speci-
fies whether the k-mer comes from the starting or ending
part and whether it is close to head or tail boundary of
exon respectively. Since the closer a k-mer is to the back-
splicing junction point, the higher possibility the k-mer
can be contained by the supported reads, we call the node
with tag H/T as premium node. The data structure dis-
cussed above can help to distinguish repetitive k-mers in
the same exon or multiple exons, because it records all
possible exon positions and the neighbors of each node. In addition, since we only extract k-mer from the bound-
ary side of each exon, the number of collected k-mers is
not large. With the help of additional messages recorded
in each node, the maximum length of 16 bps is enough to
distinguish the majority part of those k-mers. Here is an
example of how we save the exon info for one node: sup-
pose one k-mer is found in the valid part of exon 1 and
exon 2, and it appears one time in exon 1 and two times in
exon 2. Then, the k-mer is converted into an integer and
set as the key of this node. Build de Bruijn graph
d We create de Bruijn graph for each chromosome
separately, and use them in parallel with RNA sequence
reads for circular RNA detection. All the k-mers used to
create de Bruijn graph come from exon, and the k-mers
from reads will be used to track the path in the graph for
circular RNA detection. We use 2 bits to present each base
in k-mer, and integer 32 is used to save the value of k-
mer. Therefore, the maximum length of k-mer presented
by each node in graph is 16 bps. For each chromosome,
only the exons that contain back splicing signal (GT-AG)
are considered. The exons with length shorter than the
chosen k-mer length are ignored. Since the back-splicing
only occurs near the boundary of exon, and one read For example, if the length of reads and k-mer is 101
and 15 respectively, based on the equation, the length of
extraction is 81, which contains 67 k-mers theoretically. Given an exon with the length of 1000 bps, two sequences
will be extracted from the beginning part (1 to 81) and
ending part (920 to 1000) respectively. All k-mers from the boundary parts of sequences are
processed sequentially and converted into integers as the
values of nodes in the graph. The edge of each node repre-
sents its next or previous neighbors. The procedure of cre-
ating de Bruijn graph is illustrated in Fig. 3. Since the node
may be shared by multiple exons or appear multiple times Page 4 of 11 Li and Wu BMC Genomics 2019, 21(Suppl 1):749 Fig. 3 a Three exons with the back-splicing signal (AG-GT/AC-CT) are chosen. b The k-mers from the beginning part and ending part of exon 1 and
exon 3 are collected, while all k-mers from exon 2 are selected since Lenexon2 < 2 × Lseq. c The graph is constructed by these collected k-mers: exon
1/exon 3 are represented by 2 separate green/blue paths in the graph, and exon 2 is represented by one red path Fig. 3 a Three exons with the back-splicing signal (AG-GT/AC-CT) are chosen. b The k-mers from the beginning part and ending part of exon 1 and
exon 3 are collected, while all k-mers from exon 2 are selected since Lenexon2 < 2 × Lseq. Build de Bruijn graph
d Two exon information are asso-
ciated with this node: exon 1 info contains node position
and one link, while exon 2 info contains node position and
two links. Each link includes the key of its previous and
next node. Find potential donor/acceptor sites We first obtain the circRNA-related reads by using the
similar strategy of CircMarker: filter out reads that none
of sampled k-mers from the read has matches in the
graph. Both original reads and its reverse complemen-
tary are considered. If the original reads is failed to be
found back in graph, CircDBG will consider its reverse
complementary. In order to identify the back splicing of circRNAs, we
need to search for the potential donor and acceptor sites. The donor side comes from the ending part of the exon,
which is contributed by the starting part of the reads,
while the acceptor side comes from the starting part of the
exon, which is contributed by the ending part of the reads. p
p
The donor side comes from the ending part of the exon,
which is contributed by the starting part of the reads,
while the acceptor side comes from the starting part of the
exon, which is contributed by the ending part of the reads. To find potential donor candidates, we sample four k-
mers from the beginning to the end of the reads, and
search for each k-mer’s hit in the graph. A valid hit means
the k-mer can be found in the graph and its next neighbor
in graph can be found in the reads. The exon supported
by at least two valid hits with tag T/E are collected as the
donor candidate. For the potential acceptor candidates,
we sample four k-mers from the end to the beginning of
the reads, and apply the similar procedure as that of the
donor candidate. There are two differences here: its pre-
vious neighbor is tracked and the valid hit should contain
the tag H/S. We also collect two additional k-mers from
reads for quality control. We try all combinations from the
donor and acceptor candidates. If the donor and acceptor Page 5 of 11 Page 5 of 11 Li and Wu BMC Genomics 2019, 21(Suppl 1):749 brief paths should be long enough or contain more than
two premium nodes by each of them. come from the same exon, we think this is the poten-
tial self-circle case. Otherwise, it belongs to regular-circle
case if the donor and acceptor come from the same tran-
script in the order of back to front. Circular RNA detection For each circRNA candidate, we try to find the first k-mer
in the reads from the beginning to the end that can find
hits in the graph with the same donor information iden-
tified in the current candidate. Then, we view this hitting
node as an anchor and iteratively search for its next neigh-
bor in the graph continuously. Once we get the path, we
save a “brief-path-donor” by only keeping the first three
nodes, the nodes with index divisible by 3 and the last
node which contains the terminal signal in its next neigh-
bor, as shown in Fig. 5. This brief path can speed up the
later comparison while keeping the same accuracy. Here,
when we check the full path in the graph, the search is ter-
minated if the path is longer than the length of the reads. In addition, the candidate is ignored if the length of the full
path is too short. Similar procedure is applied to extract
the “brief-path-acceptor” by tracing the previous neigh-
bor continuously from the anchor node, which is the first
valid hit case with the same acceptor info from the end to
the beginning of the reads. The total length of these two Finally, we merge similar candidates that share similar
boundary of both donor and acceptor site with maximum
8 bps differences by using the candidate with the shortest
summary length of donor and acceptor to represent the
final result. Find potential donor/acceptor sites If there are more than
one candidate for each circle case, we only consider the
candidate supported by the maximum number of quality
control nodes. See Fig. 4 for an illustration. Note that for
the regular-circle candidate, if donor and acceptor nearby
each other with the same sequence value, they may be
from genome repeats and this candidate is ignored. Once those two brief paths are prepared, we check
if the nodes in “brief-path-donor” can find hits in the
reads from the beginning to end sequentially. We perform
“perfect match” for regular nodes. For the premium nodes,
we perform“loose match”: the node sequence is divided
into three parts and is viewed as a hit if at least two
parts could be matched perfectly. If the number of hits
is larger than our pre-defined threshold, we consider that
the donor side is well supported by current reads. Other-
wise, we apply a weak threshold if the hit nodes contain at
least one premium node to guarantee the donor junction
point to be covered by reads. The procedure of acceptor
verification is similar to that of donor case. The only dif-
ference is that we try to check if the nodes in “brief-path-
acceptor” can find hits in reads from the end to beginning
sequentially. Then, we check the distance between two last
mapping nodes in reads from those two brief paths respec-
tively. The circRNA candidate is kept only if the distance
is < 3 bps. See Fig. 5 for an illustration. Results The blue node with symbol # is the anchor node of donor and the blue
path in graph represents the donor (Exon 2) by tracing backward from the anchor node. “Brief-path-donor” contains eight blue nodes in path,
including four premium nodes. Green path in graph represents the acceptor (Exon 1) by tracing forward from anchor node. “Brief-path-acceptor”
contains eight green nodes in path, including four premium nodes. Six nodes from the brief path of donor and acceptor can find hits in reads
respectively. Since there are more than 70% hits and the distance between nodes 6 and 7 is shorter than 3 bps, this reads is considered to support
the back splicing from Exon 2 to Exon 1 tool and others respectively. The larger overlap means the
lower bias. In addition, although several circular RNA databases
have been released recently, such as circRNADb [16] and
CircBase [17], the data in these databases come from pub-
lished papers which are obtained from existing circRNA
detection tools and only a few of those data have been ver-
ified through biological experiments. In this paper, we use
four different strategies for evaluation. All of these strate-
gies calculate the accuracy and sensitivity of each tool as
follows, where T is the total called circRNAs by a tool,
Thit is the number of called circRNAs which find matches
in the benchmark. “Benchmark” is prepared in different
ways for each strategy. In the third one, for real data, if two different datasets
come from the same tissue with different experimental
libraries, the intersection between the results of those two
datasets could be considered as the reliable results for
each tool, and the circRNAs supported by at least two
tools could be viewed as the benchmark. In the last strategy, we use the intersection between
the results of RNase R treated and untreated data to get
the reliable results for each tool, and the circRNAs sup-
ported by at least 2 tools are viewed as benchmark. See
Additional file 1 (A. Benchmark used for comparison) for
details on benchmark.
Accuracy = |Thit|
|T| , where Thit = T ∩Benchmark
Sensitivity =
|Thit|
|Benchmark|
Accuracy = |Thit|
|T| , where Thit = T ∩Benchmark
Sensitivity =
|Thit|
|Benchmark| Our experiments show that no single tool always has the
highest accuracy and sensitivity. Results Comparing different circRNA detection methods is not
straightforward. The field lacks a gold standard for assess-
ing the accuracy of their genome-wide predictions [5]. Fig. 4 a Each node stands for k-mer. Four blue nodes represent the k-mers chosen from the head part of reads which are related to donor, while
four green nodes represent the k-mers from the tail part which correspond to acceptor. Two purple nodes with symbol # represent the quality
control nodes. Two self-circle cases and two regular-circle cases are found by the combination of donor and acceptor candidates. b Check whether
or not quality control nodes support each circular case. c “Regular-Circle Candi 1” and “Self-Circle Candi 2” are kept, since they are supported by
more quality control nodes than others in their case group respectively Fig. 4 a Each node stands for k-mer. Four blue nodes represent the k-mers chosen from the head part of reads which are related to donor, while
four green nodes represent the k-mers from the tail part which correspond to acceptor. Two purple nodes with symbol # represent the quality
control nodes. Two self-circle cases and two regular-circle cases are found by the combination of donor and acceptor candidates. b Check whether
or not quality control nodes support each circular case. c “Regular-Circle Candi 1” and “Self-Circle Candi 2” are kept, since they are supported by
more quality control nodes than others in their case group respectively Li and Wu BMC Genomics 2019, 21(Suppl 1):749 Page 6 of 11 Fig. 5 Back splicing occurs from Exon 2 to Exon 1 which generates a circle. The blue node with symbol # is the anchor node of donor and the blue
path in graph represents the donor (Exon 2) by tracing backward from the anchor node. “Brief-path-donor” contains eight blue nodes in path,
including four premium nodes. Green path in graph represents the acceptor (Exon 1) by tracing forward from anchor node. “Brief-path-acceptor”
contains eight green nodes in path, including four premium nodes. Six nodes from the brief path of donor and acceptor can find hits in reads
respectively. Since there are more than 70% hits and the distance between nodes 6 and 7 is shorter than 3 bps, this reads is considered to support
the back splicing from Exon 2 to Exon 1 Fig. 5 Back splicing occurs from Exon 2 to Exon 1 which generates a circle. Results Thus, we focus on com-
paring the balance between those two indicators by using
F1 score. In the first strategy, we use simulated data for compari-
son where the simulated circRNAs are benchmark. The F1 score is calculated by 2 × Precision×Recall
Precision+Recall. In our
cases, since there is no true negatives, and all non-true
positives are viewed as false positive, the precision and
recall are equal to accuracy and sensitivity respectively. So
the F1 score is equal to 2 × Accuracy×Sensitivity
Accuracy+Sensitivity. We choose public database circRNADb in the second
strategy, and all records in database are viewed as reliable
circRNA. There are two goals of this comparison. First,
we want to examine how well the public database is sup-
ported by each tool. The larger coverage in database the
results from a tool has, the better the tool can support
the database. Second, we evaluate the bias of each tool by
checking the overlap between the results of the current Accuracy+Sensitivity
Since some tools depend on annotation files whereas
some others don’t, and the majority part of back splicing
comes from the exons in genome, we choose circRNAs Li and Wu BMC Genomics 2019, 21(Suppl 1):749 Page 7 of 11 Page 7 of 11 with junction points identified by annotated genome for
comparison. tool by taking the intersection between two differently
prepared libraries from the same tissue. The results that
are supported by both libraries are viewed as reliable. RNA-seq reads SRR4095542 and SRR5133906 are used
for data analysis. The first library (43,488,788 paired-end
reads) is prepared by 3 glioma and paired normal brain
tissue. Forthesecondlibrary (54,732,199 paired-end reads),
ten human glioblastoma samples are mixed as tumor
group, and their periphery normal tissues are mixed as
control group. Total RNAs in the second library are
extracted and treated with RNase R to remove the linear
RNAs. The results of the second strategy are shown in
Additional file 1 (B. Real data: circRNADb with tissue
H9 hESCs). CircDBG finds more circRNAs recorded in
database than other tools, and it always gets the largest
coverage (20 of 23) in each chromosome respectively. Moreover, CircDBG is the best tool with the lowest bias
(contain majority results of other tools) and the fastest
running time. The results of other three strategies are
presented below. The length of reads is 150 bp in both libraries. Results Recall
that the intersection of the called circRNAs from two
libraries is used as the final result for each tool, and
circRNAs supported by at least 2 tools are viewed as
benchmark. Simulated data We use the latest simulator released by CIRI2 [8] to
simulate circRNAs and RNA-seq reads. The length of sim-
ulated paired-end reads is 101 bps, and the coverages of
circRNA and linear RNA are 10x and 80x respectively. The error rate is 1%. The major/minor normal distribution
insertion length is 320/550, and the percentage of splicing
for skipping exon is 40%. The reference and annotation file
come from human chromosome 1 (GRCh37, version 18). Our results are shown in Fig. 7. CircDBG has the highest
F1 score (0.9539). In addition, the accuracy of CircDBG is
98.65% with the highest sensitivity and the fastest running
time. The simulated paired-end reads contain 1,115,738
pairs. 295 circular RNAs are simulated as benchmark. Accuracy, sensitivity and F1 scores are calculated for each
tool. Real data: RNase R treated and untreated samples This comparison is performed with RNase R treated and
untreated libraries. We collect two sets of treated and
untreated reads from homo sapiens and mus musculus
respectively. A circRNA is viewed as reliable if it can
be found by both RNase R treated and untreated reads
(linear RNAs tend to degrade by RNase R treatment). SRR1636985 (treated, 13,309,745 paired-end reads) and
SRR1637089 (untreated, 44,933,450 paired-end reads)
from HeLa cells are used for human. As shown in Fig. 6, both accuracy and sensitivity of
CircDBG are around 94%, and it gets the highest F1 score
(0.9406). This means that CircDBG is the best tool for
balancing accuracy and sensitivity. CircDBG also has the
fastest running time. Real data: two different prepared libraries from same tissue
This comparison is based on two different libraries from
the same issue. Intuitively, true circRNAs should be called
for both libraries. We get the reliable results for each Real data: two different prepared libraries from same tissue
This comparison is based on two different libraries from
the same issue. Intuitively, true circRNAs should be called
for both libraries. We get the reliable results for each The length of reads in both libraries is 101 bps. For mouse libraries, SRR2219951 (treated, 22,330,976
paired-end reads) and SRR2185851 (untreated, 32,939,809 Fig. 6 F1 score and running time on human simulated data a F1 score. Dotted line represents a fixed F1 score. The closer to top right, the higher F1
score it is. b Running time (in minutes) Fig. 6 F1 score and running time on human simulated data a F1 score. Dotted line represents a fixed F1 score. The closer to top right, the higher F1
score it is. b Running time (in minutes) Page 8 of 11 Li and Wu BMC Genomics 2019, 21(Suppl 1):749 Fig. 7 F1 score and running time on human glioblastoma samples a F1 score. Dotted line represents a fixed F1 score. The closer to top right, the
higher F1 score it is. b CIRCExplorer takes more than 36 hrs and is not shown here Fig. 7 F1 score and running time on human glioblastoma samples a F1 score. Dotted line represents a fixed F1 score. The closer to top right, the
higher F1 score it is. b CIRCExplorer takes more than 36 hrs and is not shown here the results based on treated and untreated reads and build
the benchmark by choosing the circRNAs supported by at
least two tools. paired-end reads) are selected, which are prepared by
mouse brain at the age of 8 to 9 weeks. The length of reads in the two groups varies with the
maximum 100 bps. For each species, we obtain final
results for each tool by taking the intersection between As shown in Fig. 8, CircDBG has the highest F1 score
and the fastest running time in both human (F1 Score: Fig. 8 F1 score and running time on human and mouse samples a F1 score on human HEK293 real data b Running time on human HEK293 real
data. c F1 score on Mus Musculus real data and d Running time on Mus Musculus real data. CIRCExplorer takes 15 hrs and 21 hrs for human and
mouse respectively, and is not shown in b and d Fig. Case study: chimeric circular RNA We develop a novel evaluation scheme, which analyzes
what reads contribute to the calling of circRNAs. In this
scheme, CircRNA reference is generated by linking the
ending part of donor exon with starting part of acceptor,
and the circRNAs detected by CircDBG are classified into
5 categories based on the alignment result of reads. See
Additional file 1 (C. Classification of circRNA by reads)
for details. We notice that there is a special case when classify-
ing the detected circRNA with real data: some reads not
only support regular-circle case, but also support the self-
circle case for the exon contained by current regular-circle
case, which is illustrated in Fig. 9. This case may relate
to chimeric phenomenon in circular RNA which can be
comprehended as “circle in circle”. We collect this type of chimeric circular RNA in real
data and find this phenomenon exists in all real data we
analyze. For example, in H9 hESCs, 102 chimeric cases can
be found among the total 11,931 circular RNA. Although
the percentage seems to be small, the number of the
chimeric cases is significant. Regarding time consumption, CircDBG belongs to
k-mer-based method. Generally speaking, k-mer-based
tools perform better than reads-mapping-based tools. This is because reads-mapping-based tools are often com-
putationally inefficient, since mapping all reads can be
slow and many RNA-seq reads are irrelevant to circRNA. Differently k-mer-based tools only look for the circRNA-
related reads for detection, and they do not depend on
any third-party mapping tool. Compared with the existing
k-mer-based methods, CircDBG also outperforms oth-
ers. This is because the existing tools scan each k-mer
of the reads to extract their affiliated messages from
k-mer table, while circDBG only scan some of k-mers
sequentially from the reads by taking advantage of “brief-
path-donor” and “brief-path-acceptor” to find the qual-
ified paths of circRNA, which is much faster and more
reliable. Real data: two different prepared libraries from same tissue
This comparison is based on two different libraries from
the same issue. Intuitively, true circRNAs should be called
for both libraries. We get the reliable results for each 8 F1 score and running time on human and mouse samples a F1 score on human HEK293 real data b Running time on human HEK293 real
data. c F1 score on Mus Musculus real data and d Running time on Mus Musculus real data. CIRCExplorer takes 15 hrs and 21 hrs for human and
mouse respectively, and is not shown in b and d Page 9 of 11 Page 9 of 11 Li and Wu BMC Genomics 2019, 21(Suppl 1):749 0.9589) and mus musculus (F1 Score: 0.9424). The accu-
racy of CircDBG in human and mouse are 98.24% and
99.25% respectively. CircDBG and CircMarker are the top
two tools that get the highest sensitivity in both human
and mouse libraries. the unordered map is applied to store the graph, which is
different from other methods. Finally, CircDBG does not
scan the whole graph but only tracks the interesting path
to find potential circular RNA. The literature of the algorithm on creating de Bruijn
graph is broad [18–21], however, it is hard to compare the
performance of de Bruijn graph created by CircDBG sep-
arately with other state-of-art methods. This is because
the purpose of our de Bruijn graph is to find circular
RNA rather than doing normal assembly. Therefore, the
de Bruijn graph needs to be designed and used in a dif-
ferent way as we mentioned above to make it compatible
with the whole algorithm for circular RNA detection. The
high efficiency data structure introduced by other state-
of-art methods cannot be applied to detect circular RNA
directly. In addition, since CircDBG is the first algorithm
that uses de Bruijn graph for circular RNA detection, we
do not compare the performance of de Bruijn graph sep-
arately with others. Moreover, since our purpose is to
find circular RNA, we only compare the performance of
the whole algorithm with other similar methods for cir-
cular RNA detection. Based on comparison results, the
de Bruijn graph created by CircDBG could let the whole
algorithm perform better than others. Discussion Since all of circular RNAs that contain the exon shorter
than k-mer will be ignored by CircDBG, we calculate two
statistic results, including the number of exon shorter
than k-mer and the number of circular RNA called by
different algorithms that contains the exon shorter than k-
mer. The sample comes from RNase R treated real data of
HeLa cells (SRR1636985). The default length of k-mer is
15 bps. The results show that there are total 612294 exons
contained by annotation file (homo sapiens GRCh37.75),
among which 3652 exons are shorter than 15 bps. 12533
circular RNA are detected in total by the tools that we
used for comparison, and 6 circular RNA contain the exon
shorter than 15 bps. CircDBG sets 5 and 16 as the number of continuous k-
mers and the length of k-mer (as shown in equation 1)
respectively by default. Theoretically, we can increase or
decrease the number of continuous k-mers. If this value is
increased, some of circular RNA, which are supported by
the short part of reads (imbalance cases), may be missing. If this value is decreased, we may gain some false positives. Based on our empirical study, “K+5” (6 k-mers) could give
us the reasonable results. As similar as the number of con-
tinuous k-mers, the length of k-mer can also be changed. If the length is increased, some reads that do not match
well with the donor/acceptor site of circular RNA may be
discarded because the capability of error tolerance turns
to be weak. If the length is decreased, it may cause a large
number of unexpected repeats, which makes it’s hard for
us to identify the valid path in graph. Based on our testing,
15 bps length of k-mer could give us the reasonable result. CircMarker. Suppose we have an exon and a read as below,
and the length of k-mer is 5. Therefore, the total number
of k-mer in reads is 20, and the k-mer “GTGAT” repeats
three times. Acknowledgements g
We thank professor Ion Mandoiu for his discussions and help with this work. We thank professor Ion Mandoiu for his discussions and help with this work. ⎧
⎨
⎩
Exon : GTGATATGTGGGGTTGGTGATTTTCTCTGTGATCAGTGATGGG
Reads : GTGATATGGTGATGGGGTGATTTT ⎧
⎨
⎩
Exon : GTGATATGTGGGGTTGGTGATTTTCTCTGTGATCAGTGATGGG
Reads : GTGATATGGTGATGGGGTGATTTT For CircDBG, the maximum length of detected valid
path is 4, which means the maximum number of contin-
uous k-mer in exon detected by CircDBG is 4 (“GTGAT”,
“TGATA”, “GATAT” and “ATATG”). For CircMarker, since
we only consider how many k-mers of reads could be
found back in exon, the number of valid k-mer is 17,
which means there are 17 k-mers in reads could be found
back in exon. Since the matching status calculated by
CircDBG is 4 out of 20, which is too low, this match-
ing case (potential site of circular RNA) will be discarded
by CircDBG. This is correct because this read does not
well match the given exon. However, since the matching
status calculated by CircMarker is 17 out of 20, which
means most of k-mers from reads (without consider their
orders) could be found back in exon, CircMarker views
this read as the strong supporter of this exon, which is
incorrect. Since all of circular RNAs that contain the exon shorter
than k-mer will be ignored by CircDBG, we calculate two
statistic results, including the number of exon shorter
than k-mer and the number of circular RNA called by
different algorithms that contains the exon shorter than k-
mer. The sample comes from RNase R treated real data of
HeLa cells (SRR1636985). The default length of k-mer is
15 bps. The results show that there are total 612294 exons
contained by annotation file (homo sapiens GRCh37.75),
among which 3652 exons are shorter than 15 bps. 12533
circular RNA are detected in total by the tools that we
used for comparison, and 6 circular RNA contain the exon
shorter than 15 bps. Conclusion In this paper, we develop a new method called CircDBG
for circular RNA detection, which is based on de Bruijn
graph. The graph represents the relationship between k-
mers in original exon and reads. This contributes to more
accurate results and runs much faster compared with
the existing k-mer-based methods. CircDBG is the stand
alone tool and does not depend on any other third party
tools. CircDBG can be downloaded from: https://github. com/lxwgcool/CircDBG. Most of reads-mapping-based circular RNA detec-
tion methods are pipeline, which require other third-
party tools such as BWA, Bowtie and Tophat-fusion. As a result, the RAM usage for those methods is scat-
tered. Generally speaking, the k-mer-based methods, such
as CircMarker and CircDBG, consume more memory
than mapping-based approaches. However, since high-
performance computing (HPC) is wildly used to run
the software in the research field of bioinformatics, the
size of RAM that we can use in HPC is much larger
than personal computer. For our testing cases, we run
CircDBG in one computing node of HPC, and the
memory size of each computing node is 128 GB. The
peak RAM costs of CIRC2, Find-circ, CIRCExplorer,
CircRNAFinder, CircDBG and CircMarker are around
10.4%, 2.1%, 12.8%, 34.1%, 13.3% and 12.7% respectively
for the whole-genome analysis based on RNase R treated
sample of HeLa cells (SRR1636985, 13,309,745 paired-end
reads). Since the size of RAM in HPC is much larger
than what we need, sacrificing RAM to gain better perfor-
mance of analysis is reasonable. Supplementary information Supplementary information
Supplementary information accompanies this paper at
https://doi.org/10.1186/s12864-019-6154-7. Supplementary information
Supplementary information accompanies this paper at
https://doi.org/10.1186/s12864-019-6154-7. Additional file 1: Supplemental materials for “Detecting Circular RNA
from High-throughput Sequence Data with de Bruijn Graph”. Discussion CircDBG uses a specialized designed de Bruijn graph
to call circular RNA. First of all, the de Bruijn Graph
designed for regular usage, such as assembly, only con-
tains limited information. Differently, the graph created by
CircDBG contains a bunch of additional messages, such
as genome ID, transcript ID, its neighbors and part tag. Specifically, multiple groups of those messages will be
recorded if the k-mer appeared in multiple different exons
or multiple different positions in one exon. Secondly, in
order to speed up the whole algorithm, CircDBG does
not use all k-mers from exons to create the graph, and Fig. 9 The first and the second part of reads support the regular-circle RNA case from exon 3 back to exon 1, while the first and the third part of
reads support the self-circle RNA case of exon 3 Fig. 9 The first and the second part of reads support the regular-circle RNA case from exon 3 back to exon 1, while the first and the third part of
reads support the self-circle RNA case of exon 3 Page 10 of 11 Li and Wu BMC Genomics 2019, 21(Suppl 1):749 CircDBG sets 5 and 16 as the number of continuous k-
mers and the length of k-mer (as shown in equation 1)
respectively by default. Theoretically, we can increase or
decrease the number of continuous k-mers. If this value is
increased, some of circular RNA, which are supported by
the short part of reads (imbalance cases), may be missing. If this value is decreased, we may gain some false positives. Based on our empirical study, “K+5” (6 k-mers) could give
us the reasonable results. As similar as the number of con-
tinuous k-mers, the length of k-mer can also be changed. If the length is increased, some reads that do not match
well with the donor/acceptor site of circular RNA may be
discarded because the capability of error tolerance turns
to be weak. If the length is decreased, it may cause a large
number of unexpected repeats, which makes it’s hard for
us to identify the valid path in graph. Based on our testing,
15 bps length of k-mer could give us the reasonable result. Abbreviations BWA: Burrows-Wheeler aligner; CircRNA: Circular RNA; HPC: High-performance
computing; RAM: Random access memory; RNA: Ribonucleic acid Availability of data and materials 19. Chikhi R, Rizk G. Space-efficient and exact de bruijn graph representation
based on a bloom filter. Algoritm Mol Biol. 2013;8(1):22. 19. Chikhi R, Rizk G. Space-efficient and exact de bruijn graph representation
based on a bloom filter. Algoritm Mol Biol. 2013;8(1):22. The software is available under the GPLv3 licence at https://github.com/
lxwgcool/CircDBG. The software is available under the GPLv3 licence at https://github.com/
lxwgcool/CircDBG. 20. Belazzougui D, Gagie T, Mäkinen V, Previtali M, Puglisi SJ. Bidirectional
variable-order de Bruijn graphs. In: LATIN 2016: Theoretical Informatics. Springer; 2016. p. 164–178. The sample data sets used in this paper are available at European Nucleotide
Archive (https://www.ebi.ac.uk/ena) with identification numbers SRR4095542,
SRR5133906, SRR1636985, SRR1637089, SRR2219951, SRR2185851 and
SRR901967. The sample data sets used in this paper are available at European Nucleotide
Archive (https://www.ebi.ac.uk/ena) with identification numbers SRR4095542,
SRR5133906, SRR1636985, SRR1637089, SRR2219951, SRR2185851 and
SRR901967. 21. Belazzougui D, Gagie T, Mäkinen V, Previtali M. Fully dynamic de Bruijn
graphs. In: International Symposium on String Processing and
Information Retrieval. Springer; 2016. p. 145–152. 21. Belazzougui D, Gagie T, Mäkinen V, Previtali M. Fully dynamic de Bruijn
graphs. In: International Symposium on String Processing and
Information Retrieval. Springer; 2016. p. 145–152. The circular RNA database used in this paper is available at circRNADb (http://
202.195.183.4:8000/circrnadb/circRNADb.php). The circular RNA database used in this paper is available at circRNADb (http://
202.195.183.4:8000/circrnadb/circRNADb.php). The reference data and annotation files used in this paper are available at
ensembl (http://useast.ensembl.org/index.html). The reference data and annotation files used in this paper are available at
ensembl (http://useast.ensembl.org/index.html). About this supplement pp
This article has been published as part of BMC Genomics, Volume 20 This
article has been published as part of BMC Genomics, Volume 21 Supplement
1, 2020: Selected articles from the 14th International Symposium on
Bioinformatics Research and Applications (ISBRA-18): genomics. The full
contents of the supplement are available at https://bmcgenomics. biomedcentral.com/articles/supplements/volume-21-supplement-1 Finally, since both CircDBG and CircMarker belong to
k-mer-based method, there is an example to demonstrate
CircDBG could avoid some false positives compare with Page 11 of 11 Page 11 of 11 Li and Wu BMC Genomics 2019, 21(Suppl 1):749 Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Funding
h
k g
This work is supported in part by grants IIS-1447711, IIS-1526415 and
CCF-1718093 from US National Science Foundation to YW. The publication
costs are funded by IIS-1526415 and CCF-1718093 from US National Science
Foundation. Funding agency has no role in design of the study and collection,
analysis, and interpretation of data and in writing the manuscript 17. Glažar P, Papavasileiou P, Rajewsky N. circbase: a database for circular
rnas. RNA. 2014;20(11):1666–70. 17. Glažar P, Papavasileiou P, Rajewsky N. circbase: a database for circular
rnas. RNA. 2014;20(11):1666–70. 18. Bowe A, Onodera T, Sadakane K, Shibuya T. Succinct de Bruijn graphs. In:
International Workshop on Algorithms in Bioinformatics. Springer; 2012. p. 225–35. 18. Bowe A, Onodera T, Sadakane K, Shibuya T. Succinct de Bruijn graphs. In:
International Workshop on Algorithms in Bioinformatics. Springer; 2012. p. 225–35. Received: 9 September 2019 Accepted: 30 September 2019
Published: 5 March 2020 Received: 9 September 2019 Accepted: 30 September 2019
Published: 5 March 2020 Authors’ contributions
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Characterizing the social support and functioning of a low-threshold medication for opioid use disorder treatment cohort at intake
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Abstract Background: Despite the growing morbidity and mortality rates associated with opioid use disorder, a large gap
still exists between treatment need and capacity. Low-threshold clinics utilizing medication for opioid use disorder
(MOUD) treatment can increase treatment access but are understudied, and little is known about how patient demo-
graphic characteristics are associated with their social support and functioning in these settings. Methods: We used multivariate regression to estimate associations between demographic characteristics and self-
reported social support or functioning indicators among patients receiving MOUD in a low-threshold clinic using
several validated instruments administered at intake: Behavior and Symptom Identification Scale, Brief Pain Inventory,
and Life Events Checklist for DSM-5. Patients initiating MOUD treatment between April 1 and December 31, 2017, with
complete surveys were included (N=582). Results: Patients were primarily male (62%), aged 34 or older (53%), non-Hispanic White (79%), separated or not mar-
ried (86%), and unemployed (64%). Over 20% did not live in a house or apartment in the past month. Women were
more likely to “get along” with people outside their family or in social situations and to identify their partner as their
source of support. Women, non-White, and older patients were at higher risk of social functioning-disrupting events
(physical/sexual assaults or experiencing chronic pain), while employment and housing were protective against
exposure to these trauma-related events. However, employment and housing also decreased the odds of talking with
others about substance use. The aforementioned results were obtained from multivariate logistic regression models
and were significant to p<0.05. Conclusions: Variation in support and functioning by demographic characteristics suggests that treatment facilities
may benefit from adopting strategies that take baseline disparities in support and functioning into account. rds: Medication for opioid use disorder, Social support, Social functioning, Low-threshold model © 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. Open Access William Oles1†, Marcus Alexander1*†, Navin Kumar1,2, Benjamin Howell3,4, Patrick G. O’Connor5,
Lynn M. Madden5,6 and Declan T. Barry6,7 William Oles1†, Marcus Alexander1*†, Navin Kumar1,2, Benjamin Howell3,4, Patrick G. O’Connor5,
Lynn M. Madden5,6 and Declan T. Barry6,7 Oles et al. BMC Psychiatry (2022) 22:236
https://doi.org/10.1186/s12888-022-03884-5 Oles et al. BMC Psychiatry (2022) 22:236
https://doi.org/10.1186/s12888-022-03884-5 Characterizing the social support
and functioning of a low‑threshold medication
for opioid use disorder treatment cohort
at intake William Oles1†, Marcus Alexander1*†, Navin Kumar1,2, Benjamin Howell3,4, Patrick G. O’Connor5,
Lynn M. Madden5,6 and Declan T. Barry6,7 William Oles1†, Marcus Alexander1*†, Navin Kumar1,2, Benjamin Howell3,4, Patrick G. O’Conno
Lynn M. Madden5,6 and Declan T. Barry6,7 Background Drug overdose deaths in the United States (US) have
increased in the past two decades, with overdoses involv-
ing illicit and prescription opioids being the primary
driver of the epidemic [1]. Across the 12-month period
ending in January 2021, the CDC reported over 94,000 *Correspondence: marcus.alexander@yale.edu
†William Oles and Marcus Alexander contributed equally to this work. 1 Human Nature Lab, Yale Institute for Network Science, Yale University,
New Haven, CT, USA
Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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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. Oles et al. BMC Psychiatry (2022) 22:236 Page 2 of 10 and stress [19]. Two such examples are physical/sexual
assault and chronic pain experiences, both of which can
have lasting impacts on people’s ability to meet their life’s
responsibilities and engage in support networks [20, 21]. drug overdose deaths, an increase of approximately 30%
compared to the previous 12-month period [2]. In the
wake of disruptions to health services and social distanc-
ing that have followed the COVID-19 pandemic, rates of
opioid use and overdose-related morbidity and mortality
have accelerated, with the largest monthly increases in
drug overdose deaths since 2015 occurring in the months
following statewide lockdown responses to COVID-19
[2, 3]. Although some studies have investigated the role of
social support in MOUD treatment [14–16], fewer stud-
ies have related both positive and negative indicators of
social support and functioning to patient demographic
characteristics at treatment initiation, and no study has
done so in a low-threshold treatment setting. While
we make no prior assumptions about how patients in a
low-threshold treatment setting may differ from other
populations, we believe the current study adds value by
extending the investigation of social support and func-
tioning in the literature on MOUD to low-threshold
treatment settings. This study utilizes validated instru-
ments administered at MOUD initiation over the course
of nine months. The aims of the study are twofold: 1) to
describe the demographic characteristics of a cohort of
patients receiving MOUD in a low-threshold setting,
and 2) to relate the cohort’s demographic characteristics
to both positive and negative indicators of social sup-
port and social functioning. This analysis will allow cli-
nicians and other administrators in the MOUD space to
gain an idea of the support needs of their patient popula-
tions, which can not only strengthen existing programs
through mechanisms of quality improvement but also
expand planning for the scope of future MOUD treat-
ment programs. Medications for opioid use disorder (MOUD)—such
as methadone, buprenorphine, or buprenorphine-nalox-
one—curb opioid cravings and decrease overdose events
[4]. MOUD treatment programs have become a key pub-
lic health strategy in combatting the opioid epidemic
[5, 6], particularly during the COVID-19 pandemic [7]. Despite the efficacy of MOUD, a significant gap has been
estimated between treatment need and treatment capac-
ity for opioid use disorder nationwide [8, 9]. The gap
exists, in part, due to barriers to access, such as health
insurance coverage, waiting lists, lack of transportation,
and treatment stigma [10]. Barriers exist on the pre-
scriber side as well, with physicians facing prior authori-
zation requirements from insurance companies and
waiver training mandates from the federal government
in order to prescribe many MOUD therapies [11]. Low-
threshold treatment models seek to directly address bar-
riers to care, and therefore reduce the treatment gap, in a
number of ways. In contrast to traditional treatment set-
tings, which may have rigid requirements for admission
and continuation, low-threshold treatment clinics often
offer same-day treatment entry, flexible medication for-
mulations, and endorse a harm reduction approach that
does not require abstinence [12]. Data source and sample p
Data from 588 MOUD treatment patients was gath-
ered through surveys administered at intake to the APT
Foundation in New Haven, Connecticut (CT). APT
Foundation is a private, non-profit, community-based
organization that specializes in treatment of opioid use
disorder and which organizes a low-threshold program
by walk-in admission, having locations accessible to pub-
lic transportation, and treating those underinsured or
uninsured [22]. Of the 588 patients, 582 had complete
survey responses, with one record missing ethnicity, two
records missing marital status, and three records missing
employment status after data processing. Patients using
MOUD at APT Foundation in New Haven were asked
to complete a set of validated instruments at treatment
initiation, including the Behavior and Symptom Identifi-
cation Scale (BASIS-24), Brief Pain Inventory (BPI), and
Life Events Checklist for DSM-5 (LEC-5). Records of
all complete patient intakes from April 1, 2017 through
December 31, 2017 were included. Survey measures were
selected for analysis from each instrument by experts
specializing in substance use disorders (authors BH, Social support—which can be conceptualized as emo-
tional or tangible resources shared within social relation-
ships including expressions of sympathy, offers of advice,
and physical/financial aid [13]—is associated with good
health outcomes generally and substance use disorder
treatment outcomes specifically. Among people who use
opioids, perceived support from family or peers is related
to earlier treatment seeking and longer MOUD treatment
adherence [14–16]. Engaging in networks of social sup-
port is one domain of a broader concept of social func-
tioning—the ability to “successfully engage with life and
fulfill personal roles” in society [17]. The disruption of
social functioning in the settings of work, school, home,
or recreational life is a DSM-5 diagnostic criterion of opi-
oid use disorder [17, 18], and improving patients’ abilities
to meet responsibilities in these settings is often a target
of MOUD programs. Impairment of social functioning
may not only be a consequence of long-term opioid use
but a precursor, as said disruptions are also intertwined
with the experience of and coping with traumatic events Oles et al. BMC Psychiatry (2022) 22:236 Oles et al. BMC Psychiatry (2022) 22:236 Oles et al. BMC Psychiatry (2022) 22:236 Page 3 of 10 Table 1 APT
Foundation
2017
cohort
demographic
characteristics (N=582 complete survey). Residential status BASIS-24 is a measure of self-reported psychiatric dis-
tress and functioning and can be used as a metric of
mental health treatment outcomes [23]. The instrument
has been demonstrated to be reliable (internal consist-
ency α values ranged from 0.75 to 0.91) and discrimi-
nately valid in clinical populations [24]. Responses were
drawn from three of the six survey domains (Depres-
sion and functioning, Interpersonal relationships, and
Substance use). We also drew on BASIS-24 responses
to abstract demographic information, including sex,
age, race, ethnicity, marital status, employment status,
and past-month residential status. For the purposes of
analysis, we recoded sex to female/not female, race to
non-White/White, ethnicity to Hispanic/non-Hispanic,
marital status to married/not married, employment
status to unemployed/part-time/full-time, and residen-
tial status to lived in house or apartment/did not live
in house or apartment. The decision to encode demo-
graphic information in this way was made to reduce the
number of comparisons and ensure model convergence
given the unbalanced distribution of responses across
the study cohort (Table 1). Wording for the original
questions and answer choices is summarized in Supple-
mental Table 1 (see Additional file 1). positive indicators of social functioning and were rated
on a 5-item Likert scale ranging from “None of the time”
to “All of the time”. It is worth noting that labels of ‘posi-
tive’ and ‘negative’ social support or functioning were
assigned by experts who specialize in substance use dis-
orders and are familiar with survey instrument structure
(authors BH, PGO, LMM, DTB). These labels are meant
to help conceptualize the expected valence of each meas-
ure. For example, reporting getting along with one’s fam-
ily suggests a higher quality social support or functioning
in that domain per it’s inclusion in the ‘Interpersonal
relationships’ BASIS-24 subscale and is therefore consid-
ered a positive indicator in the current study. Additionally, we chose to analyze questions about how
often the respondent felt close to another person, felt
that they had someone to turn to for help, tried to hide
their drinking or drug use, and how often others talked
to them about their drinking or drug use, which were
rated on a 5-item Likert scale ranging from “Never” to
“Always”. Data source and sample Overall, % (N)
Sex
Female
38.1 (222)
Male
61.9 (360)
Age in years, Mean (SD)
36.8 (10.5)
Race
White
79.0 (460)
Non-White
21.0 (122)
Ethnicity
Non-Hispanic
87.5 (509)
Hispanic
12.5 (73)
Marital status
Not married
85.7 (499)
Married
14.3 (83)
Employment status
Unemployed
64.0 (372)
Part-time
16.1 (94)
Full-time
19.9 (116)
Residential status
Didn’t live in house/apartment past 30 days
22.0 (128)
Lived in house/apartment past 30 days
78.0 (454) PGO, LMM, DTB), who made selections based on each
measure’s pertinence to patient social support or social
functioning. Although data was collected for each instru-
ment in full as part of a standardized intake protocol,
we chose to analyze individual survey items, rather than
composite survey scores, since the composite scores
on the whole were not necessarily designed to measure
social support or functioning. Ethical considerationsh The study involving a medical chart review was approved
by the APT Foundation Board of Directors as well as the
Human Investigations Committee at the Yale School of
Medicine. All participants provided informed consent for
data collection. All methods were performed in accord-
ance with the relevant guidelines and regulations set
forth by the APT Foundation Board of Directors and the
Human Investigations Committee at the Yale School of
Medicine. Residential status Participants also indicated from a list which
relationship was their main source of social support,
which was recoded into three binary variables: main
source of support is a partner, main source of support is
family, or main source of support is a friend. The ques-
tion about how often respondents tried to hide their
drinking or drug use was reverse coded and interpreted
as how often they did not hide their use from others, and i
In addition to individual demographic characteristics,
eight survey questions were chosen for analysis from
BASIS-24 which are also summarized in Supplemental
Table 1 (see Additional file 1). Each item is based on the
past week of the respondent’s life and includes questions
about the amount of time the respondent got along with
family, got along with those outside their family, and got
along in social situations, all of which were considered Oles et al. BMC Psychiatry (2022) 22:236 Oles et al. BMC Psychiatry (2022) 22:236 Page 4 of 10 gathered in the intake survey (authors BH, PGO, LMM,
DTB): sex, age, race, ethnicity, marital status, employ-
ment status, and residential status. Six participant
records (1%) were excluded after data cleaning for the
purposes of our analysis due to missing demographic
data. Missingness in indicators of social support and
functioning varied but was less than 1% for any given
indicator (Supplemental Table 1, see Additional file 1). Each model was compared to a null with no covariates
to assess improved goodness of fit in predictions, which
was confirmed by significant reduction in deviance for
all models (Supplemental Tables 3 and 4, see Additional
file 2). Statistical significance was set at p<0.05. Statistical
analyses were performed using R version 3.5.2. these set of questions were considered positive indicators
of social support. It should be noted, however, that being
talked to by others about drinking of drug use is not
always a positive indicator of support and that the impact
of being talked to about those matters is often context-
and relationship-dependent. The BPI was designed to assess the severity of physical
pain and the impact of pain on daily functioning [25]. The
instrument has been used in a number of clinical settings
and is shown to be valid and reliable (internal consist-
ency α values ranged from 0.83 to 0.96) [26]. Group characteristics Of 588 patients entering a low-threshold MOUD treat-
ment program, 582 had complete baseline surveys and
were considered for analysis. 222 (38%) were female, 460
(79%) were White, 73 (13%) were Hispanic, and the aver-
age age was 36.8 (SD=10.5). Additionally, 499 patients
were not married (86%), 372 were unemployed (64%),
and 128 (22%) reported not residing in a house or apart-
ment in the past month. The demographic characteristics
for the sample are summarized in Table 1. Significant
adjusted odds ratios showing changes in probability of
both positive and negative indicators of social support
and functioning by demographic characteristics are sum-
marized in Fig. 1. All results are reported in Supplemen-
tal Tables 3 and 4 (see Additional file 2). Residential status We assessed
a single measure, rated 0–10, which asked how often
pain interfered with the respondents’ relationships with
people in the past week as a negative indicator of social
functioning.h Results The LEC-5 was developed at the National Center for
PTSD in tandem with the Clinician Administered PTSD
Scale (CAPS) to assess exposure to potentially traumatic
events [27], and it has been shown to be both valid and
reliable as a measure of trauma history (intraclass cor-
relation coefficients ranged from 0.62 to 0.64 for events
experienced) [28]. For each traumatic event, patients
indicated their experience on a nominal scale, which
we recoded as a binary variable indicating whether the
respondent reported that the event “Happened to me”
(Supplemental Table 1, see Additional file 1). We selected
five items from the LEC-5 that are negative predictors of
social functioning: physical assault, assault with weapon,
sexual assault, unwanted sexual experience, and the sud-
den and unexpected death of someone close. Positive social support and social functioning indicators Note: Age scaled up by a
factor of 10 those employed full-time were 36% less likely to report
pain interference (aOR=0.64, 95% CI=0.42–0.96). Each
additional 10 years in age was associated with a 20%
increase in likelihood of pain interfering with social rela-
tionships (aOR=1.20, 95% CI=1.04–1.39). drug or alcohol use from others (Part-time employment:
aOR=0.49, 95% CI=0.31–0.75; Full-time employment:
aOR=0.43, 95% CI=0.28–0.65), while living in a house or
apartment was associated with 49% decrease in the likeli-
hood that the patient reported not hiding their use from
others (aOR=0.51, 95% CI=0.35–0.76). Living in a house or apartment was associated with
a 55% decrease in the likelihood of experiencing sex-
ual assault (aOR=0.45, 95% CI=0.27–0.75) and a 49%
decrease in the likelihood of having an unwanted sex-
ual experience (aOR=0.51, 95% CI=0.30–0.85). Those
that resided in a house or apartment were also 42%
less likely to report being assaulted with a weapon than
those that did not (aOR=0.58, 95% CI=0.38–0.89). Hav-
ing a full-time job was associated with a 59% decrease
in the probability of having an unwanted sexual experi-
ence (aOR=0.41, 95% CI=0.18–0.85). Those with a full-
time job were 49% less likely than those unemployed to
report experiencing physical assault (aOR=0.51, 95%
CI=0.32–0.80). Patients also provided information about their pri-
mary source of social support. Women (aOR=1.86, 95%
CI=1.26–2.77), those that lived in a house or apartment
(aOR=1.70, 95% CI=1.04–2.86), and those that were
married (aOR=14.25, 95% CI=7.80–27.71) were about
1.9, 1.7, and 14.3 times more likely to list their partner
as their source of support, respectively. Each additional
10 years in a patient’s age was associated with a 21%
decrease in the probability they reported having family as
their primary support (aOR=0.79, 95% CI=0.66–0.93). Negative social support and social functioning indicators
Women were about 67% more likely than men to report
experiencing physical assault (aOR=1.67, 95% CI=1.18–
2.37) and over 10 times as likely to report sexual assault
(aOR=10.76, 95% CI=6.81–17.37) or another unwanted
sexual experience (aOR=10.40, 95% CI=6.53–17.06). Women were also 2.3 times more likely than men to
have experienced the sudden death of someone they
knew (aOR=2.25, 95% CI=1.31–3.93), while non-White
patients were 83% less likely than White patients to have
experienced the sudden death of another (aOR=0.17,
95% CI=0.02–0.69). Discussionh This study extends the current literature by identifying
associations between demographic characteristics and
positive and negative indicators of social support and
functioning among patients entering a low-threshold
MOUD program. The probability of reporting trauma
experiences, social functioning disruptions, or poor net-
work support varied across the demographic subgroups. Positive social support and social functioning indicators Non-White patients were about 2.1
times more likely than White patients to report that they
felt their experiences of pain interfered with their social
relationships (aOR=2.13, 95% CI=1.25–3.62), while Positive social support and social functioning indicators The objectives of our analysis were to summarize patient
characteristics and to identify which characteristics are
associated with reports of social support and function-
ing. Descriptive statistics (means, standard deviations,
proportions) were calculated to examine the cohort’s
demographic characteristics. For social support or func-
tioning measures that are reported as a frequency, we
employed multivariate ordinal logistic regression, which
allowed us to model the log odds of each change in the
response scale as a linear combination of key demo-
graphic characteristics (Supplemental Table 3, see Addi-
tional file 2). For social support or functioning measures
about support relationships or experiencing a traumatic
event, we employed multivariate binomial logistic regres-
sion, which models the log odds of the binary relation-
ship or event with a linear combination of covariates
(Supplemental Table 4, see Additional file 2). Results
are reported as odds ratios to ease interpretability. Each
model included the following covariates, which were
selected by our panel of experts specializing in substance
use disorders based on the availability of information Positive social support and social functioning indicators
Patients with full-time jobs were 32% less likely than
those unemployed to report getting along with their
family (adjusted odds ratio [aOR]=0.68, 95% confidence
interval [CI]=0.46–0.997), and women were 36% more
likely than men to report getting along with people out-
side their family (aOR=1.36, 95% CI=1.002–1.86) and
60% more likely to report getting along in social situa-
tions (aOR=1.60, 95% CI=1.18–2.18). Married patients
were 41% less likely to report feeling close to another per-
son compared to non-married patients (aOR=0.59, 95%
CI=0.38–0.91). Concerning employment and age, those with full-time
jobs were 45% less likely to report being talked to about
their alcohol or drug use (aOR=0.55, 95% CI=0.37–
0.82), and each additional 10 years in a patient’s age was
associated with a 22% decrease in the likelihood that
they reported being talked to about their alcohol or drug
use by others (aOR=0.78, 95% CI=0.67–0.91). Being
employed was associated with at least a 51% decrease
in the likelihood that a patient reported not hiding their Oles et al. BMC Psychiatry (2022) 22:236 Page 5 of 10 Fig. 1 Significant adjusted odds ratios of indicators of social support and functioning by demographic characteristics. Note: Age scaled up by a
factor of 10 Fig. 1 Significant adjusted odds ratios of indicators of social support and functioning by demographic characteristics. Sex Sixty-two percent of our cohort was male. We noted
differences by sex in self-reported social support and Oles et al. BMC Psychiatry (2022) 22:236 Oles et al. BMC Psychiatry (2022) 22:236 Page 6 of 10 functioning. Women were more likely than men to
report getting along with people outside their family or
in social situations and were also more likely to identify
their partner as their primary source of support, despite
similar rates of marriage between men and women. This
may indicate that men initiating MOUD treatment are
less likely to be engaged with a social support network. Women are more likely than men to rely on social sup-
port to manage problems and emotional stress, and they
are also more likely to have a larger selection of drug-
free network support when receiving MOUD treatment
[29]. However, severe loneliness has been associated
with higher odds of illicit opioid use among women in
MOUD treatment programs while the same effect was
not observed for men, suggesting that lack of social sup-
port may have a disproportionate impact [30]. Addition-
ally, our results are consistent with a more general finding
that women experiencing OUD are more likely to have
experienced past physical, sexual, or emotional traumas
[31]. More work is needed to link the differences by sex
in social support and functioning to potential disparities
in MOUD treatment outcomes and to understand how
MOUD treatment providers might leverage this evidence
to tailor support network and trauma-informed care pro-
gramming to their patient populations. both diagnosis and treatment and maintaining emotional
support throughout the treatment timeline. MOUD
treatment providers might consider incorporating strat-
egies to build support networks for patients experienc-
ing social isolation and to address potentially modifiable
factors precipitating isolation, such as living arrange-
ment and social participation [35]. Finally, our results
also highlight the need for future work to address the
role of chronic pain management among older patient
populations. Race and ethnicityh The majority of our cohort was White and non-Hispanic;
however, the proportion of patients who self-identified
as Hispanic (13%) was greater than other traditional
and low-threshold MOUD programs [32, 33]. However,
no social support or functioning indicators examined in
the current study varied significantly by Hispanic ethnic-
ity, suggesting parity between those identifying as His-
panic and non-Hispanic in measures of social support
and functioning when adjusting for other demographic
characteristics. Compared to White patients, non-White patients were
more likely to report that pain interfered with their rela-
tionships but less likely to have experienced the sudden
death of someone they knew. The finding of disparity
in pain interference is consistent with recent studies of
patients experiencing chronic pain, which found that
Black patients were significantly more likely than White
patients to report pain interference on the BPI and that
this disparity was moderated by perceived social sta-
tus [36]. Racial disparities in the experience of sudden
death in this setting have not been studied. Our finding
that non-White patients were less likely to have experi-
enced the sudden death of someone they knew may be
explained by the greater rates of opioid-related mortality
among White individuals compared to non-White indi-
viduals [37]. However, this result should also be inter-
preted with caution. Socioeconomic status has been
shown to mediate the effects of race on probability of
mortality, and since the time of data collection for this
study, racial disparities in mortality have widened paral-
lel to the progression of the COVID-19 pandemic [37,
38]. Black individuals experiencing opioid use disorder
are less likely to be initiated on MOUD treatment com-
pared to White individuals [39], and among those receiv-
ing MOUD treatment, Black individuals have lower odds
of being retained in treatment for at least one year or
completing treatment [40, 41]. These racial disparities
in MOUD treatment are driven by structural racism and
systemic inequities which lead to differential diagnosis
and treatment of non-White patients, so we interpret our Ageh The mean age of our cohort was 36.8 years and about 13%
were over the age of 50, which is a smaller proportion of
older patients than other traditional and low-threshold
MOUD programs [32, 33]. Although older patients are
typically insured and have similar if not better treatment
outcomes as younger patients, older adults who use opi-
oids are less likely to perceive their drug use as problem-
atic and initiate treatment [31, 34]. Additionally, older
patients in low-threshold MOUD programs may have dif-
ferent treatment needs than both older patients in other
settings and younger patients in low-threshold programs. We found that as the age of patients increased, the
probability of someone talking to them about substance
use or having family as primary social support decreased,
while the probability of pain interfering in their relation-
ships increased. Our findings regarding conversations
about substance use and family as social support are
consistent with broader epidemiological data showing a
higher prevalence of social isolation among older adults
relative to younger adults [35]. Given the surge in older
adults seeking treatment for OUD in the United States
[34], future work might focus on connecting the impact
of social isolation among older adults to MOUD treat-
ment outcomes and identifying the specific challenges
imposed by isolation such as accessing healthcare for The mean age of our cohort was 36.8 years and about 13%
were over the age of 50, which is a smaller proportion of
older patients than other traditional and low-threshold
MOUD programs [32, 33]. Although older patients are
typically insured and have similar if not better treatment
outcomes as younger patients, older adults who use opi-
oids are less likely to perceive their drug use as problem-
atic and initiate treatment [31, 34]. Additionally, older
patients in low-threshold MOUD programs may have dif-
ferent treatment needs than both older patients in other
settings and younger patients in low-threshold programs. We found that as the age of patients increased, the
probability of someone talking to them about substance
use or having family as primary social support decreased,
while the probability of pain interfering in their relation-
ships increased. Our findings regarding conversations
about substance use and family as social support are
consistent with broader epidemiological data showing a
higher prevalence of social isolation among older adults
relative to younger adults [35]. Employment statush The majority of our cohort was unemployed. A greater
proportion of women were unemployed relative to men. Both full- and part-time employment were associated
with hiding one’s drinking or drug use from others, while
full-time employment was also associated with not get-
ting along with one’s family and not being talked to by
others about substance use. These associations suggest
that holding a job may disincentivize drug use disclosure,
possibly due to stigma or fear of retribution by employers
or because there are fewer opportunities to talk with oth-
ers due to time spent at work. However, full-time employment was also negatively
associated with reports of experiencing physical assault
or unwanted sexual experiences. Since employment is a
source of income and structure, it is often a target out-
come of MOUD treatment programs as a way to bol-
ster social functioning and has been associated with Ageh Given the surge in older
adults seeking treatment for OUD in the United States
[34], future work might focus on connecting the impact
of social isolation among older adults to MOUD treat-
ment outcomes and identifying the specific challenges
imposed by isolation such as accessing healthcare for Oles et al. BMC Psychiatry (2022) 22:236 Oles et al. BMC Psychiatry (2022) 22:236 Page 7 of 10 improved outcomes such as heroin abstinence and longer
retention in treatment [41, 45, 46]. Consequently, assist-
ing patients with obtaining employment is a potentially
important target for MOUD treatment programs [47]. While our study provides no evidence for causality, the
finding that employment was associated with not getting
along with one’s family and hiding substance use from
others warrants further consideration by those develop-
ing employment-based interventions. Additionally, exist-
ing literature has primarily focused on linking patient
employment with MOUD treatment outcomes, with less
work exploring how employment is related to measures
of social support or functioning among patients using
MOUD. Future work may provide further support for the
efficacy of employment as a target outcome by investigat-
ing the psychosocial causes and consequences of employ-
ment among individuals using MOUD. finding of differences in pain interference with relation-
ships in the context of other structurally-borne barriers
to treatment success for non-White patients. Future work
might further investigate how racial disparities in social
support or functioning are related to treatment outcomes
and how structural barriers to care can be addressed in
the MOUD treatment setting. Residential status Twenty-two percent of our cohort reported not living in a
house or apartment in the past month, which represents
a greater proportion than some other traditional and
low-threshold MOUD programs [32, 33]. Additionally,
of the remainder of our cohort who reported living in a
house or apartment, nearly 7% reported living in multiple
locations in the past month, suggesting that the preva-
lence of unstable housing may be underestimated. People
experiencing transient housing or chronic homelessness
are among the most socially disenfranchised groups
experiencing OUD and are therefore a target of low-
threshold treatment models. Patients receiving MOUD
who resided in a house or apartment in the past month
were more likely to report having a partner as a source
of social support and less likely to report not hiding
their substance use from others, ever be assaulted with a
weapon, ever be sexually assaulted, or ever have another
unwanted sexual experience. Our findings suggest that
those experiencing past-month housing instability seem
less likely to have a support network and more likely to
have experienced physical and sexual trauma. These
results corroborate a larger literature relating the lack of
social support and unstable housing to drug use and psy-
chiatric comorbidities [48]. Accessible MOUD treatment
approaches tailored to this subgroup are needed and have
the potential to improve outcomes by increasing treat-
ment access and providing opportunities to build drug-
free networks [48, 49]. Marital status A small minority of our cohort was married. We found
that married patients were more likely to indicate their
partner as their primary source of support, but less likely
to report feeling close to another person. While no other
study has investigated differences in perceived closeness
to others among a population of married patients receiv-
ing MOUD, recent work using a sample of heterosexual
couples has shown that relationship quality is negatively
correlated with MOUD-treatment-related stress for both
men and women, and that women’s positive family and
friend ties outside of the partnership are linked to lower
stress for both partners [42]. Other work has also indi-
cated that marriage decreases the odds of patients using
cocaine or heroin during treatment and that the effect is
strengthened if there is a high level of perceived close-
ness to the partner in the marriage [43]. This association
has provided a basis for the development of behavioral
couples therapy interventions in substance use disorder
treatment settings, many of which have shown promise
with respect to treatment outcomes [44]. Our results
indicate that partnerships play an important role in per-
ceived support and that interventions framed around
increasing trust, understanding, and support among cou-
ples might further improve treatment outcomes. Limitations Our study has several limitations. Data for this study
comes from surveys administered as part of a standard-
ized intake process rather than for the investigation of Oles et al. BMC Psychiatry (2022) 22:236 Oles et al. BMC Psychiatry (2022) 22:236 Page 8 of 10 our research question. Survey measures were then cho-
sen retrospectively for analysis based on their relevance
to this study. Future studies may utilize a broader range
of validated instruments. The survey items chosen for
this study were obtained from validated instruments that
each have a unique composite scoring system; however,
we chose to analyze survey items individually outside of
the context of their parent instruments’ composite scores. Our analysis was cross-sectional, and further work is
needed to determine causal relationships. We assessed
associations between social support, functioning, and
various demographic factors but did not test the asso-
ciation between such factors and treatment outcomes. Additionally, while our finding of differences in social
support and functioning by demographic characteristics
may have implications for ongoing trends in opioid use
and overdose during the COVID-19 pandemic, data for
this study was collected in 2017 and should therefore be
interpreted in this context. Future work might also use an
intersectional approach to investigate systematically the
interactions between demographic characteristics and
social support and functioning among patients receiving
MOUD. Finally, although we attempt to adjust for a vari-
ety of demographic characteristics, the majority of our
cohort was non-Hispanic White and represents a single
clinic. Therefore, our findings should be read in-line with
these generalizability limitations. LEC-5: Life Events Checklist for DSM-5; CAPS: Clinician Administered PTSD
Scale. Conclusions Our low-threshold MOUD cohort represented demo-
graphic subgroups that traditionally experience social dis-
enfranchisement, such as those with past-month housing
instability. Even when controlling for other demographic
covariates, both positive and negative indicators of social
support and functioning varied significantly by sex, age,
race, marital status, employment, and residential status. Variation in support and functioning by demographic
characteristics suggests that MOUD treatment programs
may benefit from adopting strategies which take base-
line disparities in support and functioning into account
and generate desired treatment outcomes by aligning
therapies and support resources offered with their patient
populations. Future work may benefit from linking meas-
ures of social support and functioning among patients
receiving MOUD to treatment outcomes to gain a more
complete picture of how each is related to the treatment
timeline. Availability of data and materials The datasets generated during and analyzed during the current study are not
publicly available due to limitations of ethical approval involving patient data
but are available from the corresponding author (MA) on reasonable request. Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1186/s12888-022-03884-5. Additional file 1: Supplemental Table 1. Demographic and social sup-
port/functioning items chosen from validated instruments. Supplemen-
tal Table 2. APT Foundation 2017 cohort demographic characteristics, all
categories listed on BASIS-24 included with test statistics for comparison
by sex. Additional file 2: Supplemental Table 3. Multivariate ordinal analysis
results of social support and social functioning indicators by demographic
characteristics of patients entering low-threshold MOUD treatment. Supplemental Table 4. Multivariate binomial analysis results of social
support and social functioning indicators by demographic characteristics
of patients entering low-threshold MOUD treatment. Funding g
NK was supported by The Horowitz Foundation for Social Policy, Institu-
tion for Social and Policy Studies, Yale University for this work. NK has also
received funding from the Foundation for a Smoke-Free World for a separate
project. DTB was supported by the National Institutes of Health (NIH) [NIH
U01 HL150596-01] for this work. The funding bodies had no role in the design,
analysis, or interpretation of the data in the study. Consent for publication Consent for publication
Not applicable. Not applicable. Acknowledgements
Th
h
i h
h The authors wish to thank all participants, administrative, and clinical staff of
the APT Foundation, New Haven, CT for making this study possible. Competing interests The authors have declared no competing interest. Authors’ contributions WO, MA, NK, BH, PGO, LMM, and DTB conceptualized the study and helped to
collect and validate the data. WO, MA, and NK came up with the methodol-
ogy, implemented the formal analysis, wrote the original draft, and created
visualizations of results. WO, MA, NK, BH, PGO, LMM, and DTB helped to review
and edit the final draft. The authors read and approved the final manuscript. Author details
1 1 Human Nature Lab, Yale Institute for Network Science, Yale University, New
Haven, CT, USA. 2 Department of Sociology, Yale University, New Haven, CT,
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cine and Yale-New Haven Hospital, New Haven, CT, USA. 4 VA Connecticut
Healthcare System, New Haven, CT, USA. 5 Section of Internal Medicine, Yale
University School of Medicine and Yale-New Haven Hospital, New Haven, CT, Abbreviations
MOUD: Medication for opioid use disorder; US: United States; CT: Connecticut;
BASIS-24: Behavior and Symptom Identification Scale; BPI: Brief Pain Inventory; Ethics approval and consent to participate The study involving a medical chart review was approved by the APT Founda-
tion Board of Directors as well as the Human Investigations Committee at
the Yale School of Medicine. All participants provided informed consent for
data collection. All methods were performed in accordance with the relevant
guidelines and regulations set forth by the APT Foundation Board of Directors
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https://openalex.org/W3092951883
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https://digital.csic.es/bitstream/10261/237821/1/BerenguerJ_NitrateRespiration.pdf
|
English
| null |
Nitrate Respiration in Thermus thermophilus NAR1: from Horizontal Gene Transfer to Internal Evolution
|
Genes
| 2,020
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cc-by
| 11,677
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Received: 13 October 2020; Accepted: 2 November 2020; Published: 4 November 2020 Abstract: Genes coding for enzymes of the denitrification pathway appear randomly distributed
among isolates of the ancestral genus Thermus, but only in few strains of the species
Thermus thermophilus has the pathway been studied to a certain detail. Here, we review the
enzymes involved in this pathway present in T. thermophilus NAR1, a strain extensively employed as a
model for nitrate respiration, in the light of its full sequence recently assembled through a combination
of PacBio and Illumina technologies in order to counteract the systematic errors introduced by the
former technique. The genome of this strain is divided in four replicons, a chromosome of 2,021,843 bp,
two megaplasmids of 370,865 and 77,135 bp and a small plasmid of 9799 pb. Nitrate respiration is
encoded in the largest megaplasmid, pTTHNP4, within a region that includes operons for O2 and
nitrate sensory systems, a nitrate reductase, nitrate and nitrite transporters and a nitrate specific
NADH dehydrogenase, in addition to multiple insertion sequences (IS), suggesting its mobility-prone
nature. Despite nitrite is the final product of nitrate respiration in this strain, the megaplasmid
encodes two putative nitrite reductases of the cd1 and Cu-containing types, apparently inactivated by
IS. No nitric oxide reductase genes have been found within this region, although the NorR sensory
gene, needed for its expression, is found near the inactive nitrite respiration system. These data
clearly support that partial denitrification in this strain is the consequence of recent deletions and IS
insertions in genes involved in nitrite respiration. Based on these data, the capability of this strain to
transfer or acquire denitrification clusters by horizontal gene transfer is discussed. Keywords: denitrification; evolution; thermophile; horizontal gene transfer; nitrate respiration;
PacBio sequencing genes
G C A T
T A C G
G C A T genes
G C A T
T A C G
G C A T genes Review Mercedes Sánchez-Costa 1, Alba Blesa 2
and José Berenguer 1,* 1
Center for Molecular Biology Severo Ochoa (CBMSO), Autonomous University of Madrid-Spanish Nationa
Research Council (UAM-CSIC), 28049 Madrid, Spain; mercedes.sanchez@cbm.csic.es
2
Department of Biotechnology, Faculty of Experimental Sciences, Francisco de Vitoria University,
28223 Madrid, Spain; alba.blesa@ufv.es 1
Center for Molecular Biology Severo Ochoa (CBMSO), Autonomous University of Madrid-Spanish National
Research Council (UAM-CSIC), 28049 Madrid, Spain; mercedes.sanchez@cbm.csic.es
2
Department of Biotechnology, Faculty of Experimental Sciences, Francisco de Vitoria University,
28223 Madrid, Spain; alba.blesa@ufv.es *
Correspondence: jberenguer@cbm.csic.es; Tel.: +34-911964498 1. Introduction In addition to being used as frequent sources of enzymes of biotechnological interest, few isolates
of the genus Thermus have been employed as study model according to their easy adaptation to fast
growth under laboratory conditions, in both complex and mineral media, and their highly efficient
natural competence system [21], which has allowed the development of quite a complete set of genetic
tools [20,22–25]. Among these laboratory-adapted models, the T. thermophilus HB8, HB27 and NAR1 strains have
been extensively used. The first two were the first Thermus strains for which complete genome
sequences were available [26]. Both are non-fermentative, obligate aerobes that harbor a highly
syntenic 1.8 Mbp chromosome, with less than 6% of differential genes [27]. They also share a
megaplasmid named pTT27 of 232,605 and 256,992 bp for HB27 and HB8 strains, respectively,
allocating the highest gene diversity, with variable regions containing differential strain-specific
genes. The HB8 strain contains another 81,151 bp megaplasmid (pVV8) that has been lost in many
laboratory-adapted derivatives [28], and a smaller (9322 bp) plasmid named pTT8, of higher copy
number, both absent in the HB27 strain. While the HB8 strain has been used mainly for structural
genomic analysis (http://www.thermus.org/e_index.htm), the employment of HB27 strain has been
focused on cell-physiology analysis and as a host for biotechnological applications due to its higher
transformability compared to HB8, reaching constitutive DNA uptake rates up to 40 kbp per cell and
second [29]. In contrast to the HB8 and HB27 strains, T. thermophilus NAR1 strain is able to grow
anaerobically with nitrate, with final production of nitrite, which is accumulated in the medium. The genes encoding this property have been cloned and sequenced following classic methods, allowing
the identification of the main enzymes implicated in this process (reviewed in [17]). Further partial
sequencing of this strain based on Illumina technology allowed the confirmation of these data, although
the structure of the coding region within the genome or the presence of genes putatively involved in
further steps of the denitrification process could not verified due to the apparent massive presence
of insertion sequences, which generated a quite fragmented genome. The recent application of new
single-molecule sequencing methods based on the PacBio technology [30], in combination with previous
Illumina data, has allowed the generation of a full assembled genome for this strain. 1. Introduction Many prokaryotes use denitrification as their main energy generation respiratory mechanism
in the absence of oxygen or when this preferential electron acceptor is scarce in the environment [1]. The canonic process has been well described in excellent reviews [2–5] and involves four steps of
successive reductions (nitrate > nitrite > nitric oxide > nitrous oxide > dinitrogen), encoded by
the corresponding membrane or periplasmic reductases (Nar/Nas, Nir, Nor, Nos) expressed either
by a single organism or by different ones sharing a common environment, allowing, as a whole,
the displacement to the atmosphere of soluble nitrogen species [2,6,7]. However, in many cases, the last
step of the pathway is less efficient and nitrous oxide, a powerful greenhouse gas, is the final product
of the process, being accumulated in the atmosphere [8–11]. Indeed, the denitrification process has been studied in great detail for many years but using
essentially model proteobacteria [3,12–14]. Nonetheless, there is also available relevant information Genes 2020, 11, 1308; doi:10.3390/genes11111308 www.mdpi.com/journal/genes www.mdpi.com/journal/genes Genes 2020, 11, 1308 2 of 16 about this process in Gram-positives (Firmicutes) [15], and in few extremophiles, both archaea [16] and
bacteria [17]. Among these last ones, denitrification genes and actual functionality have been described
for phylogenetically diverse species, many of which are thermophiles [18]. The genus Thermus is one of
the few ancestral phylogenetic groups for which the denitrification process has been studied to some
extent (reviewed in [17]). This genus belongs to the ancient clade Deinococcus–Thermus and includes hundreds of moderate
to extreme thermophile strains, isolated from both natural and man-made environments, currently
grouped into 27 species (https://lpsn.dsmz.de/genus/thermus), among which Thermus aquaticus and
Thermus thermophilus are the most studied. The genus itself was originally defined as aerobic, and,
actually, many strains are unable to grow in the absence of oxygen. However, anaerobic growth with
nitrogen oxides has been described for few strains of the genus, ones growing only with nitrate and
others able to grow too with nitrite as electron acceptors, leading to the final production of nitrous
oxide in some of these strains [17,19,20]. 1. Introduction The analysis of
this consensus sequence, described for the first time in this work, reveals a quite complex evolutionary
history of the denitrification pathway in T. thermophilus NAR1. 2.1. DNA Preparation and Sequencing T. thermophilus NAR1 (TthNAR1) is a strain which was originally mistaken with the strain HB8. It was renamed as NAR1 once the sequence of HB8 was available and the presence of specific genes,
including the ability to grow on nitrate, was revealed [17]. TthNAR1 was grown overnight at 65 ◦C
under aerobic conditions in 20 mL of Thermus broth [31]. Genomic DNA isolation was carried out 3 of 16 Genes 2020, 11, 1308 following the standard protocol of phenol-chloroform, including a RNAse A treatment. After quality
control assessment on agarose gel and quantification by ND1000 Nanodrop spectrophotometer, sample
was submitted to whole genome sequencing, which was performed following 2 approaches: PacBio RS
II SMRT cells (PacBio) and mate-pair Illumina (Solexa). PacBio sequencing was done for single reads
(1X 16,500) at NSC Centre (Norway), and Illumina sequencing was carried out following the mate-pair
system (Miseq, read type 2 × 36 −251) at Secugen (Scientific Park, Campus UAM, Madrid, Spain). 2.3. Annotation of TthNAR1 Replicons The scaffold genome obtained from Illumina reads was annotated using RAST [39]. PacBio genome
assembly annotation was carried out using 3 software tools, following different automated approaches
in order to ensure consonant functional annotation. PROKKA [40], RAST [39] and AAMG [41] were
executed and the results obtained were compared using BEACON [42] and then filtered to avoid
duplications. Finally, functional annotation of the final consensus genome assembly integrating the
PacBIO genome assembly supplemented with short-accurate Illumina reads was executed using
PROKKA. Genomic maps of the detected replicons were visually represented with DNAplotter
(http://www.sanger.ac.uk/science/tools/dnaplotter). The sequences employed in this study were extracted from the whole-genome project 506,402
which have been deposited in EMBL/GenBank under the accession number PRJEB29203 (https:
//www.ncbi.nlm.nih.gov/bioproject/506402). Data from this PacBio de novo genome assembly described
herein (GCA_900604845.1) were verified using Illumina sequences from a prior genome sequencing
project of this strain (unpublished). Sequences used can be checked here (https://www.ncbi.nlm.nih. gov/bioproject/PRJEB29203/). 2.2. Genome Sequence Analysis and Assembly Illumina reads were trimmed using Trimmomatic [32], and quality sequence analysis were
performed over reads using FastQC software [33]. Sequence alignment and preliminary assembly was
performed using Newbler Assembler and CLC Genomics Workbench 4.0.3 (CLCBio). Contigs were
aligned and sorted against reference genome using an in-house script of BLAST. Scaffold sequences
were obtained using Scaffold builder (https://github.com/metageni/Scaffold_builder) which sorted the
307 contigs generated by draft sequencing, filled gaps and small overlaps following alignment with
Needleman–Wunsch algorithm. This was also done using LAST aligner (http://last.cbrc.jp/). PacBio sequencing results encompassed 60,929 reads with a medium size of 11,306 bp which were
de novo aligned using HGAP3 (Pacific Biosciences, SMRT Analysis Software v2.3.0) and compared to
the one provided by the sequencing center using QUAST (http://bioinf.spbau.ru/quast). Reads were
aligned with pbalign following BLASR method [34] and coverage of each alignment was performed
with GenomeCoverageBed (http://bedtools.readthedocs.io/en/latest/content/tools/genomecov.html)
and visually represented using GNUplot (http://gnuplot.info/). As reported elsewhere [35–37], after the assembly and annotation of the PacBio sequences, several
indels errors were spotted by manual inspection and comparison with Illumina-based sequences. Therefore, PacBio assembly had to be corrected using the Illumina aforementioned reads. Briefly,
Illumina reads were aligned to the PacBio assembly with Bowtie2 aligner (http://bowtie-bio.sourceforge. net/bowtie2/index.shtml) and detection of indels was performed using Pilon [38] and PacBio utilities. A polished consensus genome assembly of the PacBio information supplemented with the Illumina
sequences led to the selection of 4 contigs corresponding to the 4 replicons found in TthNAR1 strain. Coverage of each alignment was performed with GenomeCoverageBed and visually represented using
DNAplotter (https://www.sanger.ac.uk/tool/dnaplotter/). 2.4. Phylogenomic Assessments T. thermophilus NAR1 consensus genome assembly was compared to fully assembled genomes
from other available genomes of Thermus spp. at the NCBI database using an in-house script for BLAST, 4 of 16 Genes 2020, 11, 1308
2.4. Phylogenom and genes’ synteny was checked by filtering for coverage higher than 80%. A multiple alignment using
Mauve [43] for chromosome replicon and Gepard (http://cube.univie.ac.at/gepard) and MumMmer
(http://mummer.sourceforge.net/) for the plasmids was executed, the latter visually represented by
dotplots. Presence of denitrification genes was verified using the raw Illumina reads and homologs
were searched using BLAST (NCBI). from other available genomes of Thermus spp. at the NCBI database using an in-house script for
BLAST, and genes’ synteny was checked by filtering for coverage higher than 80%. A multiple
alignment using Mauve [43] for chromosome replicon and Gepard (http://cube.univie.ac.at/gepard)
and MumMmer (http://mummer.sourceforge.net/) for the plasmids was executed, the latter visually
represented by dotplots. Presence of denitrification genes was verified using the raw Illumina reads
and homologs were searched using BLAST (NCBI). Genes 2020, 11, 1308
gene bank, a
indicated as D Genes 2020, 11, 1308
gene bank, a
indicated as D 5 of 16
is The larger contig, of 2,019,399 bp, corresponds to the chromosome which shares most of its
genes with the chromosome of the HB8 and HB27 strains. Moreover, the order of chromosomal genes
of TthNAR1 is similar to that of TthHB27, whereas an inversion is detected in the chromosome of
HB8 (Figure 2A). The second replicon in size corresponds to a megaplasmid of 370,488 bp which
shares many common genes with pTT27, despite the presence of strain-specific genes and two large
inversions (Figure 2B,C). Based on the coverage, this plasmid has a similar copy number to that of
the chromosome (a ratio of 1.3:1). A smaller plasmid of 77,040 bp was also detected in TthNAR1,
which showed no similarities with pTT27 but actually shared few genes that are present in plasmid
pVV8, the third plasmid of TthHB8, present in old stocks of the strain, but not in laboratory-adapted
derivatives [28]. However, several regions of this plasmid were shared with plasmids pTTJL1801
and pTHTHE1601 from the strains TthJL18 and TthSG0.5, respectively (Figure S1D,E). Interestingly,
the sequence coverage reveals a lesser copy number for this plasmid than that of the chromosome
(0.38:1), suggesting that it could be lost in part of the population. Finally, a smaller plasmid with a
copy number apparently similar to that of the chromosome (1:1.3) is also present in TthNAR1 (Table 1). This plasmid shows scarce similarity to the small plasmid pTT8 described in TthHB8 (Figure S1F). The larger contig, of 2,019,399 bp, corresponds to the chromosome which shares most of its genes
with the chromosome of the HB8 and HB27 strains. Moreover, the order of chromosomal genes of
TthNAR1 is similar to that of TthHB27, whereas an inversion is detected in the chromosome of HB8
(Figure 2A). The second replicon in size corresponds to a megaplasmid of 370,488 bp which shares
many common genes with pTT27, despite the presence of strain-specific genes and two large
inversions (Figure 2B,C). Based on the coverage, this plasmid has a similar copy number to that of
the chromosome (a ratio of 1.3:1). A smaller plasmid of 77,040 bp was also detected in TthNAR1,
which showed no similarities with pTT27 but actually shared few genes that are present in plasmid
pVV8, the third plasmid of TthHB8, present in old stocks of the strain, but not in laboratory-adapted
derivatives [28]. 3.1. Sequence of the TthNAR1 Strain
3.1. Sequence of the TthNAR1 Strain The PacBio-generated sequences of TthNAR1 strain shows a GC content similar to other Thermus
strains (68.79%) and higher similarity to TthJL18 genome (84.5% homology) followed by that of
TthHB8 strain (78.7%). The genome was divided into four contigs that could be circularized in the
corresponding replicons (Figure 1). The PacBio-generated sequences of TthNAR1 strain shows a GC content similar to other Thermus
strains (68.79%) and higher similarity to TthJL18 genome (84.5% homology) followed by that of
TthHB8 strain (78.7%). The genome was divided into four contigs that could be circularized in the
corresponding replicons (Figure 1). Figure 1. Genome of TthNAR1. Circular maps of the four replicons detected in TthNAR1 strain,
elaborated using DNAPlotter using the consensus genome assembly achieved from supplementing
PacBIO sequences with Illumina data. This strain harbors a highly conserved 2 Mbp chromosome (A)
Figure 1. Genome of TthNAR1. Circular maps of the four replicons detected in TthNAR1 strain,
elaborated using DNAPlotter using the consensus genome assembly achieved from supplementing
PacBIO sequences with Illumina data. This strain harbors a highly conserved 2 Mbp chromosome (A)
and plasmids of different sizes: a pTT27-like megaplasmid of 370 kbp (B) and two smaller plasmids of
77 kbp (C) and 9.8 kbp (D). These last two plasmids have little relation to other plasmids in the gene
bank, as described in Figure S1. Localization of the nitrate and nitrite respiration cluster is indicated as
DI cluster in the pTT27-like megaplasmid (B). Figure 1. Genome of TthNAR1. Circular maps of the four replicons detected in TthNAR1 strain,
elaborated using DNAPlotter using the consensus genome assembly achieved from supplementing
PacBIO sequences with Illumina data. This strain harbors a highly conserved 2 Mbp chromosome (A)
Figure 1. Genome of TthNAR1. Circular maps of the four replicons detected in TthNAR1 strain,
elaborated using DNAPlotter using the consensus genome assembly achieved from supplementing
PacBIO sequences with Illumina data. This strain harbors a highly conserved 2 Mbp chromosome (A)
and plasmids of different sizes: a pTT27-like megaplasmid of 370 kbp (B) and two smaller plasmids of
77 kbp (C) and 9.8 kbp (D). These last two plasmids have little relation to other plasmids in the gene
bank, as described in Figure S1. Localization of the nitrate and nitrite respiration cluster is indicated as
DI cluster in the pTT27-like megaplasmid (B). Genes 2020, 11, 1308
gene bank, a
indicated as D Region
(CRISPR)
rRNA
tRNA
IS
Chromosome
2,021,843
339.8
2220
0
6
51
70
pTTHNP2
9799
448.2
12
0
0
0
19
pTTHNP3
77,135
130.5
106
2 (questionable)
0
0
102
pTTHNP4
370,865
464.9
429
4
0
0
100 A further manual analysis of the PacBio genome revealed the presence of mutations that truncated
genes encoding for essential proteins. As an example, the DNA gyrase genes gyrA and gyrB were
truncated at three positions, leading to two and three ORFs in what should correspond to the subunits
A and B of the DNA gyrase, respectively. The comparison of these sequences with that of Illumina
reads did not show any of these apparent mutations, supporting the existence of errors in the PacBio
assembly. Interestingly, these apparent mutations were not randomly distributed along the genome,
but concentrated and reiterative at specific points, where more than 90% of the PacBio reads (coverage
above X300 except for pTTHNP3) contained the same error (a G deletion) and, thus, were incorporated
by the software to the corresponding consensus PacBio sequence. By contrast, 90% of Illumina reads
at the same point (coverage X50) showed no interruptions in the coding frame and encoded the
corresponding potentially functional wild type proteins. Due to the identification of these systematic errors, a thorough genome-wide search for differences
between PacBio-based and Illumina-based reads in TthNAR1 was carried out. This analysis revealed the
existence of 2927 deletions along the PacBio genome and a single insertion in PacBio sequences respect
to the Illumina counterpart that essentially affected homopolymers of 5–6 units of G or C. This number
of errors corresponds to around one deletion every 1000 bases, thus suggesting the presence of a great
number of artifactual apparent mutants (1297 ORFs) in the initial PacBio sequence annotation of the
strain. Interestingly, these mutations were not distributed along all the G/C homopolymers types but
affected only 1.64% of those present in the genome, suggesting the existence of additional sequence
features that could be affecting the activity at these points of the PacBio single molecule reading system. Nonetheless, a search for putative sequence pattern using as template a 20 bp sequence upstream
and downstream from the systematic reading errors did not reveal any significant sequence pattern
consensus that could justify it. Genes 2020, 11, 1308
gene bank, a
indicated as D However, several regions of this plasmid were shared with plasmids pTTJL1801 and
pTHTHE1601 from the strains TthJL18 and TthSG0.5, respectively (Figure S1D,E). Interestingly, the
sequence coverage reveals a lesser copy number for this plasmid than that of the chromosome
(0.38:1), suggesting that it could be lost in part of the population. Finally, a smaller plasmid with a
copy number apparently similar to that of the chromosome (1:1.3) is also present in TthNAR1 (Table
1). This plasmid shows scarce similarity to the small plasmid pTT8 described in TthHB8 (Figure S1F). Figure 2. Synteny of TthNAR1 replicons with other Tth strains. (A) Alignment of (top to bottom) Tth
strains NAR1, HB27 and HB8 chromosomes with Mauve (no connection lines). The sequence has been
divided in four section colors corresponding to syntenic regions to facilitate the visualization. Dotplots of TthNAR1 370 kbp megaplasmid against pTT27 megaplasmids found in HB8 (B) and HB27
(C). Figure 2. Synteny of TthNAR1 replicons with other Tth strains. (A) Alignment of (top to bottom) Tth
strains NAR1, HB27 and HB8 chromosomes with Mauve (no connection lines). The sequence has been
divided in four section colors corresponding to syntenic regions to facilitate the visualization. Dotplots
of TthNAR1 370 kbp megaplasmid against pTT27 megaplasmids found in HB8 (B) and HB27 (C). Figure 2. Synteny of TthNAR1 replicons with other Tth strains. (A) Alignment of (top to bottom) Tth
strains NAR1, HB27 and HB8 chromosomes with Mauve (no connection lines). The sequence has been
divided in four section colors corresponding to syntenic regions to facilitate the visualization. Dotplots of TthNAR1 370 kbp megaplasmid against pTT27 megaplasmids found in HB8 (B) and HB27
(C)
Figure 2. Synteny of TthNAR1 replicons with other Tth strains. (A) Alignment of (top to bottom) Tth
strains NAR1, HB27 and HB8 chromosomes with Mauve (no connection lines). The sequence has been
divided in four section colors corresponding to syntenic regions to facilitate the visualization. Dotplots
of TthNAR1 370 kbp megaplasmid against pTT27 megaplasmids found in HB8 (B) and HB27 (C). 6 of 16 Genes 2020, 11, 1308 Table 1. Summary of features of the consensus TthNAR1 replicons. CDS, RNA and tRNA were
identified with PROKKA, and IS using the ISFinder database. Contig
Bases
Coverage
CDS
Rep. Genes 2020, 11, 1308
gene bank, a
indicated as D It is also relevant to note that a similar effect of systematic errors in PacBio-based sequences
compared to Illumina reads was detected for Mycolicibacteriun hassiacum [44], another genome harboring
high G + C content, although the number of systematic errors found in this genome was one tenth
of that observed for TthNAR1 (481 in a 5 Mbp genome, roughly one in 10,000 bases). In addition,
this case, no apparent sequence pattern around the points where the reads systematically failed could
be found. In any case, these results clearly support that a combination of technologies is needed to
obtain an accurate genome sequence, at least for organisms showing high G + C content, and the users
of the databases should be aware of the methodologies followed to obtain the published sequences. In the TthNAR1 case, the errors detected in the PacBio sequences were corrected with the Illumina
reads, and the final genome was assembled and annotated, showing no present indels in any essential
gene. In the corrected sequence, the analysis of the genome (Materials and Methods) showed the
presence of six copies of the ribosomal RNAs, 51 tRNA genes and 2767 CDS coding for putative
proteins (Table 1). In addition, using the IS finder program with default search values, many ISs,
some of them putatively active and others truncated, were found along the four replicons. In addition,
four CRISPR arrays were found, allocated in the megaplasmid pTTHNP4, and two putative ones were
also detected in pTTHNP3. Genes 2020, 11, 1308 7 of 16 7 of 16 3.2. The Nitrate Reductase Gene Cluster The gene cluster coding for nitrate respiration (nar) is localized between positions 140,059 and
158,930 of the pTTHNP4 megaplasmid. This cluster includes 15 ORFs encoding functional proteins;
for most of them, their participation has been experimentally verified at different steps of nitrate
respiration (Figure 3). A pseudogene harboring three frameshifts that originally encoded a putative
iron transporter is also found within the cluster, suggesting its lost in a host context where this element
is efficiently transported. Genes 2020, 11, x FOR PEER REVIEW
8 of 16 Figure 3. Comparison of the denitrification clusters of TthNAR1 and other Thermus spp. Genetic
context of nitrate and nitrite respiration genes found in TthNAR1, showing homolog genes and its
order in the closely related strains TthSG05 JP17-16 and T. scotoductus SA-01 (Tsco SA01). Genetic maps
were performed according to fully assembled genomes available at NCBI database. Grey empty
arrows represent genes not related with nitrate/nitrite respiration. Filled grey arrows represent
transposases or fragments of transposases (indicated as tnp). Genes anchored with an asterisk indicate
truncated sequences (pseudogenes). Percentages of identities are indicated within the grey boxes and
length of the arrows is proportional to the size of the CDS. Figure 3. Comparison of the denitrification clusters of TthNAR1 and other Thermus spp. Genetic context
of nitrate and nitrite respiration genes found in TthNAR1, showing homolog genes and its order in
the closely related strains TthSG05 JP17-16 and T. scotoductus SA-01 (Tsco SA01). Genetic maps were
performed according to fully assembled genomes available at NCBI database. Grey empty arrows
represent genes not related with nitrate/nitrite respiration. Filled grey arrows represent transposases
or fragments of transposases (indicated as tnp). Genes anchored with an asterisk indicate truncated
sequences (pseudogenes). Percentages of identities are indicated within the grey boxes and length of
the arrows is proportional to the size of the CDS. Figure 3. Comparison of the denitrification clusters of TthNAR1 and other Thermus spp. Genetic
context of nitrate and nitrite respiration genes found in TthNAR1, showing homolog genes and its
order in the closely related strains TthSG05 JP17-16 and T. scotoductus SA-01 (Tsco SA01). Genetic maps
were performed according to fully assembled genomes available at NCBI database. Grey empty
arrows represent genes not related with nitrate/nitrite respiration. Filled grey arrows represent
transposases or fragments of transposases (indicated as tnp). Genes anchored with an asterisk indicate
truncated sequences (pseudogenes). 3.3. The Respiratory Nitrate Reductase (NR) of TthNAR1 and Other Thermus spp. As commented above, the core of the nitrate respiration cluster includes five genes conserved in
all the nitrate respiring strains of Thermus spp. so far described, instead of the four genes typically
described for the NR of mesophilic model organisms [46]. The additional gene is expressed at the 5′
extreme of the mRNA and encodes a membrane-bound periplasmic cytochrome with two heme C
groups [47]. Mutants lacking this protein are unable to grow anaerobically under nitrate conditions and
further biochemical analysis revealed that they produced a soluble and immature form of NarG [48],
the protein subunit where nitrate is reduced, suggesting the requirement of NarC for the maturation
and membrane attachment of the enzyme. A similar behavior was found for mutants lacking NarI,
the bi-heme B membrane cytochrome, conserved in all the respiratory nitrate reductases characterized
so far. Furthermore, bacterial two-hybrid assays supported the existence of strong interactions between
the two cytochromes NarC and NarI, as well as among NarI, NarH and NarG, whereas the NarJ
chaperone was only shown to interact with NarG [48]. All these data clearly support the existence of a
heterotetrameric nitrate reductase that contains a periplasmic cytochrome C as an additional subunit,
absent in the NR reductases described in other model bacteria. In addition to being required for the synthesis and maturation of an active NR, the putative
participation of NarC cytochrome in the electron transport within nitrate respiration is not well defined
yet. However, it could probably be working as an electron-transport branching system, deriving
electrons to other acceptors in case of nitrate scarcity. In this sense, it is noteworthy to mention the
presence of a very similar gene cluster (to the Thermus one) in nitrifying bacteria such as Nitrobacter
hamburguensis, where the encoded enzyme function as nitrite oxidoreductase, involved in the oxidation
of nitrite to nitrate by the NarG-homolog, and the electrons generated going through the cytochrome B
(NarI homologue) to the periplasmic NarC homolog, with final destination in complex III or complex IV
(the cytochrome oxidase) [1]. Interestingly, in denitrifying strains of T. thermophilus such as TthPRQ16,
the lack of NarC makes the cell unable to grow with nitrite or NO as electron acceptors [49] in a context
where the expression of the complex III is inhibited, suggesting the involvement of NarC as an electron
bridge towards the periplasmic nitrite reductase or the membrane nitric oxide reductase (Figure 4). Genes 2020, 11, 1308 Genes 2020, 11, 1308 8 of 16 When the sequence of the nar cluster from TthNAR1 was compared to that found in other
nitrate respiring strains of Thermus spp., such as T. thermophilus SG0.5JP17-16 (TthSG) or T. scotoductus
SA01 (Tsco), two main differences became clear. On the one hand, the dispensability of the NADH
dehydrogenase system (Nrc): in TthSG it is absent except for the nrcD ferredoxin and a pseudogene
deletion form of nrcE; and in Tsco it is separated into two group of genes, nrcD on one side and nrcDF
in the other, also lacking nrcE (Figure 3). On the other hand, the nitrate/nitrite transporters, where the
tandem NarK–NarT is found in TthNAR1 and Tsco, are replaced by a single transporter of a different
MFS subfamily (NarO) in TthSG. Interestingly, NarO can surrogate the pair NarKT when expressed in
trans in denitrifying derivatives of the strain T. thermophilus HB27 [45]. By contrast, the sequence and synteny of the genes encoding the nitrate reductase (narCGHJI) and
that of the regulators (dnrST and drpAB) is highly conserved, supporting that they constitute the core
enzyme and the regulatory control systems for nitrate respiration. Interestingly, this region can be
horizontally transferred to aerobic strains of Thermus spp. such as T. thermophilus HB27 by conjugation
or by natural competence, allowing the new host to grow anaerobically with nitrate [17]. 3.2. The Nitrate Reductase Gene Cluster Percentages of identities are indicated within the grey boxes and
l
th f th
i
ti
l t th
i
f th CDS
Figure 3. Comparison of the denitrification clusters of TthNAR1 and other Thermus spp. Genetic context
of nitrate and nitrite respiration genes found in TthNAR1, showing homolog genes and its order in
the closely related strains TthSG05 JP17-16 and T. scotoductus SA-01 (Tsco SA01). Genetic maps were
performed according to fully assembled genomes available at NCBI database. Grey empty arrows
represent genes not related with nitrate/nitrite respiration. Filled grey arrows represent transposases
or fragments of transposases (indicated as tnp). Genes anchored with an asterisk indicate truncated
sequences (pseudogenes). Percentages of identities are indicated within the grey boxes and length of
the arrows is proportional to the size of the CDS. 3.3. The Respiratory Nitrate Reductase (NR) of TthNAR1 and Other Thermus spp. As commented above, the core of the nitrate respiration cluster includes five genes conserved in
all the nitrate respiring strains of Thermus spp. so far described, instead of the four genes typically
described for the NR of mesophilic model organisms [46]. The additional gene is expressed at the 5′
extreme of the mRNA and encodes a membrane-bound periplasmic cytochrome with two heme C
groups [47]. Mutants lacking this protein are unable to grow anaerobically under nitrate conditions
and further biochemical analysis revealed that they produced a soluble and immature form of NarG
[48], the protein subunit where nitrate is reduced, suggesting the requirement of NarC for the
Proteins required for nitrate respiration are expressed in four transcriptional units (Figure 3). The largest one is the narCGHJIKT operon which codes for the conserved components of the nitrate
reductase (NarGHI), a chaperone (NarJ) needed for the insertion of the diMGD cofactor in NarG,
a cytochrome C (NarC) absent in Nar operon in mesophiles, and two nitrate/nitrite transporters (NarK
and NarT). A four-gene operon, nrcDEFN, encodes a NADH dehydrogenase that shows structural
similarities to succinate dehydrogenase and is transcribed only under anoxia in the presence of nitrate. Finally, two bigenic operons, dnrST and drpAB, encoding regulatory proteins that respond to oxygen
(DnrS and DnrT) or to nitrate (DrpA and DrpB), are located upstream and downstream, respectively,
of the narCGHJIKT operon. 3.4. A Putative Nitrate Respirasome in the NAR1 Strain The presence of a NADH dehydrogenase bound to the membrane through a protein complex in
the nar cluster of TthNAR1 suggests the putative existence of a “nitrate respirasome” in this strain
(Figure 4). The nrcDEFN operon encodes a ferredoxin (NrcD) that in bacterial two-hybrid assays
strongly interacts with the polytopic membrane protein NrcE [50]. The cytoplasmic C terminus of
NrcE concomitantly interacts with an iron-sulfur containing protein (NrcF) similar to that found in
succinate:quinone oxidoreductases, and with NrcN, a flavoenzyme that shows type-II like NADH
dehydrogenase activity when overexpressed and purified from E. coli [50,51]. In addition, in TthNAR1,
bacterial two-hybrid assays revealed strong interactions between NrcF and NrcD. At physiological
levels, mutants in nrcE, nrcF or nrcN (nrcD was too small so as to get insertion mutants) showed
slower growth rates than the wild type strain under nitrate respiration conditions, and the membrane
fractions from mutants lacking any of these proteins showed a significantly decreased NADH oxidation
capacity in the presence of nitrate compared to the wild type performance. All these data, together
with those figures regarding the parallel transcriptional induction of the nrcDEFN and narGDHJIKT
operons under nitrate respiration, pointed to the existence of a highly efficient transfer of electrons
between both complexes, likely through the formation of a nitrate respiratory supercomplex (Figure 4). It is important to note that the existence of such a complex has not been biochemically proven yet,
but bacterial two-hybrid assays strongly support the existence of interactions between both protein
complexes through their membrane units NrcE and NarI, which could ease the exchange of electrons
between them, either directly or through the menaquinones. 3.3. The Respiratory Nitrate Reductase (NR) of TthNAR1 and Other Thermus spp. In this context, complementation experiments showed the greater relevance of the distal heme C group
in the process [49]. If this were the actual role of the fourth component of the Thermus NR, it should be
deduced that the ability to respire nitrite and NO has to be the rule and not the exception in nitrate
respiring strains. In other words, nitrate respiration has to be biochemically linked to nitrite respiration
in odds to an efficient denitrification performance. 9 of 16
which Genes 2020, 11, 1308
interactions betw
ld
th Figure 4. A putative nitrate respirasome in TthNAR1. This scheme shows the likely structure of
the nitrate respirasome, including the Nrc NADH dehydrogenase complex and the nitrate reductase. Activity of the complex will separate protons. Interactions detected by two-hybrids assays are indicated
as double arrowheads lines. Heme groups are indicated as black hexagons and iron sulfur clusters as
diamonds. The role of the NarC component of the NR as electron transporter towards NOR and NOS
enzymes is indicated as discontinuous arrows. Figure 4. A putative nitrate respirasome in TthNAR1. This scheme shows the likely structure of
the nitrate respirasome, including the Nrc NADH dehydrogenase complex and the nitrate reductase. Activity of the complex will separate protons. Interactions detected by two-hybrids assays are indicated
as double arrowheads lines. Heme groups are indicated as black hexagons and iron sulfur clusters as
diamonds. The role of the NarC component of the NR as electron transporter towards NOR and NOS
enzymes is indicated as discontinuous arrows. 3.5. Regulation of Nitrate Respiration in TthNAR1 As in other model bacteria, the synthesis of the enzymes for nitrate respiration depends on two
concomitant environmental signals: the decrease in oxygen below a threshold level and the presence
of nitrate in the growth medium [3]. In most of these model systems, oxygen is sensed through a
transcriptional regulator of the cAMP family, named FNR, that contains a [4S-4Fe] iron–sulfur cluster
as the oxygen sensor [52]. Upon oxidation of the cluster, FNR behaves as a monomer and lacks affinity 10 of 16 Genes 2020, 11, 1308 for its binding sites. In the genome of TthNAR1, there are no FNR homologs that could play such
a role, so oxygen sensing has to be displayed through a different mechanism. On the other hand,
due to its membrane impermeability, in most denitrifying Proteobacteria, nitrate sensing is carried
out by two-component systems of the NarX/NarL family, where the nitrate sensory component, NarX,
dimerizes upon detection of nitrate at the periplasmic side of the membrane, and a phosphorylation
system leads to the phosphorylation of the response regulator of the NarL family, which subsequently
binds to a heptameric sequence, found in diverse repeats upstream the regulated genes, which also
usually contain binding sites for the FNR dimers [3,53]. As with the oxygen sensing, TthNAR1 does not
encode homologs to NarX/NarL system despite its responsiveness to nitrate, supporting the existence
of a different class of nitrate sensors in this ancient bacterial group. The identification of the sensory systems for oxygen and nitrate within the nar cluster was
the consequence of horizontal gene transfer experiments towards the aerobic TthHB27 strain [31,54]. In these experiments, the oxygen and nitrate sensors were transferred along the nitrate reductase operon
within an approximately 30 kbp DNA fragment. Subsequently, it was shown that mutants lacking any
of the Dnr proteins (DnrS and DnrT, formerly RegA and RegB in [55]), encoded immediately upstream
of the nar operon (Figure 3), were defective in anaerobic growth with nitrate due to their requirement for
the transcription of the narCGHJIKT operon. However, only DnrT (and not DnrS) was required for the
transcription of the nrcCEFN operon, supporting the existence of functional differences among the role of
these proteins. 3.5. Regulation of Nitrate Respiration in TthNAR1 Further, these assays demonstrated that DnrT (a transcriptional factor of the CRP family)
was insensitive to oxygen—actually it has no iron–sulfur cluster—or nitrate signals and was acting in a
concentration-dependent manner through the binding to sites located approximately at −43 position
from the starting site of the regulated promoters (i.e., CCTTCACCTTACTCCTTGACCCCGGTCAT in
Pnrc), where it was used to recruit the RNA polymerase [55]. In contrast, the conformation of DnrS was
sensitive to the presence of oxygen, as revealed by its increased sensitivity to proteases under aerobic
conditions. Despite these data showing DnrS oxygen sensitivity, the actual oxygen sensor in DnrS
is not known, but it is likely dependent on the GAF domain found at its N-terminal region. Besides,
its putative DNA binding capability could not be assayed, likely due to the mentioned sensitivity to
oxygen. Despite these uncertainties yet to be solved, it seems clear that the dnrST operon, conserved in
all the nitrate reductase clusters described for Thermus spp., is involved in oxygen detection. In this
context, it is also worth mentioning the role of DnrT, not only as a transcription activator factor for all
the promoters involved in the expression of genes required for nitrate respiration, but also as repressor
of the pbc1 promoter, responsible for the expression of the complex III in T. thermophilus, suggesting
that another electron transporter replaces this complex during anaerobic growth. The nitrate detection system was also difficult to assess because of the high sensitivity of the
TthNAR1 strain to this compound, which required the use of rich media prepared with the same
yeast extract and bactopeptone stocks, as some of these commercial products had enough nitrate
amounts in their composition to activate the system in the absence of additional nitrate. In any case,
the nitrate sensory system was also genetically linked to the nitrate respiration cluster, as observed
in the horizontal gene transfer experiments, and the focus was set on the drpAB operon located
immediately downstream of the narCGHJIKT operon in all the Thermus spp. so far sequenced [17]. By employing specific antisera in Western blot assays, it was found that DrpA was periplasmic, whereas
DprB was cytoplasmic, although attached to the membrane [56]. 3.5. Regulation of Nitrate Respiration in TthNAR1 Despite the lack of knowledge of
the actual mechanism of nitrate sensing by these proteins, it is clear that the transcription of the
nar, nrc and dnrST operons solely depends on the absence of oxygen in mutants lacking both DrpA
and DrpB proteins, and is completely signal independent—i.e., constitutive—in the absence of DrpB,
suggesting a repressor role for DrpB protein, in contrast to the transcription activator role played
by phosphorylated NarL [56]. However, as the sequence of DrpB does not show any putative DNA
binding motif, its action could be likely dependent on an interaction with the master regulators DnrS
or/and DnrT (Figure 5), in a way similar to what was proposed for the nitrate/oxygen co-sensing protein
NreA of Staphylococcus aureus [57]. Concomitantly, the expression of DrpA and DrpB was dependent 11 of 16
rs DnrS
i Genes 2020, 11, 1308
p
p
y
binding motif, its on the concentration of DnrT, but not on DnrS, supporting the existence of an induction/repression
circuit that keeps the concentration of the four regulators under control, allowing the re-setting of the
system [56]. A speculative model for the regulatory circuit that controls nitrate respiration in TthNAR1
is shown in Figure 5, in which the presence of oxygen and absence of nitrate keeps DrpB active to
inhibit DnrS and/or DnrT, whereas presence of nitrate along absence of oxygen inactivates it, allowing
DnrS and DnrT to activate the nitrate respiration clusters. protein NreA of Staphylococcus aureus [57]. Concomitantly, the expression of DrpA and DrpB was
dependent on the concentration of DnrT, but not on DnrS, supporting the existence of an
induction/repression circuit that keeps the concentration of the four regulators under control,
allowing the re-setting of the system [56]. A speculative model for the regulatory circuit that controls
nitrate respiration in TthNAR1 is shown in Figure 5, in which the presence of oxygen and absence of
nitrate keeps DrpB active to inhibit DnrS and/or DnrT, whereas presence of nitrate along absence of
oxygen inactivates it allowing DnrS and DnrT to activate the nitrate respiration clusters Figure 5. Regulatory circuitry controlling nitrate respiration. DnrS is the oxygen sensor of the system,
being active in its absence and allowing its own expression and that of cotranscribed gene dnrT, as
well as acting in the transcription of the nar operon. 3.5. Regulation of Nitrate Respiration in TthNAR1 DnrT is a general activator needed for transcription
of all the operons for nitrate respiration as well as to transcribe nir, nor and nrs operons needed for
nitrite respiration in denitrifying strains of Tth, a process in which DnrS is also needed. NorR is a
transcription factor that stimulates transcription of nrc, nirJ and its own operon, but represses nirS
promotion in the presence of NO. DrpB activity on transcription is likely to occur through interaction
with the master regulator proteins in the absence of nitrate, and releases this activity by an unknown
mechanism (?) when DrpA detects nitrate in the periplasm (see the text for details). 3.5. Regulation of Nitrate Respiration in TthNAR1 DnrT is a general activator needed for
transcription of all the operons for nitrate respiration as well as to transcribe nir, nor and nrs operons
needed for nitrite respiration in denitrifying strains of Tth, a process in which DnrS is also needed. NorR is a transcription factor that stimulates transcription of nrc, nirJ and its own operon, but
represses nirS promotion in the presence of NO. DrpB activity on transcription is likely to occur
through interaction with the master regulator proteins in the absence of nitrate, and releases this
activity by an unknown mechanism (?) when DrpA detects nitrate in the periplasm (see the text for
Figure 5. Regulatory circuitry controlling nitrate respiration. DnrS is the oxygen sensor of the system,
being active in its absence and allowing its own expression and that of cotranscribed gene dnrT, as well
as acting in the transcription of the nar operon. DnrT is a general activator needed for transcription
of all the operons for nitrate respiration as well as to transcribe nir, nor and nrs operons needed for
nitrite respiration in denitrifying strains of Tth, a process in which DnrS is also needed. NorR is a
transcription factor that stimulates transcription of nrc, nirJ and its own operon, but represses nirS
promotion in the presence of NO. DrpB activity on transcription is likely to occur through interaction
with the master regulator proteins in the absence of nitrate, and releases this activity by an unknown
mechanism (?) when DrpA detects nitrate in the periplasm (see the text for details). Figure 5. Regulatory circuitry controlling nitrate respiration. DnrS is the oxygen sensor of the system,
being active in its absence and allowing its own expression and that of cotranscribed gene dnrT, as
well as acting in the transcription of the nar operon. DnrT is a general activator needed for
transcription of all the operons for nitrate respiration as well as to transcribe nir, nor and nrs operons
needed for nitrite respiration in denitrifying strains of Tth, a process in which DnrS is also needed. NorR is a transcription factor that stimulates transcription of nrc, nirJ and its own operon, but
represses nirS promotion in the presence of NO. 3.5. Regulation of Nitrate Respiration in TthNAR1 DrpB activity on transcription is likely to occur
through interaction with the master regulator proteins in the absence of nitrate, and releases this
activity by an unknown mechanism (?) when DrpA detects nitrate in the periplasm (see the text for
Figure 5. Regulatory circuitry controlling nitrate respiration. DnrS is the oxygen sensor of the system,
being active in its absence and allowing its own expression and that of cotranscribed gene dnrT, as well
as acting in the transcription of the nar operon. DnrT is a general activator needed for transcription
of all the operons for nitrate respiration as well as to transcribe nir, nor and nrs operons needed for
nitrite respiration in denitrifying strains of Tth, a process in which DnrS is also needed. NorR is a
transcription factor that stimulates transcription of nrc, nirJ and its own operon, but represses nirS
promotion in the presence of NO. DrpB activity on transcription is likely to occur through interaction
with the master regulator proteins in the absence of nitrate, and releases this activity by an unknown
mechanism (?) when DrpA detects nitrate in the periplasm (see the text for details). Figure 5. Regulatory circuitry controlling nitrate respiration. DnrS is the oxygen sensor of the system,
being active in its absence and allowing its own expression and that of cotranscribed gene dnrT, as
well as acting in the transcription of the nar operon. DnrT is a general activator needed for
transcription of all the operons for nitrate respiration as well as to transcribe nir, nor and nrs operons
needed for nitrite respiration in denitrifying strains of Tth, a process in which DnrS is also needed. NorR is a transcription factor that stimulates transcription of nrc, nirJ and its own operon, but
represses nirS promotion in the presence of NO. DrpB activity on transcription is likely to occur
through interaction with the master regulator proteins in the absence of nitrate, and releases this
activity by an unknown mechanism (?) when DrpA detects nitrate in the periplasm (see the text for
Figure 5. Regulatory circuitry controlling nitrate respiration. DnrS is the oxygen sensor of the system,
being active in its absence and allowing its own expression and that of cotranscribed gene dnrT, as well
as acting in the transcription of the nar operon. details).
3.6. Genes for Nitrite Respiration in TthNAR1 3.6. Genes for Nitrite Respiration in TthNAR1
Nitrite respiration in denitrifying strains such as TthPRQ25 and TthPRQ16 depends on the
expression of the nirSJM operon, which codes for the periplasmic cd1 type nitrite reductase (NirS), a
protein involved in the synthesis of the heme d cofactor (NirJ) and a periplasmic cytochrome C550
Nitrite respiration in denitrifying strains such as TthPRQ25 and TthPRQ16 depends on the
expression of the nirSJM operon, which codes for the periplasmic cd1 type nitrite reductase (NirS),
a protein involved in the synthesis of the heme d cofactor (NirJ), and a periplasmic cytochrome C550
(NirM) proposed to act as electron transporter towards NirS [58,59]. protein involved in the synthesis of the heme d cofactor (NirJ), and a periplasmic cytochrome C550
(NirM) proposed to act as electron transporter towards NirS [58,59]. p
p
p
On the other hand, the presence of a periplasmic cytochrome C (NarC), as a fourth subunit of
the nitrate reductase of Thermus spp. discussed above, suggests that the capability of this enzyme to
replace the complex III during further steps of the denitrification pathway [49] is a general property
that allows the transfer of electrons to compatible external acceptors in case of nitrate scarcity. Actually,
in complete denitrifying strains such as TthPRQ25, nitrite accumulates in the medium until nitrate
becomes scarce, and then the former is used as electron acceptor, likely employing the NarC component
of the NR as surrogate to the complex III, repressed under these conditions. This suggests that nitrate
and nitrite respiration are not simultaneous but are functionally linked. The genomic proximity of
the genes encoding both processes in those denitrifying strains has already been sequenced, and the
requirement of the master regulators DnrS and DnrT for nitrite respiration [59] clearly support the
existence of a complete denitrification island. This denitrification island can be one-step transferred
to the aerobic TthHB27 strain, leading to the selection of a denitrifying derivative (TthHB27d) [17]. Genes 2020, 11, 1308 12 of 16 12 of 16 Interestingly, the nitrate respiration cluster of the nitrate respiring TthNAR1 strain can control the
nitrite respiration cluster from other strains [17], supporting that inability of TthNAR1 to use nitrite
happens to be an anomaly. Interestingly, the nitrate respiration cluster of the nitrate respiring TthNAR1 strain can control the
nitrite respiration cluster from other strains [17], supporting that inability of TthNAR1 to use nitrite
happens to be an anomaly. details).
3.6. Genes for Nitrite Respiration in TthNAR1 Indeed, the availability of the complete genome sequence of TthNAR1 allowed us to confirm this
unicity. Executing BLASTP searches with the TthSG NirS, NirJ and NirM protein sequences, we found
homologs (98–100% of identity) for the three proteins in TthNAR1 pTTHNP4 megaplasmid (positions
170,194 to 171,804; 168,964 to170,112 and 167,260 to 167,694, respectively), allocated near the nitrate
respiration cluster, as if they were part of an old denitrification island. In addition, in that same region,
TthNAR1 encodes a putative nitrite reductase of the Cu-Nir type (NirK), similar (84.6% of identity) to
that of T. scotoductus [60]. The presence of two types of nitrite reductase (cd1 and Cu-types) in a single
organism is quite unusual, but it strongly suggests that a recent ancestor of TthNAR1 strain displayed
nitrite respiration capability. The reasons by which TthNAR1 cannot use nitrite despite the presence of genes coding for
two types of nitrite reductases seemed clear when a detailed analysis of the sequences was carried
out. On the one hand, a transposase pseudogene of the IS256 family, fragmented into three ORFs
(TTHNP4_186/187/188), is found inserted between the nirJ and the nirM genes, which could limit or
abolish the expression of the latter (Table 2, Figure S2). On the other hand, the putative Cu-Nir (NirK,
TTHNP4_0191) lacks 49 N-terminal amino acids respect of that of Tsco due to the insertion of another IS
(IS982-like, TTHNP4_192). The lost region of NirK includes the signal peptide needed for periplasmic
secretion of this protein and also the putative promoter signals required for its expression, supporting
that the protein is not synthesized at all. Table 2. Putative transposases located within the denitrification cluster of TthNAR1 strain. Typology
of the transposases have been deduced according to the ISfinder database (https://isfinder.biotoul.fr/). Position
ORF
IS-Type
163912–162710
TthNP4_176 + TthNP4_177
ISTth4
167718–168215
TthNP4_186 *
ISTth4
168356–168670
TthNP4_187 *
ISTth4
168631–168915
TthNP4_188 *
ISTth4
173562–174395
TthNP4_192
IS982-like
* Transposases that can belong to a unique ORF. Table 2. Putative transposases located within the denitrification cluster of TthNAR1 strain. Typology
of the transposases have been deduced according to the ISfinder database (https://isfinder.biotoul.fr/). In conclusion, the expression of Cu-Nir (NirK) has been lost by the insertion of an IS, and the nirJSM
cluster has been divided into two fragments, with high chances of no expression of the cytochrome
nirM. details).
3.6. Genes for Nitrite Respiration in TthNAR1 Further analysis suggests that these insertion events would have likely occurred a long time
ago, as the corresponding transposases are pseudogenes due to the presence of several mutations
within them. Acknowledgments: The technical assistance of Esther Sánchez is also acknowledged. The next-generation
sequencing (NGS) data analysis was performed by the Genomics and NGS Core Facility at the Centro de
Biología Molecular Severo Ochoa (CBMSO, CSIC-UAM) which is part of the CEI UAM + CSIC, Madrid,
Spain—http://www.cbm.uam.es/genomica. 3.7. Genes for NO Respiration 3.7. Genes for NO Respiration Nitrite respiration generates NO and leads to the concomitant requirement for an active NO
reducing system, either to use it as electron acceptor for respiration, or at least to remove this toxic
compound in an effective manner. To this purpose, close to the nitrite reductases’ sequences, all the
denitrifying Thermus strains so far studied show an heterotrimeric nitric oxide reductase [54,61]
encoded by the norCBH operon and a NO-sensory system encoded by the nrsRST operon [62]. The genome of TthNAR1 does not encode the norCBH operon, neither in the region close to
the nitrite reductase genes nor in other regions of the genome. Actually, no residual coding regions
corresponding to these genes were located at the expected positions, suggesting that these genes were
lost a long time ago, or that another unrelated enzyme develops this function. However, homologs to
the NO-responsive regulator NsrR and its associated proteins NsrS and NsrT form a cluster almost
identical in sequence to that of TthPRQ25 or TthSG, supporting that the system is functional for
NO detection despite the absence of the norCBH genes. In this context, it is interesting to note that, 13 of 16 13 of 16 Genes 2020, 11, 1308 immediately upstream of the nsrRST operon, and encoded in the same transcriptional sense, two genes
are found that code for proteins annotated as cytochrome-oxidase subunits CbcB (TTHNP4_195) and
CbcA (TTHNP4_196). Keeping in mind the similarities and common phylogenetic origins between
NOR and cytochrome oxidases, and the presence of these two genes in a denitrification genomic context,
it is tempting to speculate that these enzymes actually use NO as substrate. It is also worth noting
the presence of a similar cbcBA cluster immediately upstream the sequence of NirK in T. scotoductus,
although in TthNAR1, an IS, is integrated in between them, suggesting a putative inactivation of the
corresponding promoter. Author Contributions: Conceptualization, J.B.; methodology, A.B. and M.S.-C.; resources, J.B.; data curation,
J.B. and M.S.-C.; writing—original draft preparation, J.B.; writing—review and editing, A.B.; visualization, A.B.,
M.S.-C.; supervision, J.B.; project administration, J.B.; and funding acquisition, J.B. All authors have read and
agreed to the published version of the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Supplementary Materials: The following data are available online at http://www.mdpi.com/2073-4425/11/11/
1308/s1, Figure S1: Plasmids synteny. Figure S2: Presence of transposases within the denitrification cluster. Funding: This research was funded by the Spanish Ministry of Science and Innovation, grant num
BIO2016-77031-R and PID2019-109073RB-I00. 4. Conclusions The current availability of whole genome sequences from many Thermus spp. has allowed
the identification of genes involved in different metabolic processes. Nonetheless, in the case of
denitrification, the existence of complete and partial denitrifying strains as well as aerobic ones has
been puzzling. The employment for many years of the TthNAR1 strain as a model to study nitrate
respiration demanded a complete and verified genome in order to achieve a better understanding,
and re-interpretation when needed, of those experimental results. Upon sequencing of the TthNAR1 strain with the PacBio technology, we identified systematic errors
in the reads that affected many genes, actually absent when the Illumina sequences were overlayed,
so a significant conclusion from these bioinformatic analysis arises: double method sequencing should
be mandatory for whole genome sequencing of high G + C content organisms. Once the sequences of
TthNAR1 were corrected, we were able to assemble four circular contigs, one of which, a megaplasmid
with a scaffold shared with the pTT27 megaplasmids of the aerobic strains HB27 and HB8, encoded the
enzymes involved in nitrate respiration, a metabolic trait extensively studied in this TthNAR1 strain. Moreover, thanks to the availability of the whole consensus sequence, we found a group of genes
encoding enzymes for nitrite respiration upstream the nitrate respiration cluster in what constitutes a
whole denitrification island. This element includes genes for nitrite respiration and a regulator for NO
sensing, but no genes encoding a NOR enzyme were found, despite their presence in other denitrifying
strains of the Thermus genus. In addition, we found a panoply of IS elements, apparently inserted in a
random manner, that either separated genes within a cluster (nirM from nirSJ) or inactivated them
(nirK) (Table 2), suggesting that the ability to respire nitrite was lost in an ancestor of the TthNAR1
strain and discarding the hypothesis that suggested the acquisition of the nitrate reductase cluster by
an aerobic ancestor to render the nitrate respiring strain. Supplementary Materials: The following data are available online at http://www.mdpi.com/2073-4425/11/11/
1308/s1, Figure S1: Plasmids synteny. Figure S2: Presence of transposases within the denitrification cluster. Author Contributions: Conceptualization, J.B.; methodology, A.B. and M.S.-C.; resources, J.B.; data curation,
J.B. and M.S.-C.; writing—original draft preparation, J.B.; writing—review and editing, A.B.; visualization, A.B.,
M.S.-C.; supervision, J.B.; project administration, J.B.; and funding acquisition, J.B. All authors have read and
agreed to the published version of the manuscript. 4. Conclusions ding: This research was funded by the Spanish Ministry of Science and Innovation, grant numbers
2016-77031-R and PID2019-109073RB-I00. Acknowledgments: The technical assistance of Esther Sánchez is also acknowledged. The next-generation
sequencing (NGS) data analysis was performed by the Genomics and NGS Core Facility at the Centro de
Biología Molecular Severo Ochoa (CBMSO, CSIC-UAM) which is part of the CEI UAM + CSIC, Madrid,
Spain—http://www.cbm.uam.es/genomica. Conflicts of Interest: The authors declare no conflict of interest. 14 of 16 14 of 16 Genes 2020, 11, 1308 References 1. Simon, J.; Klotz, M.G. Diversity and evolution of bioenergetic systems involved in microbial nitrogen
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network required for Pseudomonas aeruginosa nitrate respiration. J. Bacteriol. 2007, 189, 4310–4314. [CrossRef] 53. Schreiber, K.; Krieger, R.; Benkert, B.; Eschbach, M.; Arai, H.; Schobert, M.; Jahn, D. The anaerobic regulatory
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54. Alvarez, L.; Bricio, C.; Gomez, M.J.; Berenguer, J. Lateral transfer of the denitrification pathway among 53. Schreiber, K.; Krieger, R.; Benkert, B.; Eschbach, M.; Arai, H.; Schobert, M.; Jahn, D. The anaerobic regulatory
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54. Alvarez, L.; Bricio, C.; Gomez, M.J.; Berenguer, J. Lateral transfer of the denitrification pathway among
Thermus thermophilus strains. Appl. Environ. Microbiol. 2011, 77, 1352–1358. [CrossRef] 54. Alvarez, L.; Bricio, C.; Gomez, M.J.; Berenguer, J. Lateral transfer of the denitrification pathway among
Thermus thermophilus strains. Appl. Environ. Microbiol. 2011, 77, 1352–1358. [CrossRef] 55. Cava, F.; Berenguer, J. Biochemical and regulatory properties of a respiratory island encoded by a conjugative
plasmid in the extreme thermophile Thermus thermophilus. Biochem. Soc. Trans. 2006, 34, 97–100. [CrossRef] 56. Chahlafi, Z.; Alvarez, L.; Cava, F.; Berenguer, J. The role of conserved proteins DrpA and DrpB in nitrate
respiration of Thermus thermophilus. Environ. Microbiol. 2018, 20, 3851–3861. [CrossRef] 57. Nilkens, S.; Koch-Singenstreu, M.; Niemann, V.; Gotz, F.; Stehle, T.; Unden, G. Nitrate/oxygen co-sensing by
an Nrea/Nreb sensor complex of Staphylococcus carnosus. Mol. Microbiol. 2014, 91, 381–393. [CrossRef] 58. Alvarez, L.; Bricio, C.; Hidalgo, A.; Berenguer, J. References Parallel pathways for nitrite reduction during anaerobic
growth in Thermus thermophilus. J. Bacteriol. 2014, 196, 1350–1358. [CrossRef] 59. Cava, F.; Zafra, O.; da Costa, M.S.; Berenguer, J. The role of the nitrate respiration element of Thermus
thermophilus in the control and activity of the denitrification apparatus. Environ. Microbiol. 2008, 10, 522–533. [CrossRef] 60. Opperman, D.J.; Murgida, D.H.; Dalosto, S.D.; Brondino, C.D.; Ferroni, F.M. A three-domain copper-nitrite
reductase with a unique sensing loop. IUCrJ 2019, 6, 248–258. [CrossRef] 61. Bricio, C.; Alvarez, L.; Gomez, M.J.; Berenguer, J. Partial and complete denitrification in Thermus thermophilus:
Lessons from genome drafts. Biochem. Soc. Trans. 2011, 39, 249–253. [CrossRef] 62. Alvarez, L.; Quintans, N.G.; Blesa, A.; Baquedano, I.; Mencia, M.; Bricio, C.; Berenguer, J. Hierarchical control
of nitrite respiration by transcription factors encoded within mobile gene clusters of Thermus thermophilus. Genes 2017, 8, 361. [CrossRef] Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional
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Combination of adsorption/desorption and photocatalytic reduction processes for PFOA removal from water by using an aminated biosorbent and a UV/sulfite system
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Environmental research
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Environmental Research Combination of adsorption/desorption and photocatalytic reduction
processes for PFOA removal from water by using an aminated biosorbent
and a UV/sulfite system Combination of adsorption/desorption and photocatalytic reduction
processes for PFOA removal from water by using an aminated biosorbent
and a UV/sulfite system Zhongfei Ren a,*, Ulrich Bergmann b, Jean Noel Uwayezu c, Ivan Carabante c, Jurate
Tore Lejon c,d, Tiina Leivisk¨a a Ren a,*, Ulrich Bergmann b, Jean Noel Uwayezu c, Ivan Carabante c, Jurate Kumpiene
on c,d Tiina Leivisk¨a a a Chemical Process Engineering, University of Oulu, P.O. Box 4300, FIN-90014, Oulu, Finland
b Department of Biochemistry and Biocenter, University of Oulu, Oulu, FIN-99020, Finland
c Waste Science and Technology, Luleå University of Technology, Luleå, Sweden
d Department of Chemistry, UiT-The Arctic University of Norway, Norway Handling Editor: Aijie Wang Keywords:
Perfluorooctanoic acid
Advanced reduction process
Adsorption/ desorption
Biomass sorbent
UV/Sulfite system that in the contaminated site (ΣPFAS 32.0 μg/L) (Beale et al., 2022). In
addition, PFAS have also been detected in human urine, blood, nails,
hair and breast milk (Jian et al., 2018; Macheka-Tendenguwo et al.,
2018). Human exposure to PFAS has been proven to be associated with
many diseases, such as kidney cancer, testicular cancer and thyroid
disease (Jian et al., 2018; Sunderland et al., 2019). Perfluorooctanoic
acid (PFOA) and PFOS are two representative PFAS listed as persistent
organic pollutants (POPs) in the Stockholm Convention to limit their
production and application (Stockholm Convention, 2019). In June
2022, the US Environmental Protection Agency (USEPA) issued an
interim updated health advisory for PFOA and PFOS in drinking water, * Corresponding author.
E-mail address: zhongfei.ren@oulu.fi (Z. Ren). Environmental Research 228 (2023) 115930
Combination of adsorption/desorption and photocatalytic reduction
processes for PFOA removal from water by using an aminated biosorbent
and a UV/sulfite system
Zhongfei Ren a,*, Ulrich Bergmann b, Jean Noel Uwayezu c, Ivan Carabante c, Jurate Kumpiene c,
Tore Lejon c,d, Tiina Leivisk¨a a
a Chemical Process Engineering, University of Oulu, P.O. Box 4300, FIN-90014, Oulu, Finland
b Department of Biochemistry and Biocenter, University of Oulu, Oulu, FIN-99020, Finland
c Waste Science and Technology, Luleå University of Technology, Luleå, Sweden
d Department of Chemistry, UiT-The Arctic University of Norway, Norway
Contents lists available at ScienceDirect
Environmental Research
journal homepage: www.elsevier.com/locate/envres Environmental Research 228 (2023) 115930
Combination of adsorption/desorption and photocatalytic reduction
processes for PFOA removal from water by using an aminated biosorbent
and a UV/sulfite system
Zhongfei Ren a,*, Ulrich Bergmann b, Jean Noel Uwayezu c, Ivan Carabante c, Jurate Kumpiene c,
Tore Lejon c,d, Tiina Leivisk¨a a
a Chemical Process Engineering, University of Oulu, P.O. Box 4300, FIN-90014, Oulu, Finland
b Department of Biochemistry and Biocenter, University of Oulu, Oulu, FIN-99020, Finland
c Waste Science and Technology, Luleå University of Technology, Luleå, Sweden
d Department of Chemistry, UiT-The Arctic University of Norway, Norway
Contents lists available at ScienceDirect
Environmental Research
journal homepage: www.elsevier.com/locate/envres Environmental Research 228 (2023) 115930 Contents lists available at ScienceDirect A R T I C L E I N F O Per- and polyfluoroalkyl substances (PFAS) are stable organic chemicals, which have been used globally since the
1940s and have caused PFAS contamination around the world. This study explores perfluorooctanoic acid
(PFOA) enrichment and destruction by a combined method of sorption/desorption and photocatalytic reduction. A novel biosorbent (PG-PB) was developed from raw pine bark by grafting amine groups and quaternary
ammonium groups onto the surface of bark particles. The results of PFOA adsorption at low concentration
suggest that PG-PB has excellent removal efficiency (94.8%–99.1%, PG-PB dosage: 0.4 g/L) to PFOA in the
concentration range of 10 μg/L to 2 mg/L. The PG-PB exhibited high adsorption efficiency regarding PFOA, being
456.0 mg/g at pH 3.3 and 258.0 mg/g at pH 7 with an initial concentration of 200 mg/L. The groundwater
treatment reduced the total concentration of 28 PFAS from 18 000 ng/L to 9900 ng/L with 0.8 g/L of PG-PB. Desorption experiments examined 18 types of desorption solutions, and the results showed that 0.05% NaOH
and a mixture of 0.05% NaOH + 20% methanol were efficient for PFOA desorption from the spent PG-PB. More
than 70% (>70 mg/L in 50 mL) and 85% (>85 mg/L in 50 mL) of PFOA were recovered from the first and second
desorption processes, respectively. Since high pH promotes PFOA degradation, the desorption eluents with NaOH
were directly treated with a UV/sulfite system without further adjustment. The final PFOA degradation and
defluorination efficiency in the desorption eluents with 0.05% NaOH + 20% methanol reached 100% and 83.1%
after 24 h reaction. This study proved that the combination of adsorption/desorption and a UV/sulfite system for
PFAS removal is a feasible solution for environmental remediation. Available online 17 April 2023
0013-9351/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
https://doi.org/10.1016/j.envres.2023.115930
Received 19 January 2023; Received in revised form 4 April 2023; Accepted 15 April 2023 * Corresponding author.
E-mail address: zhongfe Available online 17 April 2023
0013-9351/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
* Corresponding author.
E-mail address: zhongfei.ren@oulu.fi (Z. Ren).
https://doi.org/10.1016/j.envres.2023.115930
Received 19 January 2023; Received in revised form 4 April 2023; Accepted 15 April 2023 2.1. Chemicals and reagents Sodium chloride, sulfuric acid, sodium hydroxide, hydrochloric acid,
absolute ethanol and methanol were purchased from VWR. Sodium
sulfite, polyethylenimine (PEI, ≥99%, Mw 25 000 by LS, branched) and
glycidyltrimethylammonium chloride (GTMAC, ≥90%), trifluoroacetic
acid (TFA, >99.0%), pentafluoropropionic acid (PFPrA, >98%), hep
tafluorobutyric acid (PFBA, >99%), nonafluoropentanoic acid (PFPeA,
>98%),
undecafluorohexanoic
acid
(PFHxA,
>98%),
trideca
fluoroheptanoic acid (PFHpA, >96%) and PFOA (>95%) were all pur
chased from Sigma-Aldrich. The fluoride ionic strength adjustment (ISA)
powder pillows and fluoride standard solutions (0.5, 1 and 2 mg/L) from
Hach were used for fluoride analysis. A stock solution of PFOA (2 g/L)
was prepared by dissolving PFOA in Milli-Q water (Merck Millipore). HCl, NaOH and H2SO4 solutions were used for pH adjustment. Unless
otherwise specified, all chemicals used were of analytical grade. i
The raw material, pine bark (PB), was collected from the RoihuPuu
Company (Oulu, Finland). After drying at 80 ◦C for 24 h, the dried PB
was then ground and sieved to obtain a 90–250 μm fraction for further
modification. The groundwater sample was collected close to a hot spot
where PFAS that contained aqueous film forming foams (FFF) had been
applied in firefighting training activities. The common separation technologies applied for PFAS removal
include surface-active foam fractionation (SAFF) (Burns et al., 2021),
membrane treatment (Lee et al., 2022) and sorption (Boyer et al.,
2021a). Of the various separation methods, sorption is a well-studied
approach for removing PFAS due to its relatively low cost and ease of
operation. The most investigated sorbents include activated carbon
(AC), commercial resins and biosorbents. AC is one of the most versatile
and cost-effective adsorbents applied for PFAS treatment (Yuan et al.,
2022). However, despite the high adsorption capacity in relation to
PFAS, the regeneration of spent ACs by conventional desorption eluents
is challenging (Du et al., 2014). One possible method for the regenera
tion of ACs is to use high temperature to decompose the adsorbed PFAS,
although this consumes more energy. For example, DiStefano et al. re
ported the reactivation of PFAS laden granular activated carbon (GAC)
by thermal treatment at 1650–2000 ◦F (899–1093 ◦C), which achieved
>99.99% of PFAS destruction (DiStefano et al., 2022). An alternative
choice to ACs is anion exchange resins (AER), which was reported to
remove both long- and short-chain PFAS efficiently in various concen
trations (Boyer et al., 2021a). Moreover, the regeneration of PFAS laden
resins by various salt solutions (e.g. 1. Introduction Per- and poly-fluoroalkyl substances (PFAS) have been widely used
globally, leading to extensive contamination around the world (Li et al.,
2020; Xu et al., 2021). Due to the high stability of the C–F bond, PFAS
are extremely resistant to oxidation and are recognized as persistent
pollutants. Besides, PFAS have been proven to have significant bio
accumulation effects and toxicity to the environment (Ghisi et al., 2019;
Helmer et al., 2022). It has been reported that the serum per
fluorooctanesulfonate (PFOS) concentration (889 ± 56 ng/mL) of tur
tles exposed to PFAS-contaminated water was 235 times higher than Z. Ren et al. Environmental Research 228 (2023) 115930 which recommends a level of 0.004 parts per trillion (ppt) for PFOA and
0.02 ppt for PFOS (US EPA, 2022). Thus, it is critical to develop effective
techniques for purification water contaminated with PFAS. photocatalytic reduction system was successfully applied for PFOA
removal. A novel biosorbent (PG-PB) was synthesized from pine bark
with surface modification using polyethylenimine (PEI) and glycidyl
trimethylammonium chloride (GTMAC). The properties of PG-PB were
analysed, and its adsorption kinetics, isotherms, and the effects of pH on
PFOA removal were investigated. Then this study focused on testing
various desorption solutions for PG-PB regeneration, which would allow
the reuse of the adsorbent and reduce disposal problems. The resulting
PFOA-rich effluent was directly treated with the UV/sulfite system in
order to permanently destroy the PFOA. i
In recent decades, various destructive technologies, such as
advanced oxidation processes (AOPs), advanced reduction processes
(ARPs), electrocatalysis and photodecomposition, have been investi
gated for the elimination of PFAS (cleavage of the C–F bond) from water
(Lu et al., 2020; Uwayezu et al., 2021). Among these techniques, AOPs
have shown good degradation performance for PFAS. However, AOPs
typically do not completely degrade long-chain PFAS, leading to the
formation of more stable short-chain PFAS and resulting in low
defluorination efficiency. (Chen and Zhang, 2006; Qian et al., 2016). In
contrast, ARPs have exhibited much higher defluorination (up to 99%)
and decomposition efficiency (up to 100%) by cleaving C–F bonds with
highly reactive radicals and hydrated electrons (eaq
−, E = −2.9 V) (Bentel
et al., 2019; Ren et al., 2021). Hydrated electrons can be generated from
water or from certain source chemicals such as iodide, sulfite or indole
under UV radiation. (Guo et al., 2019). 2.2. Preparation of PEI-GTMAC modified pine bark (PG-PB) The modification of pine bark was done in two steps by using a
method previously developed for peat biomass (Gogoi et al., 2019): 1)
Firstly, 4 g of pine bark particles were soaked in 40 mL water with
continuous stirring. The mixture was then heated to 60 ◦C, followed by
the addition of 10 g of PEI; stirring was continued for 6 h. After that, the
intermediate product (PEI-PB) was cooled down to room temperature
and washed with Milli-Q water several times to remove the extra PEI. 2)
Next, the wet PEI-PB was soaked in 40 mL 2 g/L NaOH solution and
heated to 60 ◦C with stirring. Then, 5 mL of GTMAC was added into the
mixture and stirring was continued for 6 h. The product (PG-PB) was
filtered and washed with Milli-Q water several times to remove extra
chemicals. Finally, the wet PG-PB was dried at 80 ◦C for 24 h and stored
for further use. 1. Introduction Among these, sulfite has been
recognized as a good reductant for PFAS degradation by ARPs due its
high efficiency and robustness. For example, in the study by Song et al. (2013), PFOA was totally degraded by the UV/sulfite system in 1 h and
the defluorination ratio reached 88.5% after 24 h (Song et al., 2013). Our recent study has shown that the UV/sulfite system works efficiently
even with high concentrations of coexisting compounds (1.709 M of
chloride, 5 mM of carbonate, 5 mM of phosphate and 10 mg/L of humic
acid) (Ren et al., 2021). However, the concentration of PFAS reported in
contaminated waters is usually too low (ng to μg/L level) to be directly
treated by ARPs (Li et al., 2020; Rahman et al., 2014). Therefore, pre
treatment methods are required to concentrate PFAS before applying
ARPs. 2.1. Chemicals and reagents NaCl, NaOH, NH4Cl, methanol and
ethanol) has been proved to be efficient. In Dixit et al.‘s study, 10% NaCl
solution (W/V) was used for PFAS desorption from Purolite® A860,
which resulted in 94% and 90% recovery of PFOA and PFOS, respec
tively (Dixit et al., 2020). Aminated biosorbents are environmentally
sustainable and cost-effective alternatives to AER, as the raw materials
are renewable and abundantly available (Vu and Wu, 2020). In the study
by Deng et al. aminated rice husk was prepared and used for the removal
of PFOA, perfluorobutanoic acid (PFBA) and PFOS from water, with a
sorption capacity of 2.49, 1.70 and 2.65 mmol/g, respectively (Deng
et al., 2013). Although aminated biosorbents have been widely inves
tigated for anion removal, studies related to PFAS sorption/desorption
by aminated biosorbents remain limited. Therefore, the development of
efficient biosorbent and desorption methods is essential to achieve
sustainable workflows for PFAS removal by a combined method of
sorption/desorption and ARPs. 2.4. Adsorption and desorption of PFOA The adsorption and desorption tests were carried out in 50 mL
centrifuge tubes. In a typical adsorption or desorption test, 0.02 g of PG-
PB was introduced into a 50 mL solution (dosage: 0.4 g/L) in a centrifuge
tube and mixed using tube rotators (Fisherbrand™ Multi-Purpose Tube
Rotators) at 25 rpm. The adsorption of PFOA at low concentration was
performed without pH adjustment at initial PFOA concentrations of
0.01, 0.05, 0.1, 0.15, 0.2, 0.5, 1, 1.5 and 2 mg/L. The effect of pH on
PFOA adsorption (200 mg/L) was studied at 10 different pH values (2, 3,
4, 5, 6, 7, 8, 9, 10 and 11) to determine the optimal pH conditions for PG-
PB. The adsorption kinetic experiments were conducted in 200 mg/L
PFOA solution at pH 3.3 with different time intervals (5, 10, 20, 30, 45,
60, 120, 180, 240, 300, 360 and 540 min). The adsorption isotherm
experiments were carried out with PFOA concentrations of 10, 20, 60,
100, 200, 400, 600 and 1000 mg/L for 24 h, at pH 3.3. The analytical
methods for PFOA are given in the supporting information. For the
desorption experiments, NaOH, NaCl, methanol and mixtures of them
(mixture details are provided in Section 3.6) were used as the regener
ation solution and the desorption efficiencies of PFOA were measured. The best performing regeneration solution was selected and used for The column study was done in transparent polymethyl methacrylate
(PMMA) columns connected to a peristaltic pump. The inner diameter of
the columns was 10 mm, and the height was 150 mm. The weight of the
filled PG-PB was 0.4 g and the height was around 40 mm. The columns
were filled with quartz sand before and after filling the sorbent to
maintain a good hydraulic property. Two PFOA solutions with different
concentrations, 40 mg/L and 80 mg/L (without pH adjustment), were
selected as the feed solution. The flow rate was 0.2 mL/min and the
temperature was 21 ◦C. Due to the high adsorption capacity of PG-PB,
the sampling interval was set to 3 h during the day, and 6 h during
the night. The Thomas model (Chu, 2010) was used to fit the column
adsorption data. The PFAS-contaminated groundwater was used for the batch
adsorption tests to evaluate the PFAS removal efficiency by the PG-PB. 2.3. Characterization of PG-PB To understand the physical and chemical properties of the bio
sorbent, the raw pine bark (PB), PEI-modified pine bark (PEI-PB) and
PEI-GTMAC-modified pine bark (PG-PB) were characterized by
Brunauer-Emmett-Teller (BET) surface area analysis, Barrett-Joyner-
Halenda (BJH) pore size and volume analysis, SEM, FTIR and XPS. The surface compositions and chemical bonding of PB, PEI-PB, PG-PB
and spent PG-PB were analysed by XPS (Thermo Fisher Scientific
ESCALAB 250Xi). The specific surface areas and pore size distributions
of PB, PEI-PB and PG-PB were obtained by N2 adsorption using the
ASAPTM 2020 Accelerated Surface Area and Porosimetry system
(Micromeritics) with the Brunauer-Emmett-Teller (BET) and Barrett-
Joyner-Halenda (BJH) methods. The morphology, particle size and In this study, a combination of sorption/desorption and a 2 Z. Ren et al. Environmental Research 228 (2023) 115930 Fig. 1. EDS mapping of a) PG-PB and b) PFOA-saturated PG-PB. Fig. 1. EDS mapping of a) PG-PB and b) PFOA-saturated PG-PB. elemental distribution of PB and PG-PB were analysed with a field
emission scanning electron microscope (FESEM, Zeiss Ultra Plus),
equipped with an energy-dispersive X-ray spectroscopy (EDS) detector. Functional groups on PB, PEI-PB, PG-PB and PFOA-saturated PG-PB
were analysed with a Fourier-transform infrared (FTIR) spectrometer
(Bruker, Vertex V80/DRIFT). further experiments. All samples were filtered through a 0.45 μm
membrane (VWR, polyethersulfone) to remove small particles of PG-PB
before the PFOA analysis. i The pseudo first order (Lagergren, 1898), pseudo second order
(Blanchard et al., 1984), intra-particle diffusion (Weber and Morris,
1963), Boyd (Boyd et al., 1947) and Elovich (McLintock, 1967) equa
tions were employed to fit the experimental data of the PFOA adsorption
kinetics. The non-linear Langmuir (1918), Freundlich (1907),
Redlich-Peterson
(Redlich
and
Peterson,
1959)
and
Dubinin-Radushkevich (Dubinin, 1947) equations were applied to fit the
experimental data of the PFOA adsorption isotherm. The expressions of
the equations are listed in the supporting information in Section S1. 3.1. SEM, BET-BJH and EDS analysis SEM analysis was performed to investigate the morphology of the
biosorbent. The FESEM images of PB, PEI-PB, PG-PB and PFOA-
saturated PG-PB showed that the surface modification and introduc
tion of PFOA did not cause any visible changes to the surface of the pine
bark particles (Fig. S1). However, the BET-BJH analysis showed that the
SSA and total pore volume were reduced after surface modification
(Table S1). This could be attributed to the reaction between PEI, GTMAC
and the PB surface that blocked the microporous structure, as the
average pore diameter increased from 17.69 nm to 72.36 nm after
GTMAC modification. The low SSA of PG-PB (0.2387 m2/g) also indi
cated that the SSA is not the key factor governing the PFOA adsorption. The EDS and EDS mapping results are given in Fig.S2 and Fig. 1. The EDS
results for PG-PB show that the Cl content increased to 9.85%, which
was in agreement with the XPS results (Section 3.3, Fig. S5). Besides, the Fig. 2. FTIR spectra of PB, PEI-PB, PG-PB and PFOA-saturated PG-PB. dosages were 0.4 g/L and 0.8 g/L. Before sampling, all treated samples
were filtered with a filter paper to remove particles. The groundwater
samples were analysed by Eurofins Food & Feed Testing Sweden AB. 2.4. Adsorption and desorption of PFOA All the adsorption experiments were done in 250 mL centrifuge bottles
in a rotary mixer for 24 h at room temperature (21 ◦C). The PG-PB 3 3 Environmental Research 228 (2023) 115930 Z. Ren et al. (21 ◦C). To obtain the desorption effluent, the adsorption experiments
were firstly conducted in 100 mg/L PFOA solution with 0.4 g/L PG-PB
for 24 h, then the spent PG-PB was regenerated with 500 mL of 0.05%
NaOH solution and a mixture of 0.05% NaOH +20% MeOH for 4 h
(solid: liquid = 1 : 2500). Finally, the desorption effluent was collected
and subjected to photocatalytic degradation. To investigate the effect of
the concentration on degradation performance, two desorption effluents
were diluted 10 times before UV/sulfite degradation, whereas the UV/
sulfite system was applied directly to two desorption effluents. The
dosage of sulfite was 10 mM and pH was measured by a pH meter
(inoLab® pH 7110). Fig. 2. FTIR spectra of PB, PEI-PB, PG-PB and PFOA-saturated PG-PB. 2.5. UV/sulfite treatment for desorption effluent A cylindrical glass container (78 mm in diameter and 270 mm in
height) was used as the UV degradation reactor. A UV lamp (TUV PL-S
11 W, 254 nm, Philips) was packed in a quartz tube and placed in the
centre of the reactor. Experiments were conducted at room temperature Fig. 3. C 1s XPS spectra of a) PB, b) PEI-PB, c) PG-PB and d) PFOA-saturated PG-PB. Fig. 3. C 1s XPS spectra of a) PB, b) PEI-PB, c) PG-PB and d) PFOA-saturated PG-PB. Fig. 3. C 1s XPS spectra of a) PB, b) PEI-PB, c) PG-PB and d) PFOA-saturated PG-PB. Environmental Research 228 (2023) 115
Fig. 4. N 1s XPS spectra of a) PB, b) PEI-PB, c) PG-PB and (d) PFOA-saturated PG-PB. t al. Environmental Research 228 (2 Z. Ren et al. Environmental Research 228 (2023) 115930 Fig. 4. N 1s XPS spectra of a) PB, b) PEI-PB, c) PG-PB and (d) PFOA-saturated PG-PB. Fig. 4. N 1s XPS spectra of a) PB, b) PEI-PB, c) PG-PB and (d) PFOA-saturated PG-PB. Fig. 4. N 1s XPS spectra of a) PB, b) PEI-PB, c) PG-PB and (d) PFOA-saturated PG-PB. EDS mapping (Fig. 1a) shows that Cl was distributed homogeneously on
the PG-PB surface. This result indicates that the quaternary ammonium
groups were successfully grafted onto the surface of the biosorbent after
GTMAC modification. After the adsorption of PFOA, dense EDS signals
of F were observed on the surface of the PFOA-saturated PG-PB (Fig. 1b),
and the F content increased to 37.4% while the Cl content dropped to
0.76% (Fig. S2). It can be deduced from the above results that the ion
exchange between Cl−and PFOA anions is one of the predominant
adsorption mechanisms. Table 1
Peak table of PB, PEI-PB, PG-PB and PFOA-saturated PG-PB from XPS survey. Table 1
Peak table of PB, PEI-PB, PG-PB and PFOA-saturated PG-PB from XPS survey. Sample
Name
peak BE (eV)
Atomic %
PB
C 1s
284.9
83.3
O 1s
532.6
16.2
N 1s
401.7
0.5
PEI-PB
C 1s
285.2
75.1
O 1s
532.2
15.6
N 1s
399.6
9.8
PG-PB
C 1s
285.2
80.4
O 1s
532.3
12.6
N 1s
399.9
6.1
Cl 2p
197.8
1.0
PFOA-saturated PG-PB
C 1s
286.8
41.3
O 1s
532.8
11.0
N 1s
401.4
5.2
F 1s
689.8
42.3
Cl 2p
199.0
0.2 3.4.1. PFOA removal at low concentration 3.4.1. PFOA removal at low concentration The C 1s spectra of the four samples are shown in Fig. 3. The C 1s
spectrum of PB showed three peaks (Fig. 3a) at 284.6 eV, 286.3 eV and
288.3 eV, which could be assigned to the C–C, C–O/C–N and C––O bonds
in the PB (Su et al., 2022). After PEI modification, the peaks at 285.8 eV
and 287.4 eV (Fig. 3b) were attributed to the C–N/C–O and C––O
groups. The same peaks were also observed in the PG-PB sample
(Fig. 3c), which suggests that the grafting of amine and quaternary
ammonium groups had been achieved. Two intensive peaks were
observed at 294.0 eV and 291.6 eV in the PFOA-saturated PG-PG sample
(Fig. 3d), which could be attributed to the CF3 and CF2 components of
PFOA (Liu et al., 2018). In addition, the peak at 290.0 eV probably
originated from the O–C––O group of PFOA. The above results indicate
the successful adsorption of PFOA on the PG-PB surface, which is also
consistent with the FTIR results. To validate the applicability of PG-PB at environmentally relevant
concentrations of PFOA, adsorption experiments were carried out at
initial PFOA concentrations ranging from 0.01 to 2 mg/L. The results
(Fig. 5a) indicate that PG-PB has excellent removal efficiency (96.4%)
even at a PFOA concentration as low as 10 μg/L. As the initial PFOA
concentration increased, the qe value increased linearly. At an initial
PFOA concentration of 2 mg/L, qe reached 4.95 mg/g and the removal
efficiency was 99.1%. The results indicate that the maximum adsorption
capacity was not reached at low concentrations of PFOA, which suggests
that changes in pH may not significantly affect PFOA removal efficiency
at these concentrations. Thus, PG-PB appears to be a feasible option for
removing PFOA within the environmentally relevant concentration
range. However, considering that PG-PB has a high adsorption capacity
for PFOA, and that high concentration of PFOA is needed in subsequent
degradation processes, the pH effect, isotherms, kinetics and desorption
experiments were investigated under a higher PFOA concentration level. The peak at 399.8 eV in the N 1s spectrum originated from the
organic nitrogen in the raw pine bark (Fig. 4a). After PEI modification,
the C–N peak shifted to 399.3 eV (Fig. 4b), and the proportion of ni
trogen increased dramatically (Table 1). 3.2. FTIR analysis The FTIR spectra of PB, PEI-PB, PG-PB and PFOA-saturated PG-PB
are shown in Fig. 2. The broad band at 3412 cm−1 is associated with the
hydroxyl group and hydrogen bonds (Fleming et al., 2019). The
stretching vibration of C–H presents a strong peak at 2922 cm−1, which
derives from the natural lignin-containing materials (Herbert, 1960). After PEI modification, a band appeared at 1660 cm−1, which was
attributed to the N–H bending vibration from the large amount of amine
groups derived from PEI (Sowmya and Meenakshi, 2014). However, in
PG-PB, the peak at 1660 cm−1 had shrunk significantly due to the re
action of amine groups and GTMAC, which linked the quaternary
ammonium groups onto the biosorbent (Gogoi et al., 2019). The band
observed in the PG-PB spectrum at 1480 cm−1 was assigned to the
symmetric stretching vibration of –CH3 group from quaternary ammo
nium (Ren et al., 2017). In addition, the broad band located at
2000-2200 cm−1 was assigned to the asymmetrical bending vibration of
the quaternary ammonium group (–N+(CH3)3) and the torsional oscil
lation of the trimethylammonium group (Zou et al., 2015). This corre
sponded well to the XPS results (Fig. 4c) as quaternary ammonium
groups were detected in the N 1s spectrum. The band at 1361 cm−1 is in accordance with the single bond –OH bending vibration (Li et al., 2015),
which is derived from the –COOH group of PFOA. In addition, the strong
peak at 1689 cm−1 is ascribed to the stretching vibration of –C––O and
–COOH in the carbonyl groups (Lin et al., 2015; Xu et al., 2015), which is
in line with the XPS results and the high amount of PFOA. The sharp
band at 1250 cm−1 was assumed to correspond to the C–F vibration of
PFOA (Lin et al., 2015; Xu et al., 2015). To summarize, FTIR analysis
confirmed the successful surface modification of PG-PB by PEI and
GTMAC, and high PFOA adsorption on the surface. 3.3. XPS analysis 3.3. XPS analysis 3.3. XPS analysis The XPS survey spectra are shown in Fig. S4. The C 1s, N 1s, Cl 2p and The XPS survey spectra are shown in Fig. S4. The C 1s, N 1s, Cl 2p and 5 5 Environmental Research 228 (2023) 115930 Z. Ren et al. (
)
Fig. 5. 3.4.1. PFOA removal at low concentration This could be attributed to the
large amounts of primary amine (-NH2 group), secondary amine
(-(CHx)2NH group) and tertiary amine groups (-(CHx)3N group) from the
PEI (Fig. S3a) modification (Gogoi et al., 2019). The new peak at 402.3
eV in the N 1s spectrum (Fig. 4c) of PG-PB can be ascribed to the qua
ternary ammonium groups (-(CH3)3N+Cl−) after GTMAC modification
(Gogoi et al., 2019; Cao et al., 2016). For PFOA-saturated PG-PB, the
C–N peak shifted from 398.8 eV to 399.4 eV. This is probably due to the
adsorption of PFOA by amine groups that changed the properties of the
C–N bond. 3.2. FTIR analysis a) PFOA removal at low concentration (pH = 5.2); b) Effect of pH on PFOA adsorption (Initial PFOA concentration = 200 mg/L); (PG-PB dosage: 0.4 g/L,
contact time = 24 h, T = 21 ◦C). Fig. 5. a) PFOA removal at low concentration (pH = 5.2); b) Effect of pH on PFOA adsorption (Initial PFOA concentration = 200 mg/L); (PG-PB dosage: 0.4 g/L,
contact time = 24 h, T = 21 ◦C). entration (pH = 5.2); b) Effect of pH on PFOA adsorption (Initial PFOA concentration = 200 mg/L); (PG-PB dosage: 0.4 g/L Fig. 5. a) PFOA removal at low concentration (pH = 5.2); b) Effect of pH on PFOA adsorption (Initial PFOA concentrati
contact time = 24 h, T = 21 ◦C). exchange was partly responsible for PFOA adsorption. However, as the
NaCl solution was not effective for PFOA desorption (Section 3.6), and
the PG-PB achieved an extremely high adsorption capacity for PFOA
(Section 3.4), this implies that other adsorption mechanisms such as
hydrophobic, electrostatic attraction and PFOA self-aggregation
contribute to the adsorption. A detailed discussion is given in Sections
3.4 and 3.6. F 1s high-resolution spectra of PB, PEI-PB, PG-PB and PFOA-saturated
PG-PB, are given in Figs. 3–4 and Fig. S5-S6. The main elements
observed on the PB surface (Table 1) were carbon (83.3%), oxygen
(16.2%) and nitrogen (0.46%). After PEI modification and GTMAC
modification, the atomic percentages of N in PEI-PB and PG-PB had
greatly increased, to 9.8% and 6.1%, respectively. Besides, a Cl 2p peak
was also detected for the PG-PB sample. These results indicate that the
amine group and quaternary ammonium had been successfully grafted
onto the biomass surface. The strong F 1s peak observed in the XPS
survey of PFOA-saturated PG-PB (42.3%, Table 1) suggests that PG-PB
has a high adsorption capacity for PFOA. 3.4.3. Adsorption kinetics and isotherms The effect of contact time on PFOA adsorption by PG-PB is presented
in Fig. 6a and b. The adsorption of PFOA needs a rather long time (6 h) to
reach equilibrium. However, 81.4% of the PFOA was rapidly removed
from the water within 2 h, resulting in an adsorption capacity of ≈407
mg/g. During this period, the cationic sites (quaternary ammonium
groups and protonated amine groups) were quickly filled with PFOA
anions. Then the removal efficiency was gradually increased and finally
reached equilibrium with an adsorption capacity of ≈456 mg/g (91.3%
of the PFOA was removed) at 6 h, after which it remained >450 mg/g
throughout the tests with longer contact times (6–9 h). i The kinetic experimental data of PFOA adsorption were fitted into
non-linear PFO, PSO, intra-particle diffusion, Boyd and Elovich equa
tions. The obtained parameters of each model are given in Table S2. Among these models, the PSO model provided the best fit to the
experimental data due to its highest correlation coefficient (R2 = 0.988),
lowest chi-square value (χ2 = 11.7) and good prediction of qe (472.6 mg/
g) compared to the qexp (456.0 mg/g). This indicates that the PSO model
is adequate to describe the PFOA adsorption process by PG-PB, whereas
the intra-particle diffusion and Elovich models have a low R2 (0.748 and
0.917) and high χ2 (289.0 and 111.8), which means they are not suitable
for evaluating the PFOA adsorption process. The Elovich model is
typically used to describe the chemisorption process (McLintock, 1967). The low R2 and high χ2 values indicated that the PFOA adsorption
mechanisms were not governed fully by chemisorption. The
intra-particle diffusion model is used for identifying the reaction path
ways and adsorption mechanisms. The plot of qt against t0.5 gives two
linear regions (Fig. 6b), referring to a multistep mechanism although the
fitting to the experimental data is rather poor. The Boyd model was
employed to determine the rate-limiting step by analysing the plot of Bt
versus time (t). If the plot is linear and passes through the origin,
intra-particle diffusion is the rate-limiting step. Otherwise, film diffusion
is likely to be the rate-limiting step. The results given in the supporting
information (Fig. S8) showed that the plot is not linear and did not pass
through the origin. This suggests that adsorption was probably
controlled by film diffusion. Fig. 6. 3.4.2. Effect of pH The effect of pH on PFOA adsorption by PG-PB was studied with a
contact time of 4 h; the results are given in Fig. 5b. The results show that
the pH has a significant impact on PFOA adsorption by PG-PB. In gen
eral, PG-PB was able to adsorb more PFOA at lower pH. More than
89.2% (qe = 446.1 mg/g) PFOA was removed at pH 2. At higher pH
conditions, the adsorption performance significantly decreased,
although it was still high. At an initial pH of 6, 7 and 8, the qe values
were reduced to 293.0, 258.0 and 259.4 mg/g, respectively. When the
pH was adjusted to 11, only 30.8% (qe = 154.2 mg/g) of the PFOA was
removed from the water. The high PFOA removal efficiency at low pH
could be attributed to the protonated amine groups under acidic con
ditions (Liu et al., 2018), which provided further binding sites in addi
tion to the quaternary ammonium group. A similar trend was observed
in a previous study related to sulfate adsorption by aminated peat (Gogoi
et al., 2019). This also indicates that the hydroxide anion might compete
with the PFOA anion for adsorption sites on PG-PB, which implies that
NaOH can be used as a desorption solution for PG-PB regeneration. The
pH values after adsorption were recorded and the pH was subjected to The Cl 2p XPS spectra are shown in Fig. S5. In the PB and PEI-PB
samples, no Cl 2p peak was detected (Fig. S5a, b). After GTMAC modi
fication, the Cl 2p1/2 and Cl 2p3/2 peaks were observed at 198.7 and
197.0 eV in the PG-PB (Fig. S5c) and the binding energy separation
between Cl 2p1/2 and Cl 2p3/2 peaks was 1.7 eV, which can be related to
the chloride on the quaternary ammonium groups (-(CH3)3N+Cl−) (Ren
et al., 2015). After PFOA adsorption, the atomic percentage of Cl
dropped from 0.97% to 0.24%. In addition, a strong peak at 688.9 eV
was observed in the F 1s spectrum (Fig. S6d), which implies that ion 6 Environmental Research 228 (2023) 115930 Z. Ren et al. change during the adsorption experiment (Fig. 5b). In the experiments
Fig. 6. 3.4.2. Effect of pH a) Pseudo-first-order, pseudo-second-order, Elovich and b) intra-particle
diffusion kinetics by a non-linear model and experimental kinetics for the
sorption of PFOA onto PG-PB; c) Langmuir, Freundlich, Redlich-Peterson and
Dubinin-Radushkevich isotherms obtained with non-linear equations and
experimental data for the sorption of PFOA onto PG-PB (PFOA concentration in
kinetics: 200 mg/L, PG-PB dosage: 0.4 g/L, pH = 3.3, contact time for iso
therms = 24 h, T = 21 ◦C). Z. Ren et al. between the PG-PB surface and the aqueous medium during the
adsorption process. It has been reported that the aqueous pKa of PFOA is
in the range of −0.5 to 4.2 (Zango et al., 2023), and most likely between
0 and 1 (Rayne and Forest, 2010; Cheng et al., 2009). This means PFOA
existed as deprotonated anions in this study. Based on the results of the pH tests and the literature, the dominant
mechanism responsible for PFOA adsorption is the electrostatic inter
action between PFOA anions and quaternary ammonium groups on PG-
PB. In addition, a large amount of amine groups on PG-PB are proton
ated at low pH values (Gogoi et al., 2019; Elwakeel, 2010), and the
electrostatic attraction between cationic amine groups and PFOA anions
further enhances the PFOA adsorption. Apart from electrostatic inter
action, hydrophobic interaction most likely plays an important role in
PFOA adsorption (Dixit et al., 2021). Studies have shown that PFOA and
PFOS can form hemi-micelles on the surface of a sorbent even when the
concentration is as low as 0.1%–1% critical micelle concentration (CMC)
(CMCPFOA: 15 696 mg/L, CMCPFOS: 4573 mg/L) (Du et al., 2014; Baran
et al., 2001; Johnson et al., 2007; Yu et al., 2009). For example, once the
carboxyl head of PFOA had bonded to the positive surface, the hydro
phobic tail of PFOA could aggregate and form a micelle bi-layer or
hemi-micelle with other PFOA molecules (Liu et al., 2018; Chen et al.,
2017), which further increased the PFOA uptake capacity. In summary,
the main adsorption mechanisms that contributed to PFOA adsorption
on PG-PB were ion exchange, electrostatic attraction and hydrophobic
interaction. 3.6. Regeneration studies on PG-PB To test the reusability of PG-PB, several different desorption eluents
were preliminarily selected for regeneration experiments and the
adsorption after each regeneration step was also examined (Fig. 8a and
b). In Fig. 8a, it is clear that water and NaCl solution (5% and 10%) show
very poor desorption efficiency for the used PG-PB (up to 1.7% PFOA
recovery by 5% NaCl). In contrast, NaOH solution (1% and 2%) and
100% MeOH exhibited acceptable desorption efficiency (up to 59.1%
with 1% NaOH). Moreover, the presence of MeOH in the NaOH solution
enhances the desorption. The mixture of NaOH and MeOH solution was
effective for PFOA desorption (up to 92.5% PFOA recovery by 50%
MeOH + 0.2% NaOH). However, it can be seen in Fig. 8b that the
adsorption capacity of regenerated PG-PB by NaOH and MeOH + NaOH
mixture decreased slightly. One possible explanation is that very alka
line conditions could dissolve cellulose from the surface of the PG-PB
(Kim et al., 2016; Zhong et al., 2017), which would lead to the loss of 3.4.3. Adsorption kinetics and isotherms a) Pseudo-first-order, pseudo-second-order, Elovich and b) intra-particle
diffusion kinetics by a non-linear model and experimental kinetics for the
sorption of PFOA onto PG-PB; c) Langmuir, Freundlich, Redlich-Peterson and
Dubinin-Radushkevich isotherms obtained with non-linear equations and
experimental data for the sorption of PFOA onto PG-PB (PFOA concentration in
kinetics: 200 mg/L, PG-PB dosage: 0.4 g/L, pH = 3.3, contact time for iso
therms = 24 h, T = 21 ◦C). change during the adsorption experiment (Fig. 5b). In the experiments
with an initial pH of below 7, the pH increased after adsorption, whereas
in the experiments with an initial pH of above 7 a decrease in pH after
adsorption was observed. This could be attributed to the equilibrium i
The adsorption isotherm experiments were performed with an initial
PFOA concentration from 10 to 1000 mg/L. The non-linear Langmuir,
Freundlich, Redlich-Peterson and Dubinin-Radushkevich equations
were used to fit the experimental data of the PFOA adsorption isotherm. 7 Z. Ren et al. Environmental Research 228 (2023) 115930 (
)
9
Fig. 7. a) Concentrations and proportion of the main PFAS in the contaminated groundwater; b) adsorption results of the 6 main PFAS by PG-PB (PG-PB dosage: 0.4
and 0.8 g/L, adsorption time: 24 h, temperature: 21 ◦C, pH = 7). d groundwater; b) adsorption results of the 6 main PFAS by PG-PB (PG-PB dosage: 0.4 Fig. 7. a) Concentrations and proportion of the main PFAS in the contaminated groundwater; b) adsorption results of the 6 main PFAS by PG-PB (PG-PB dosage: 0.4
and 0.8 g/L, adsorption time: 24 h, temperature: 21 ◦C, pH = 7). Fig. 7. a) Concentrations and proportion of the main PFAS in the contaminated groundwater; b) adsorption results of the
and 0.8 g/L, adsorption time: 24 h, temperature: 21 ◦C, pH = 7). ion of the main PFAS in the contaminated groundwater; b) adsorption results of the 6 main PFAS by PG-PB (PG-PB dosage: 0.4
emperature: 21 ◦C, pH = 7). Fig. 7. a) Concentrations and proportion of the main PFAS in the contaminated groundwater; b) adsorption results of the 6 main PFAS by PG-PB (PG-PB dosage: 0.4
and 0.8 g/L, adsorption time: 24 h, temperature: 21 ◦C, pH = 7). concentrations and proportions of the first five main PFAS contaminants
in the groundwater are given in Fig. 7a. Table 2 The related parameters of the two models are shown in Table S3 and the
fitting curves are given in Fig. 6c. Of the four models, the Redlich-
Peterson model has the highest R2 (0.986) although the χ2 (270.9) of
the model was high, which means the fitting has some defects. Compared with the Redlich-Peterson model, the Freundlich model gives
a better fit as it has a high R2 (0.973) and lower χ2 (122.4), indicating the
multilayer adsorption of PFOA on the heterogenous surface of the PG-PB
(Tran et al., 2017). As shown in Fig. 6c, the maximum PFOA adsorption
capacity of PG-PB reached 1171.3 mg/g with the initial PFOA concen
tration of 1 g/L. The high adsorption capacity could be attributed to the
large number of active binding sites of PG-PB and the unique composi
tion of PFOA. Unlike common inorganic anions, PFOA anions have a
long tail with a hydrophobic property, which makes it easy to form
micelles and hemi-micelles with other PFOA anions (Du et al., 2014). The column adsorption results and discussion are given in supporting
information Section S2. The high adsorption capacity and fast kinetics of
PG-PB make it an excellent sorbent for PFAS contamination. In other studies related to PFOA adsorption, powdered activated
carbons achieved capacities of 175–524 mg/g (Du et al., 2014), granular
activated carbons achieved 112–161 mg/g (Du et al., 2014), and AER
331–1436 mg/g (Gagliano et al., 2020). Thus, the capacity of PG-PB
(1173 mg/g) is close to the maximum capacity of AER, while it is
significantly higher when compared to the non-modified ACs. The high
adsorption efficiency is attributed to the numerous amine groups present
on the surface of the materials, which facilitate electrostatic attraction
between the protonated amine groups and anionic PFOA (Du et al.,
2014). While commercial resins share many of the same characteristics
as PG-PB, one notable advantage of PG-PB is its lower cost compared
with commercial resins (Du et al., 2014). 3.4.3. Adsorption kinetics and isotherms The concentration of per
fluorohexanesulfonic acid (PFHxS) in the groundwater was detected to
be 9700 ng/L, which accounted for 51.8% of the total 28 PFAS. The
results of all the adsorption experiments are given in Fig. 7b and full
results can be found in Table S5. The distribution coefficients Kd
(equations are given in Section S1) (US EPA, 2022) of the main PFAS
were calculated to estimate their distribution between adsorbent and
water (Table 2). In general, the total concentration of the 28 PFAS was
greatly reduced, from 18 000 ng/L to 11 000 ng/L and 9900 ng/L by 0.4
g/L and 0.8 g/L dosages of PG-PB, respectively. The PG-PB showed high
removal efficiency for PFHxS and PFOA in that 59.8% PFHxS (Kd: 1.859
L/g) and 43.3% PFOA (Kd: 0.956 L/g) were removed by 0.8 g/L PG-PB. Also, despite the low PFOS concentration (120 ng/L) in the ground
water, 87.5% of the PFOS was removed by 0.8 g/L PG-PB with a high Kd
value of 8.750 L/g. This finding suggests that PG-PB is effective in
binding PFOS, even at low concentrations, and could potentially prevent
the spread of PFOS to the surrounding environment. However,
compared to PFHxS, PFOA and PFOS, other short-chain PFAS, such as
perfluorobutanesulfonic acid (PFBS), perfluorohexanoic acid (PFHxA)
and perfluoropentanesulfonic acid (PFPeS), were hardly removed at all
by PG-PB. The possible reason for this is that short-chain PFAS have
much lower hydrophobicity than long-chain PFAS, which results in
lower adsorption efficiency. Nevertheless, as the PG-PB was validated to
be highly efficient for PFAS removal at low concentration levels of PFAS,
the usage of PG-PB for PFAS-contaminated groundwater treatment is
feasible. Table 2
Distribution coefficients (Kd) of the main PFAS in the contaminated
groundwater. PFAS species
Kd (L/g), 0.4 g/L of PG-PB
Kd (L/g), 0.8 g/L of PG-PB
PFBS
0.417
0.341
PFHxA
0.139
0.069
PFHxS
2.449
1.859
PFOA
1.250
0.956
PFOS
11.136
8.750
PFPeS
0.202
0.146 Table 2
Distribution coefficients (Kd) of the main PFAS in the contaminated
groundwater. 3.5. Groundwater treatment It is essential to investigate the effectiveness of PG-PB in treating real
PFAS-contaminated water when considering a practical application. The 8 Environmental Research 228 (2023)
adsorption capacity. However, it must be borne in mind that the PG-PB
difi d i
lk li
diti
(2 /L
f N OH (0 2%)) Th
f
0.025%) and a mixture of NaOH + MeOH were chosen as the deso
l
t f
f
th
d
ti
t
t
d th
i iti l PFOA
t
Fig. 8. a) PFOA desorption by different desorption solutions; b) PFOA adsorption by regenerated PG-PB. c) desorption of PFOA from spent PG-PB and d) adso
of PFOA by regenerated PG-PB; photocatalytic degradation of PFOA desorption solution in the UV/sulfite system: e) PFOA degradation; f) defluorination
initial concentration: 20 mg/L in a and b, 100 mg/L in c-f, desorption time: 4 h, adsorption time: 24 h, PG-PB dosage: 0.4 g/L, no pH adjustment, degradatio
24 h, sulfite dosage: 10 mM, T = 21 ◦C). Z. Ren et al. Environmental Research 228 (2023) 115930 Z. Ren et al. Fig. 8. a) PFOA desorption by different desorption solutions; b) PFOA adsorption by regenerated PG-PB. c) desorption of PFOA from spent PG-PB and d) adsorption
of PFOA by regenerated PG-PB; photocatalytic degradation of PFOA desorption solution in the UV/sulfite system: e) PFOA degradation; f) defluorination (PFOA
initial concentration: 20 mg/L in a and b, 100 mg/L in c-f, desorption time: 4 h, adsorption time: 24 h, PG-PB dosage: 0.4 g/L, no pH adjustment, degradation time:
24 h, sulfite dosage: 10 mM, T = 21 ◦C). adsorption capacity. However, it must be borne in mind that the PG-PB
was modified in alkaline conditions (2 g/L of NaOH (0.2%)). Therefore,
it was considered feasible to use alkaline conditions, i.e. at least 0.2%
NaOH, for PG-PB regeneration. In the following regeneration experi
ments, the NaOH solution as well as the mixture of MeOH and NaOH
solution were used as desorption solutions and the percentage of NaOH
was reduced to prevent a loss of adsorption capacity. 0.025%) and a mixture of NaOH + MeOH were chosen as the desorption
eluent for further desorption tests, and the initial PFOA concentration
was increased to 100 mg/L. The results showed that the 1st desorption
efficiency with 0.05% NaOH (73.7%) was more effective than with
0.025% NaOH (63.2%). 4. Conclusions and perspectives eluents containing 0.05% NaOH + 10% MeOH and 0.05% NaOH + 20%
MeOH, the desorption efficiency increased to 75.9% and 82.7%,
respectively. Similar results were observed in the 2nd desorption ex
periments. It is likely that the presence of methanol weakened the hy
drophobic interaction of PFOA (Zaggia et al., 2016), which helped to
release more PFOA from the sorbent into the water and slightly
increased the desorption efficiency. Therefore, the addition of MeOH to
NaOH solution could be a feasible method to improve the desorption
performance in practical applications. However, the 2nd desorption was
able to release more PFOA from the used PG-PB compared to the 1st
desorption. A possible explanation is that PG-PB has different active sites
on its surface. Some active sites such as the quaternary ammonium
group could bind PFOA tightly and be difficult to desorb with NaOH,
whereas other sites like protonated amine groups could be easily
recovered by NaOH and release PFOA into the water. The mechanism of
desorption and UV/sulfite degradation is illustrated in Fig. S10. The contamination of water by PFAS usually involves several chal
lenges, such as low concentration, wide distribution, large volume and a
long decomposition period, which makes it difficult to achieve PFAS
mineralization in contaminated water using a single treatment method. Therefore, to overcome these challenges, a combined adsorption/
desorption and UV/sulfite degradation process for PFOA elimination
was investigated and optimized in this study. A novel biosorbent was
successfully synthesized and applied for PFOA adsorption and desorp
tion. XPS, FTIR and SEM- EDS analyses revealed that amine groups and
quaternary ammonium groups were the main functional groups grafted
onto the PB surface, and both the amine and quaternary ammonium
groups contributed to the high PFOA adsorption capacity. The devel
oped PG-PB is highly efficient in terms of PFOA uptake from water at low
or natural pH values, exhibiting great potential for the treatment of
PFOA-contaminated water. The adsorption/desorption results show that
PG-PB has a high adsorption capacity for PFOA and that it can be re
generated and reused. The photo-reductive degradation of PFOA in the
desorption effluent by a UV/sulfite system reached high levels of
degradation and defluorination efficiency, indicating that the combi
nation of adsorption/desorption and a UV/sulfite process is a feasible
method for PFOA removal. i
Fresh PG-PB was able to remove 95% of the PFOA from the water. In
the 2nd adsorption stage (Fig. Baran, J.R., 2001. Fluorinated surfactants and repellents. New York. In: Kissa, Erik,
Wilmington, D.E., Marcel Dekker (Eds.), Revised and Expanded Surfactant Science
Series, second ed., vol. 97. https://doi.org/10.1021/ja015260a xiv + 616 pp.
$195.00. ISBN 0-8247-0472-X., J. Am. Chem. Soc. 123 (2001) 8882–8882. 3.7. Photocatalytic degradation of PFOA from desorption effluent The desorption effluent was subjected to photocatalytic degradation
(UV/sulfite system). The desorption solutions were 0.05% NaOH and
0.05% NaOH + 20% MeOH as their desorption efficiencies were the
highest according to the results (Fig. 8c). The photocatalytic degrada
tion results are given in Fig. 8e and f. The average concentration of PFOA in the diluted (10-fold) desorp
tion effluents was 7.60 mg/L (0.05% NaOH) and 8.18 mg/L (0.05%
NaOH + 20% MeOH), and the pH values were around 10.9. In the non-
diluted desorption effluents, the PFOA concentration was 76.93 mg/L
(0.05% NaOH) and 82.73 mg/L (0.05% NaOH + 20% MeOH), and the
pH values were around 11.5. The results (Fig. 8e) showed that almost all
of the PFOA was removed from the diluted desorption effluents after 24
h of degradation. However, 1.56 mg/L of PFOA was detected in the non-
diluted 0.05% NaOH + 20% MeOH effluent. The defluorination effi
ciency is known as a key factor when evaluating PFAS degradation
performance. As the initial PFOA concentrations differed, the defluori
nation efficiency was calculated to evaluate the degradation perfor
mance (Fig. 8f). The average defluorination efficiencies were 88.0% and
83.3% in the diluted 0.05% NaOH and 0.05% NaOH + 20% MeOH,
respectively. In the non-diluted desorption effluents, the defluorination
efficiencies were 83.1% and 74.9% in 0.05% NaOH and 0.05% NaOH +
20% MeOH, respectively. The above results indicate that a high con
centration of MeOH may have an adverse effect on PFOA degradation in
a UV/sulfite system. This may be because the MeOH was able to react
with hydrated electrons and hence reduced the defluorination efficiency
(Bolton et al., 1976). However, a high defluorination efficiency (74.9%)
could still be achieved even in the presence of 20% MeOH. Therefore, in
practical applications, the addition of MeOH could be considered as it
could slightly increase the desorption efficiency while sacrificing some
defluorination performance. One possible solution to keep the advan
tages and eliminate the disadvantages is to recycle the MeOH by evap
oration before UV/sulfite degradation (Boyer et al., 2021b). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.envres.2023.115930. 4. Conclusions and perspectives 8d), the regenerated PG-PB was still able
to remove 93–97% of the PFOA despite the incomplete desorption. However, a further decrease in the adsorption efficiency was observed in
the 3rd adsorption stage. One possible explanation is that the surface
structure and functional groups changed during the long contact time
with NaOH due to the dissolution of hemicellulose and lignin (Kim et al.,
2016; Mwaikambo and Ansell, 2002). Thus, it is better to accept partial
regeneration than to use too strong conditions for regeneration. Nevertheless, it was confirmed that the reuse of PG-PB is possible, and
NaOH as well as a mixture of NaOH and MeOH can be used for PG-PB
regeneration. 3.5. Groundwater treatment Also, the presence of MeOH in the NaOH so
lution enhanced the desorption efficiency. In addition, the presence of
10% and 20% MeOH in 0.025% NaOH solution increased the desorption
efficiency to 67.6% and 72.5% (Fig. 8c), respectively. For the desorption According to the preliminary tests, NaOH solution (0.05% and 9 9 Z. Ren et al. Environmental Research 228 (2023) 115930 Declaration of competing interest The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper. Credit author statement Zhongfei Ren: Conceptualization, Methodology, Data curation,
Writing- Original draft preparation, Writing- Reviewing and Editing,
Software, Formal analysis, Visualization, Investigation, Validation. Ulrich Bergmann: Methodology, Data curation, Reviewing and Editing,
Jean Noel Uwayezu: Resources, Reviewing and Editing, Ivan Car
abante: Funding acquisition, Resources, Reviewing and Editing. Jurate
Kumpiene: Funding acquisition, Methodology, Resources, Reviewing
and Editing. Tore Lejon: Funding acquisition, Methodology, Resources,
Reviewing and Editing. Tiina Leivisk¨a: Supervision, Funding acquisi
tion, Conceptualization, Methodology, Resources, Writing- Reviewing
and Editing. Acknowledgements The research was conducted as part of the Less-PFAS project “Sus
tainable management of PFAS-contaminated materials (ID: 20202462)”,
funded by the European Union program Interreg Nord 2019–2022 and
the Regional Council of Lapland, Norrbotten County Council and Troms
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interactions. Carbohydr. Polym. 174, 400–408. https://doi.org/10.1016/j. carbpol.2017.06.078. Weber, W.J., Morris, J.C., 1963. Kinetics of adsorption on carbon from solution. J. Sanit. Eng. Div. 89, 31–59. https://doi.org/10.1061/JSEDAI.0000430. l Xu, C., Chen, H., Jiang, F., 2015. Adsorption of perflourooctane sulfonate (PFOS) and
perfluorooctanoate (PFOA) on polyaniline nanotubes. Colloids Surf. A Physicochem. Eng. Asp. 479, 60–67. https://doi.org/10.1016/j.colsurfa.2015.03.045. Zou, X., Zhao, X., Ye, L., 2015. Synthesis of cationic chitosan hydrogel with long chain
alkyl and its controlled glucose-responsive drug delivery behavior. RSC Adv. 5,
96230–96241. https://doi.org/10.1039/C5RA16328E. Xu, B., Liu, S., Zhou, J.L., Zheng, C., Weifeng, J., Chen, B., Zhang, T., Qiu, W., 2021. PFAS
and their substitutes in groundwater: occurrence, transformation and remediation. J. References Hazard Mater. 412, 125159 https://doi.org/10.1016/j.jhazmat.2021.125159. 12
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THE FACTOR OF CASPIAN ENERGY RESOURCES IN TURKISH-AZERBAIJANI RELATIONS
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Zenodo (CERN European Organization for Nuclear Research)
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ABSTRACT After the restoration of Azerbaijan's independence in 1991, Turkey became the first country to recognize it
on November 9, 1991. Turkey's recognition of Azerbaijan's independence as the first state and the establishment
of diplomatic relations between the parties accelerated the process of our country's integration into the world
community, increased its political weight and attractiveness for civilized countries. The Azerbaijani-Turkish
strategic partnership and brotherhood are reflected in the "One Nation - Two States" model, the author of which is
national leader Heydar Aliyev. Later, the importance of the energy resources of the Caspian Sea began to increase
in Turkish-Azerbaijani relations. It is no coincidence that Turkey is the main country in the transportation of
Azerbaijani energy resources to the world. This article touches upon the importance of the Caspian energy
resources in Turkish-Azerbaijani relations. Ключевые слова: Турция, Каспийское море, Азербайджан, нефть, газ. Keywords: Turkey, Caspian Sea, Azerbaijan, oil, gas. области Президент Азербайджанской Республики
Ильхам Алиев проявляет большую активность и
выступает совместно с главой государства Турции
Реджепом Тайипом Эрдоганом. Азербайджан и
Турция уже предпринимают серьезные шаги по
обеспечению солидарности турецко-исламского
мира. Введение. Дипломатические
отношения
между Азербайджаном и Турцией были установ-
лены 14 января 1992 года [1; 402]. Проследив вос-
ходящее развитие азербайджано-турецких отноше-
ний в 1993-2003 годах в хронологическом порядке,
можно понять, что политический архитектор и
неутомимый организатор прогресса отношений
двух государств, на новом уровне, в новом направ-
лении, именно общенациональный лидер Гейдар
Алиев. Гейдар Алиев определил форму отношений
между Азербайджаном и Турцией как «Одна нация,
два государства» [18; 74]. Особые отношения Азербайджана с Турцией и
его интеграция в международную систему с его
территориальной целостностью и богатыми энерге-
тическими ресурсами также важны для Турции. Турция отстаивала решение Ларабахской про-
блемы, защищая территориальную целостность
Азербайджана. В течение 2003-2013 годов эти отношения
быстро развивались в первые 10 лет пребывания
Президента Ильхама Алиева на посту главы госу-
дарства и охватывали ряд сфер, в том числе эконо-
мические и геополитические отношения, военное
строительство. Триумф новой нефтяной стратегии,
заложенной великим лидером Гейдаром Алиевым,
успешно продолжается с 2003 года под руковод-
ством Президента Азербайджанской Республики
Ильхама Алиева и реализуется новый геополитиче-
ский курс. Этот курс направлен на то, чтобы сде-
лать Азербайджан одной из ведущих стран мира,
которая будет влиять на мировые события. В этой Азербайджанская политика Турции связана со
многими аспектами. Один из этих аспектов связан
с транспортировкой энергоресурсов Азербайджана
на мировые рынки. АННОТАЦИЯ После восстановления независимости Азербайджана в 1991 году Турция стала первой страной, при-
знавшей его 9 ноября 1991 года. Признание Турцией независимости Азербайджана в качестве первого гос-
ударства и установление дипломатических отношений между сторонами ускорили процесс интеграции
нашей страны в мировое сообщество, усилили ее политическое весомость и привлекательность для циви-
лизованных стран. Азербайджано-турецкое стратегическое партнерство и братство нашли отражение в мо-
дели «Одна нация – два государства», автором которой является общенациональный лидер Гейдар Алиев. Позже в турецко-азербайджанских отношениях стало возрастать значение энергоресурсов Каспийского
моря. Неслучайно Турция является основной страной в транспортировке азербайджанских энергоресурсов
в мир. В данной статье затрагивается значение каспийских энергоресурсов в турецко-азербайджанских
отношениях. THE FACTOR OF CASPIAN ENERGY RESOURCES IN TURKISH-AZERBAIJANI RELATIONS Mamedli G. Academy of Public Administration under
the President of the Republic of Azerbaijan Sciences of Europe # 115, (2023) Sciences of Europe # 115, (2023) 81 Мамедли Г.Х. Академия Государственного Управления
при Президенте Азербайджанской Республики Мамедли Г.Х. Академия Государственного Управления
при Президенте Азербайджанской Республики ABSTRACT Но до этого нужно отметить
что, «Контракт века» стал важным контрактом [6],
который в последствии послужил также укрепле-
нию отношений между Турцией и Азербайджаном. На момент подписания «Контракта века» ту-
рецкая компания ТПАО владела 1,75 % акций кон-
сорциума. Однако после обращения правительства
Турции к Гейдару Алиеву 11 июля 1995 года было 82 Sciences of Europe # 115, (2023) Реализация этого проекта сделала Турцию одной из
основных стран, через которое проходят линии пе-
редачи энергоресурсов Азербайджана [16, 99]. подписано соглашение о передаче 5 процентов
доли Государственной Нефтянной Компании Азер-
байджанской Республики (SOCAR) в международ-
ном нефтяном консорциуме. Так, доля Турции в
консорциуме по разработке нефтяных месторожде-
ний Азербайджана увеличена до 6,75 процента [9,
31]. Двух трубопроводов (Баку-Новороссийск и
Баку-Супса) построенных для реализации новой
нефтяной стратегии Азербайджана, свободной и
беспрепятственной транспортировки углеводород-
ных ресурсов Каспийского моря на мировые рынки,
оказалось недостаточно. При этом, несмотря на все
давление, извне, Азербайджан осуществил проект
трубопровода БТД. 18 ноября 1999 года во время
Стамбульского саммита ОБСЕ было подписано с
участием Президента Азербайджана Гейдара Али-
ева, Президента Грузии Э. Шеварднадзе и Прези-
дента Турции Сулеймана Демиреля «Соглашение
между Азербайджаном, Грузией и Турецкой Рес-
публикой о транспортировке сырой нефти по тер-
ритории республики по трубопроводу БТД»». В то
же время президенты Азербайджанской Респуб-
лики, Грузии, Турецкой Республики, Республики
Казахстан и США подписали «Стамбульскую де-
кларацию» в поддержку этого проекта [6]. Государственная Нефтяная Компания Азер-
байджана SOCAR имеет представительства в Гру-
зии, Турции, Румынии, Австрии, Швейцарии, Ка-
захстане, Англии, Иране, Германии и Украине, а
также коммерческие компании в Швейцарии, Син-
гапуре, Вьетнаме и Нигерии. В компании работает
более 70 тысяч сотрудников. В последние годы ин-
вестиции SOCAR в Турцию начались с покупки
крупнейшего в Турции нефтехимического завода
PETKİM, расположенного в Измире. 51 процент
публичных акций Petkim Petrokimya Holding A.Ş. был продан SOCAR & Turcas Energy A.Ş. (STEAŞ)
за 2 миллиарда 40 миллионов долларов в результате
объявленного тендера, проведенного 30 мая 2008
года. В 2011 году все акции Turcas Petrol A.Ş., вла-
деющей 25 % STEAŞ, основного акционера Petkim,
были переданы SOCAR. Основанная в 1965 году
компания Petkim Petrokimya Holding A.Ş. крупней-
шая отечественная и единственная интегрирован-
ная компания нефтехимической отрасли. Прилега-
ние мощностей PETKİM к нефтеперерабатываю-
щему
заводу,
возможность
создания
новых
мощностей, большая площадь и инфраструктура,
наличие морского побережья и крупнотоннажного
порта, широкий ассортимент продукции и отсут-
ствие рыночных проблем сделали PETKİM исклю-
чительно стратегическое учреждение [20, 91]. ABSTRACT 1 августа 2002 года для строительства и управ-
ления этой линией была создана компания «BTC
Co» (БТД Ko). Оператором проекта выступила ком-
пания «BP-Азербайджан», выступившая основным
инвестором компании «BTC Co». 18 сентября 2002
года в Сангачале был заложен фундамент под стро-
ительство нефтепровода, а в состоявшейся в Баку
церемонии закладки первого камня приняли уча-
стие президенты Азербайджанской, Турецкой Рес-
публики и Грузии, министр энергетики США[19]. Владельцем нефтепровода является международ-
ный консорциум BTC Co. Акционеры BTC Co. BP (30,1%), Государственная Нефтяная Компания
Азербайджана
(ГНКАР)
(25%),
Mol Group
(8.90%), Equinor (8,71%), TPAO (6,53%), Eni (5%),
Total Energies (5%), İtochu (3,4%), İNPEX (2.5%),
ExxonMobil (2.5%), ONGC Videsh (2.36%) [4]. Экономические отношения между двумя стра-
нами приобрели новый размах, когда Государ-
ственная
Нефтяная
Компания
Азербайджана
(ГНКАР) приобрела 51 % акций PETKİM, одной из
самых авторитетных нефтяных компаний Турции. SOCAR объявила об инвестиционной стратегии на
сумму более 17 миллиардов долларов до 2018 года
в крупнейшую нефтехимическую компанию Тур-
ции PETKİM. Благодаря этому активность произ-
водственных мощностей PETKİM достигла 92 %, а
чистая прибыль в первом квартале 2021 года соста-
вила 891 757 млн турецких лир (TL). Однако за тот
же период 2020-го года - на пике пандемии - квар-
тал закончился с убытком в размере 9 781 млн ту-
рецких лир. Позже, благодаря правильному исполь-
зованию финансовых ресурсов и оптимизации опе-
рационных расходов, компания изменила ситуацию
и превратила ее в годовую чистую прибыль в раз-
мере 1,1 млрд турецких лир [15]. Инвестиции
SOCAR в Турцию не ограничились приобретением
PETKİM. Увеличение потенциала PETKİM и инве-
стиции в технологическую инфраструктуру в соот-
ветствии с мировыми стандартами осуществляются
быстрыми темпами. Основной
экспортный
нефтепровод
БТД
транспортирует азербайджанскую нефть из Кас-
пийского моря в турецкий порт Джейхан, а оттуда
на европейские рынки через Средиземное море. В
долгосрочной перспективе планируется транспор-
тировать Казахстанскую нефть и природный газ из
Туркменистана вместе с Азербайджаном по кори-
дору Восток-Запад. Соответственно, Казахстанская
нефть и природный газ из Туркменистана будут
транспортироваться в Баку по планируемой
Транскавказской магистрали по дну Каспийского
моря, а оттуда в Турцию по трубопроводу БТД. Длина линии 1768 км, диаметр линии 46/42/34
дюйма, пропускная способность 1,2 млн баррелей в
сутки [3; 29]. В экономических отношениях между Азербай-
джаном и Турцией приоритет отдавался созданию
правовой инфраструктуры с целью создания проч-
ной основы отношений. В следующем процессе
экономические
отношения
и
сотрудничество
должны развиваться и расширяться с динамичным
пониманием, более комплексными проектами в
рамках взаимных и общих интересов. ABSTRACT Наконец, 28
мая 2006 года азербайджанская нефть достигла Одним из проектов, имеющих особое значение
во взаимоотношениях двух стран является проект
Баку-Тбилиси-Джейхан. Трубопровод Баку-Тби-
лиси-Джейхан имеет большое значение не только с
экономической, но и с политической точки зрения. 83 Sciences of Europe # 115, (2023) порта Джейхан. Проект трубопровода Баку-Тби-
лиси-Джейхан, который некоторые считают вы-
мышленным проектом, который невозможно реа-
лизовать, начался со Стамбульского меморандума
о взаимопонимании 1998 года и был официально
открыт 13 июля 2006 года. Экспорт на западные
рынки начался 2 июня 2006 г. Баку-Тбилиси-Джей-
хан (БТД) как энергетический проект является все-
мирным инженерным проектом. Проект БТД явля-
ется новаторским проектом, демонстрирующим ре-
ализацию идеи «энергетического коридора» [24] Эрзурум», наряду с положительным прогрессом,
который будет наблюдаться в Азербайджане и его
экономике, экономические отношения между Азер-
байджаном и Турцией также усилились. Наблюдае-
мая стабильность и процесс развития в экономике
привели к положительному влиянию на торговые
отношения между Азербайджаном и Турцией и
приток турецкого капитала в Азербайджан. р
ур
р
TANAP, новый проект, предложенный прави-
тельством Азербайджана, представляет собой про-
ект, предусматривающий экспорт азербайджан-
ского газа в Европу в рамках Южного Газового Ко-
ридора. Природный
газ,
добываемый
на
«Шахдениз-2» и других районах к югу от него,
предполагалось транспортировать в Турцию, а за-
тем на европейские рынки по трубопроводу
TANAP. Решение о транспортировке азербайджан-
ского газа в Европу по проекту TANAP было реа-
лизовано полностью в соответствии с прямыми
инициативами, политической волей и идеями пра-
вительств Азербайджана и Турции. Президент
Азербайджана Ильхам Алиев сказал на церемонии
(17.03.2015) закладки фундамента TANAP: «Этот
проект является проектом энергетической безопас-
ности, но в первую очиредь этот проект является
проектом турецко-азербайджанского единства и
братства. Мы поддерживаем друг друга во всех
сферах. Как всегда, Азербайджан начал этот про-
ект, взяв на себя всю ответственность, показав при
необходимости совместное предприятие. Мы бу-
дем реализовывать этот проект вместе» [11]. Этот
проект помог Азербайджану экспортировать свой
газ посредством Турции. Таким образом, в формировании энергетиче-
ского коридора Восток-Запад проливы являются
наиболее важным маршрутом, повышающим гео-
политическое и геостратегическое значение Тур-
ции и обеспечивающим ее политическую значи-
мость. Азербайджано-турецкие экономические от-
ношения, конечно же, не ограничиваются участием
в крупных проектах или партнерскими отношени-
ями - страны уже давно числятся в списке основных
инвесторов друг друга [10]. Из-за этого в том числе
стабильность на Кавказе стала реальностью. В 2006
году по трубопроводу БТД было экспортировано
9,4 млн тонн нефти, в 2007 году — 28 млн тонн [17;
228]. ABSTRACT Объем ежедневного экспорта нефти из БТД в
начале 2009 года составлял 1 млн баррелей, то есть
50 млн тонн в год, хотя в последующие годы наблю-
далось частичное снижение, этот объем составлял
около 40-50 млн тонн в год [17; 228]. С 2011 года
экспорт нефти начал сокращаться. На момент вы-
хода на полную мощность примерно 1,3% мировых
поставок нефти транспортируется по БТД. С увели-
чением объема экспорта нефти, кажется, что внут-
ренние инвестиции также начали увеличиваться. Важный для энергетической безопасности Ев-
ропы проект TANAP планировалось реализовать в
течение пяти лет и будет стоить примерно 9,2 мил-
лиарда долларов США. Доли акционеров TANAP в
этом проекте составляют: ЗАО "Газовый коридор
"Джануб" - 58 процентов, "Боташ" - 30 процентов и
компания BP - 12 процентов [23]. р
у
Строительство газопровода Баку-Тбилиси-Эр-
зурум (БТЭ) для транспортировки природного газа
с месторождения «Шахдениз» началось в 2004
году. Завершить строительные работы планирова-
лось в 4 квартале 2005 года, а зимой 2006 года
начать экспорт природного газа. На первом этапе
природный газ, добытый на месторождении
«Азери», использовался для заполнения трубопро-
вода Баку-Тбилиси-Эрзурум. С июля 2007 года
начался экспорт природного газа по этому трубо-
проводу. В то время как грузинская часть проекта
газопровода Баку-Тбилиси-Эрзурум начала рабо-
тать в 2006 году, в связи с продлением строитель-
ства турецкой части линии природный газ Шах-Де-
низ смог достичь Эрзурума 3 июля 2007 года. Та-
ким образом, Турция начала получать природный
газ из Азербайджана намного дешевле, чем Россия,
по трубопроводу БТЭ. Общая длина пояса состав-
ляет 970 км, часть в Азербайджане – 442 км, часть
в Грузии – 248 км, часть в Турции – 280 км. Трубо-
провод, способный транспортировать 30 миллиар-
дов кубометров природного газа в год, начинается
от терминала в Баку и пересекает ту же террито-
рию, что и нефтепровод БТД в Турцию. Запасы ме-
сторождения Шах-Дениз, которые будут питать
БТЭ в рамках проекта «Этап-1», оценивались в 178
млрд кубометров [12; 39-60]. Проект TANAP играет важную роль в сниже-
нии зависимости Турции от природного газа и удо-
влетворении растущего спроса на газ. Разнообразие
ресурсов TANAP в Турции позволяет ее стратегии
обеспечивать надежность поставок. Мы не можем рассматривать этот проект
только как газовый проект. TANAP является важ-
ным проектом с политическими и экономическими
аспектами, который приведет к развитию экономи-
ческих и политических отношений Турции со стра-
нами региона. TANAP усилит влияние Турции в ре-
гионе. В то же время это «поднимет позиции Тур-
ции в глобальных энергетических проектах». ABSTRACT Через TAP оплачива-
ется 33 процента потребности в газе Болгарии, 20
процентов потребности Греции и до 10,5 процента
потребности Италии. Транспортировка по трубо-
проводу ТАР газа добываемая в рамках проекта
«Шах Дениз-2» в Европу первоначально составит
10 миллиардов кубометров в год. Трубопровод
начал действовать с 1 января 2021 года [22]. Генеральный
директор
TANAP
Салтук
Дюзйол сказал: «Чем больше трубопроводов про-
ходит по территории Турции, тем большую роль
они играют в обеспечении энергетических потреб-
ностей Турции. Такие инвестиции обеспечат Тур-
ции доступ к более дешевому газу. Потому что бу-
дет рынок, где газ будет конкурировать с газом [5]. Турция, которая из-за своего географического по-
ложения является энергетическим терминалом
между востоком и западом, стремится стать энерге-
тическим центром помимо этой проблемы. Проект
TANAP имеет большое значение как конкретный
проект, укрепляющий цель Турции стать центром. От проектов TANAP и TAP, играющих роль
моста в проекте по транспортировке азербайджан-
ского газа в Европу, Турция ежегодно получает
около 1,5 миллиарда долларов транспортного до-
хода [21]. Можно сделать вывод, что Турция является
примером для Азербайджана как в экономическом,
так и в плане интеграции в мировую экономиче-
скую среду. Период в торгово-экономических отно-
шениях между Турцией и Азербайджаном характе-
ризуется установлением более тесных институцио-
нальных экономических связей, привлечением
крупных турецких компаний и облегчением до-
ступа азербайджанских компаний на турецкий ры-
нок. Изречения Мустафы Кемаля Ататюрка «Ра-
дость Азербайджана – наша радость, его горе –
наша печаль» и Гейдара Алиева «Мы одна нация,
два государства» считаются самой совершенной
формулой как в Турции, так и в Азербайджане. он
выражает историю, действительность и перспек-
тивы отношений между двумя странами. Благодаря Трансанатолийскому газопроводу
(TANAP) потребности Европы в энергии были ча-
стично удовлетворены, а геостратегическое значе-
ние Турции укреплено. Напряженность между Рос-
сией и Украиной сделала проект TANAP еще более
важным. В проекте, инициированном Турцией и
Азербайджаном, азербайджанский природный газ
сыграл важную роль в обеспечении энергобезопас-
ности ЕС, а Турция также сыграла роль моста и по-
лучила значительный доход. у
Запуск
Эскишехирского
участка проекта
TANAP был согласован с началом добычи газа по
проекту «Шахдениз-2» и первая товарная поставка
газа состоялась 30 июня 2018 года. В апреле 2019
года начата пробная подача газа в рамках первого
этапа Трансанатолийского газопровода (TANAP),
являющегося частью проекта «Южный Газовый
Коридор». При участии глав государств Азербай-
джана и Турции строительство части проекта Юж-
ного Газового Коридора, проходящей через Тур-
цию, завершилось церемонией открытия части со-
единяющего с Европой газопровода TANAP-
Трансанатолийский в городе Ипсала, провинция
Эдирне, Турция [10]. ABSTRACT Поскольку азербайджанский газ, который бу-
дет поставляться через TANAP, покроет 25% расту-
щего спроса Турции на газ, он внесет важный вклад
в снижение зависимости Турции от природного
газа в России и Иране. Тот факт, что Турция явля-
ется региональным энергетическим центром, также
повысит ее значение как важной страны в обеспе-
чении безопасности энергоснабжения Европы. Правда, некоторые утверждают, что TANAP — это
геополитический проект [13]. Конечно, с реализацией нефтепроводов «Баку-
Тбилиси-Джейхан» и газопровода «Баку-Тбилиси- 84 Sciences of Europe # 115, (2023) и Европу (19,5) было переведено 40 миллиардов ку-
бометров азербайджанского газа [25]. Турция, которой принадлежит 30% акций
TANAP, будет использовать транспортируемый че-
рез ее территорию газ для собственного внутрен-
него потребления, а также будет получать плату за
транзит газа в качестве транзитной страны. Турция
покупает газ у России по высокой цене. Однако газ
Шах Дениз II, который будет транспортироваться
по TANAP, будет выгоден Турции с точки зрения
ценовой безопасности. Азербайджанский газ в Тур-
цию из TANAP будет на 12% дешевле российского
газа. Таким образом, Турция заплатит 88% цены,
которую она заплатила России за природный газ,
купленный у TANAP [8]. Еще одним важным проектом в турецко-азер-
байджанских отношениях является TAP. С вводом
в эксплуатацию указанной линии добываемый в
Азербайджане газ через Турцию поступает в Ев-
ропу. TAP является частью Южного Газового Ко-
ридора стоимостью 40 миллиардов долларов, кото-
рый строится для транспортировки природного
газа, добываемого в Азербайджане, в Европу. В за-
явлении Минэнерго говорится, что «Южный газо-
вый коридор укрепит глобальную систему энерге-
тической безопасности. ТАР запущен из Каспий-
ского региона в Европу [2]. Если мы оценим будущее проекта TANAP, то
увидим, что по этому трубопроводу в будущем
можно транспортировать не только азербайджан-
ский газ, но и газ из других стран. Проект откроет
путь для транспортировки туркменского газа по
дну Каспийского моря в Европу через Турцию. Еще
одна важная прикаспийская страна, Казахстан,
также может поставлять газ для TANAP. Проект
TANAP также является важным вкладом в между-
народное сотрудничество [14]. 17 мая 2016 года в Салониках, Греция, состоя-
лась церемония закладки фундамента TAP. В авгу-
сте 2020 года выполнено 97% работ по строитель-
ству Трансадриатического газопровода. Общая
протяженность газопровода составляет 878 км. 550
км из них покрывают территорию Греции, 215 км -
Албанию, 105 км - Адриатическое море, 8 км - Юж-
ную Италию. Самая высокая точка газопровода
находится в горах Албании (1800 м), а самая низкая
часть (-820 м) - на дне моря. 25. https://apa.az/az/senaye-ve-energetika/tanap-
la-turkiye-ve-avropaya-40-mlrd- kubmetr-azerbaycan-
qazi-catdirilib-742064 ABSTRACT В настоящее время турецко-азербайджанские
отношения строятся на сферах материального со-
трудничества, имеющих стратегическое значение
для обеих стран. В этом плане особую роль играет
«Шушинская декларация» подписанная между
Турцией и Азербайджаном 15 июня 2021 года. Пре-
зидент Азербайджана Ильхам Алиев сказал что,
«Шушинская декларация» служит расширению со-
юзнических отношений между Азербайджаном и
Турцией [7]. С момента запуска Трансанатолийского газо-
провода (TANAP) (с 30 июня 2018 г.) через нее в
рамках Южного газового коридора в Турцию (20,5) 85 Sciences of Europe # 115, (2023) 12. Jonathan Elkind, “Economic Implications of
the Baku-Tbilisi-Ceyhan Pipeline”, The Baku-Tbilisi-
Ceyhan Pipeline: Oil Window to the West, (Edited by
S. Frederick Starr and Svante E. Cornell), 2005, ss.39-
60. Внешнеэкономические связи Азербайджана с
Турцией в ближайшие годы должны развиваться по
следующим направлениям: увеличение внешнетор-
гового оборота; развитие транспортной системы;
совершенствование законодательной базы, регули-
рующей внешне экономическую деятельность; про-
ведение гибкой таможенно-тарифной политики
между двумя странами; дальнейшая либерализация
внешнеэкономической деятельности в стране; по-
вышение качества азербайджанской продукции для
дальнейшего экспорта в Турцию; развитие сотруд-
ничества в сфере туризма. По мере развития эконо-
мических возможностей Азербайджана и Турции,
сотрудничество в политической, энергетической,
торгово-экономической, транспортной и других
сферах будет развиваться и укрепляться. 13. Quliyev
F. “TANAP:
İqtisadiyyat
geosiyasətdən
irəlidir”. http://www.bbc.com/azeri/azerbaijan/2014/09/140920
_enregy_projects_analysis 14. Nuray Erdoğan, “Tanap” projesinin Türkiye
ve Azerbaycan enerji politikalarındaki yeri ve önemi. Ömer Halisdemir Üniversitesi İktisadi ve idari bilimler
fakültesi. Cild
10. 08.07.2017. https://doi.org/10.25287/ohuiibf.319259
İ 15. Nuriyev Q. İqtisadi qardaşlıq: Türkiyənin
neft-kimya
sahəsinə
Azərbaycan
dəstəyi. https://report.az/analitika/iqtisadi-qaradasliq-
turkiyenin-neft-kimya-sahesine-azerbaycan-
desteyi/ Литература 1. Aydın M. Ermenistanla ilişkiler // Türk Dış
Politikası., 3 cilddə, I cilt. İstanbul, İletişim yayınları,
2001, 415 s., s. 402. 1. Aydın M. Ermenistanla ilişkiler // Türk Dış
Politikası., 3 cilddə, I cilt. İstanbul, İletişim yayınları,
2001, 415 s., s. 402. 16. Osman Nuri Aras, Azerbaycan’ın Hazar
Ekonomisi ve Stratejisi, Derin Yayınları, İstanbul,
2008, s. 99. 2. Azerbaycan TAP üstünden Avrupa'ya ticari
gaz
sevkiyatına
başladı. https://www.internethaber.com/azerbaycan-tap-
ustunden-avrupaya-ticari-gaz-sevkiyatina-basladi-
2152739h.htm 17. Osman Nuri Aras,
Elçin Süleymanov. Azerbaycan’ın Enerji Kaynakları Gelirlerinin İhraç
Hacmindeki Yeri ve Ülke Ekonomisine Etkisi. //
international conference on Eurasian economies 2012,
s.228 https://www.internethaber.com/azerbaycan-tap- 3. Bağırzadə E. Azərbaycanın enerji ehtiyatları
və onlardan istifadənin mövcud vəziyyəti. Bakı, 2014,
s. 29. 3. Bağırzadə E. Azərbaycanın enerji ehtiyatları
və onlardan istifadənin mövcud vəziyyəti. Bakı, 2014,
s. 29. 18. Ruintən S. Azərbaycan Türk dövlətləri ilə
siyasi əlaqələr sistemində. Bakı: Adiloğlu, 2005, 236 s,
s.74 4. Baku-Tbilisi-Ceyhan
pipeline
https://en.wikipedia.org/wiki/Baku%E2%80%93Tbilis
i%E2%80%93Ceyhan_pipeline 19. Rüstəmov C. “Əsrin müqaviləsi” – 18
https://azertag.az/xeber/Asrin_muqavilesi___18-
186853 5. Düzyol
Saltuk,
“TANAP:
Türkiye
ve
Azerbaycan
İçin
Enerjide
‘Kazan-Kazan’”. //
Hürriyyət, 12 dekabr 2012. 20. Süleymanov E. Həsənov F. Azerbaycan’ın
Türkiyenin enerji sektörüne yatırımlarının Azerbaycan 6. Elçin
Zaman,
«Əsrin
müqaviləsi»:
Müstəqilliyimizin qarantı olan çox vacib layihə idi. https://ikisahil.az/post/esrin-muqavilesi-
musteqilliyimizin-qaranti-olan-chox-vacib-layihe-idi - Türkiye ekonomik ilişkilerindeki rolü. // Çankırı
Karatekin Üniversitesi Uluslararası Avrasya Strateji
Dergisi 2(2): 073-100, s.91 g
( )
,
21. TANAP
2
kez
kazandırıyor. https://www.yenisafak.com/ekonomi/tanap-2-kez-
kazandiriyor-3769534 7. Əliyev İ: “Şuşa Bəyannaməsi” Azərbaycan ilə
Türkiyə
arasında
müttəfiqlik
münasibətlərinin
genişlənməsinə
xidmət
edir”. http://interfax.az/view/848818 22. Trans
Adriatik
Boru
Xətti. https://az.wikipedia.org/wiki/Trans_Adriatik_Boru_X
ətti p
8. Gurbanov İ. “TANAP Puzzle: What stands in
the “Backstage of TANAP”?”, Erişim: https://ener-
gycorridors.wordpress.com/2012/07/11/tanap-puzzle-
what-stands-in-thebackstage-of-tanap / 23. Yaqubzadə M. “TANAP layihəsi çərçivəsində
tikinti işləri sürətlə davam etdirilir”, Xalq qəzeti. 2015. http://www.xalqqazeti.com/az/news/economy/55432 9. Heydər Əliyevin neft strategiyası. Bakı: 2001,
1-ci hissə. 491 s. s. 31. 9. Heydər Əliyevin neft strategiyası. Bakı: 2001,
1-ci hissə. 491 s. s. 31. 24. Yusuf Yazar “Enerji İlişkileri Bağlamında
Türkiye ve Orta Asya Ülkeleri”, Hoca Ahmet Yesevi
Uluslararası
Türk-Kazak
Üniversitesi,
http://www.ayu.edu.tr/static/kitaplar/enerji_raporu.pdf
, s.64 10. Heydərov
P. TANAP
tamamlandı: Azərbaycan qazı Avropa mətbəxində. https://azvision.az/news/196635/---tanap-tamamlandi-
-azerbaycan-qazi-avropa-metbexinde--tehlil---.html
11. İlham Əliyev Qarsda Transanadolu qaz boru
kəmərinin təməlinin qoyulması münasibəti ilə təntənəli
mərasimdə
iştirak
edib. (17.05.2015)
https://president.az/az/articles/view/14566 10. Heydərov
P. TANAP
tamamlandı: Azərbaycan qazı Avropa mətbəxində. https://azvision.az/news/196635/---tanap-tamamlandi-
-azerbaycan-qazi-avropa-metbexinde--tehlil---.html 25. https://apa.az/az/senaye-ve-energetika/tanap-
la-turkiye-ve-avropaya-40-mlrd- kubmetr-azerbaycan-
qazi-catdirilib-742064 11. İlham Əliyev Qarsda Transanadolu qaz boru
kəmərinin təməlinin qoyulması münasibəti ilə təntənəli
mərasimdə
iştirak
edib. (17.05.2015)
https://president.az/az/articles/view/14566 11. Литература İlham Əliyev Qarsda Transanadolu qaz boru
kəmərinin təməlinin qoyulması münasibəti ilə təntənəli
mərasimdə
iştirak
edib. (17.05.2015)
https://president.az/az/articles/view/14566
|
https://openalex.org/W4310643189
|
https://www.nature.com/articles/s41598-022-24531-9.pdf
|
English
| null |
Antibacterial efficacy of silver nanoparticles (AgNPs) against metallo-β-lactamase and extended spectrum β-lactamase producing clinically procured isolates of Pseudomonas aeruginosa
|
Scientific reports
| 2,022
|
cc-by
| 11,809
|
www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2022) 12:20685 Antibacterial efficacy of silver
nanoparticles (AgNPs)
against metallo‑β‑lactamase
and extended spectrum
β‑lactamase producing clinically
procured isolates of Pseudomonas
aeruginosa
Maria Muddassir 1, Almas Raza 1, Sadaf Munir 2, Ahmad Basirat 3, Muddassir Ahmed 4,
Mazia Shahid Butt 5, Omair Arshad Dar 6, Syed Shoaib Ahmed 7, Saba Shamim 1 &
Syed Zeeshan Haider Naqvi 1*
OPEN *email: zeeshan.haider@imbb.uol.edu.pk | https://doi.org/10.1038/s41598-022-24531-9 Scientific Reports | (2022) 12:20685 www.nature.com/scientificreports/ Abbreviations
MBL
Metallo β-lactamase
ESBL
Extended spectrum β-lactamase
GEN
Gentamicin
MEM
Meropenem
IMP
Imipenem
SCF
Cefoperazone-Sulbactam
TZP
Piperacillin/Tazobactam
CIP
Ciprofloxacin
AZT
Aztreonam
CAZ
Ceftazidime
AMK
Amikacin
CFX
Cefoxitin
UV
Ultra-violet
LB
Luria Bertani
MH
Mueller–hinton
ATCC
American Type Cell Culture
MAR index
Multiple antibiotic resistance index
MIC
Minimum inhibitory concentration
CLSI
Clinical & Laboratory Standards Institute
MHT
Modified Hodge Test
CDST
Combined Disc Synergy Test
AgNPs
Silver nanoparticles
WHO
World Health Organization
LMIC
Low income and middle income countries
MDR-PA
Multidrug resistant Pseudomonas aeruginosa
CDC
Centers for Disease Control and Prevention Multidrug-resistant Pseudomonas aeruginosa (MDR-PA) has emerged as a life-threatening opportunistic patho-
gen globally in the last two decades particularly in Pakistan, where it has become a severe concern of the health
sector becoming a leading cause of nosocomial infections, particularly in patients with postoperative surgical
injuries, intensive care units, burn and trauma units, and in those with a pre-existing pulmonary disease such
as cystic fibrosis1,2. Long-term hospitalization frequently leads to infections by Pseudomonas aeruginosa namely
malignant external otitis, endocarditis, ophthalmitis, meningitis, septicemia, and pneumonia1,2. Multidrug-
resistant Pseudomonas aeruginosa is by nature resistant to various classes of antimicrobial drugs because of
the presence of efflux pumps plus allowing low permeability of the microbial membranes2. Centers for Disease
Control and Prevention (CDC) reports that more than 32,600 clinical infections in the US are caused by Pseu-
domonas aeruginosa, causing 2700 deaths annually3. Metallo-β-lactamase (MBL) enzymes break down a wide
variety of β-lactam drugs, counting carbapenems too4. According to the Ambler classification of β-lactamase
enzymes, they are divided into four classes (A, B, C, D); MBLs belong to class B enzymes, including VIM, IMP,
NDM-1, and GIM4. Multidrug-resistant Pseudomonas aeruginosa-producing Metallo-β-lactamases are causative
for various diseases, posing a severe health issue, as resistance against multiple antibiotics is on the rise, especially
in Asian countries, including India, Pakistan, and Bangladesh therefore widespread use of antibiotics should
be discouraged5. Recently, various investigations have reported the emergence of multidrug-resistant bacterial
pathogens originating from humans, cattle, birds, and fish increasing the necessity of discovering novel, potent
and safe antimicrobials calling for vigorous routine antibiotic susceptibility testing along with the screening of
the emerging MDR strains6,7. In recent times, the slower improvement of antimicrobial agents has worsened the
situation increasing the need of searching for alternative treatment regimes as a substitute for antibiotics6,7. Antibacterial efficacy of silver
nanoparticles (AgNPs)
against metallo‑β‑lactamase
and extended spectrum
β‑lactamase producing clinically
procured isolates of Pseudomonas
aeruginosa
Maria Muddassir 1, Almas Raza 1, Sadaf Munir 2, Ahmad Basirat 3, Muddassir Ahmed 4,
Mazia Shahid Butt 5, Omair Arshad Dar 6, Syed Shoaib Ahmed 7, Saba Shamim 1 &
Syed Zeeshan Haider Naqvi 1*
OPEN Resistance to carbapenems is a global threat, especially in developing countries with limited health
resources. Prevalence, antibiogram, PCR detection of antibiotic resistance genes, and potency of
Silver Nanoparticles (AgNPs) against multidrug-resistant (MDR) Pseudomonas aeruginosa were
studied. Kirby-Bauer disc method and PCR were used to study antibiogram and drug resistance
genes respectively in 255 isolates of Pseudomonas aeruginosa obtained from a tertiary care hospital. Silver nitrate (AgNO3) precursor salts were reacted with Aspergillus flavus culture filtrate to trigger
the extracellular mycosynthesis of AgNPs. Mycosynthesis was first monitored regularly by visible
ultraviolet spectroscopy that recorded AgNP peaks of approximately 400–470 nm. Confirmation by
Transmission electron micrographs provided confirmation of AgNPs formed within a range of 5–30 nm. Individual and combined antibacterial activity of ten antibiotics and AgNPs was analyzed. Pearson
correlation coefficients (r) were calculated for phenotypic and genotypic multidrug resistance. Data
were evaluated using SPSS version 20. p-value < 0.05 was considered statistically significant. 61.5%
were carbapenemase producers (p < 0.01). The recorded frequency of blaIMP-1, blaSHV, blaVIM, blaOXA,
and blaTEM were 13%, 32%, 15%, 21%, and 43%, respectively. The reducing order of antimicrobial
activity of antibiotics and AgNPs was piperacillin/tazobactam + AgNPs (31 mm), cefoxitin + AgNPs
(30 mm) > amikacin + AgNPs (25 mm) > aztreonam + AgNPs (23 mm) > meropenem + AgNPs
(22 mm) > imipenem + AgNPs (20 mm) > gentamycin + AgNPs (17 mm) > ciprofloxacin + AgNPs
(16 mm) > cefoperazone/sulbactam + AgNPs (14 mm) ≥ ceftazidime + AgNPs (14 mm). The conjugated
effect of AgNPs plus antibiotics showed a 0.15–3.51 (average of 2.09) fold-area augmentation of
antimicrobial activity. AgNPs conjugated with antibiotics effectively inhibited MDR Pseudomonas
aeruginosa. To the best of our understanding, this is an inaugural report from Punjab Pakistan
enlisting co-expression of Metallo-β-lactamases, extended-spectrum β-lactamases, and AmpC-β-
lactamase plus activity of antibiotic-AgNPs. 1Institute of Molecular Biology and Biotechnology (IMBB), The University of Lahore, Defence Road Campus,
Lahore, Pakistan. 2Combined Military Hospital, Lahore, Pakistan. 3King Edward Medical University, Lahore,
Pakistan. 4Social Security Hospital, Gujranwala, Pakistan. 5Lahore General Hospital, Lahore, Pakistan. 6Pak
Emirates Military Hospital, Rawalpindi, Pakistan. 7Al‑Aleem Centre for Advanced Studies and Research, Gulab Devi
Educational Complex, Lahore, Pakistan. Methodology
hi
l gy
Ethical approval statement. The study was undertaken following ethical approval from the Research
Ethics Committee, IMBB The University of Lahore viz Ref # IMBB/UOL/20/138. Ethical guidelines and consent to participate. The current study was carried out following appropri-
ate guidelines and regulations followed by informed written consent from all participants. Sampling procedures. In total, 255 isolates of Pseudomonas aeruginosa obtained from multiple clinical
specimens from different departments of a tertiary care hospital were obtained including urine (27.8%), wound
swabs (34.9%), sputum (13.7%), pus (7.05%), blood (11.7%), and tissue (4.7%) were processed further. The
prevalence of Pseudomonas aeruginosa was 22.0% (255/1159). 145 isolates that tested positive for Pseudomonas
aeruginosa were from females, while 110 were from males. Bacterial isolation and identification. The clinical isolates were confirmed by culturing on Pseudomonas
cetrimide agar. The morphological characteristics of colonies were used for the identification of the isolates. Identification of isolates for Pseudomonas aeruginosa was based on bacterial culture and staining characteris-
tics. Isolates were characterized biochemically using catalase, urease, indole, citrate utilization, lactose, lysine
decarboxylation, and glucose fermentation tests18. Identification of Pseudomonas aeruginosa was by growth on
Pseudomonas cetrimide agar plus API20NE identification strips (bioMerieux, France). Identified strains were
stored in 30% glycerol broth at − 70 °C. The largest number of isolates were obtained from patients falling in
the age group of 40–49 years. Percentage of Pseudomonas aeruginosa isolated from departments counted to
surgery (36.8%), orthopedics (13.3%), gynecology (7.0%), ICU (11.4%), medicine (25.9%), and ENT (5.5%)
(p ≤ 0.001). Specimen-wise isolation was performed with wound swabs (34.9%), tissue (4.7%), blood (11.7%),
sputum (13.7%), urine (27.8%), and pus (7.05%). Resistance to imipenem was exhibited by 135 isolates while
resistance to ceftazidime was shown by 153 isolates. These isolates were further evaluated employing molecular
methodologies. Determination of antibiotic susceptibility. Antibiotic sensitivity was studied using the Kirby-Bauer
technique to evaluate antibiotic sensitivity of Pseudomonas aeruginosa isolates19. Following the recommenda-
tions of CLSI 2019 (Clinical & Laboratory Standards Institute), sensitivity testing was conducted on Mueller–
Hinton agar20. The antibiotic discs (bioMérieux, France) specific for studying gram-negative bacteria, including
Aminoglycosides (amikacin/AMK 30 µg, gentamicin/GEN 10 µg), Carbapenems (imipenem/IMP 10 µg, mero-
penem/MEM 10 µg), Monobactams (aztreonam/AZT 10 µg), Penicillins (piperacillin/tazobactam/TZP 100 µg),
Gyrase inhibitors (ciprofloxacin/CIP 5 µg), Cephalosporin (ceftazidime/CAZ 30 µg, cefoxitin/CFX 30 µg,
cefoperazone/sulbactam/SCF 75–10 µg) were used. Antibacterial efficacy of silver
nanoparticles (AgNPs)
against metallo‑β‑lactamase
and extended spectrum
β‑lactamase producing clinically
procured isolates of Pseudomonas
aeruginosa
Maria Muddassir 1, Almas Raza 1, Sadaf Munir 2, Ahmad Basirat 3, Muddassir Ahmed 4,
Mazia Shahid Butt 5, Omair Arshad Dar 6, Syed Shoaib Ahmed 7, Saba Shamim 1 &
Syed Zeeshan Haider Naqvi 1*
OPEN Summarization of E-test® interpretive criteria of MIC for Pseudomonas aeruginosa in accordance with
CLSI 201923. a Minimun inhibitory concentration measured in µg/mL. The current study is a front-runner report from Pakistan, reporting the simultaneous existence of Metallo-β-
lactamase (MBL), extended-spectrum β-lactamase (ESBL), and AmpC drug resistance genes. The current study
is also the first to report efficient effects of multiple antibiotic-AgNPs combinations against multidrug-resistant
isolates of Pseudomonas aeruginosa from Punjab, Pakistan. Antibacterial efficacy of silver
nanoparticles (AgNPs)
against metallo‑β‑lactamase
and extended spectrum
β‑lactamase producing clinically
procured isolates of Pseudomonas
aeruginosa
Maria Muddassir 1, Almas Raza 1, Sadaf Munir 2, Ahmad Basirat 3, Muddassir Ahmed 4,
Mazia Shahid Butt 5, Omair Arshad Dar 6, Syed Shoaib Ahmed 7, Saba Shamim 1 &
Syed Zeeshan Haider Naqvi 1*
OPEN A
pioneering study reported the presence of 42% blaVIM gene multidrug-resistant gram-negative rods8. Yet another
study from Pakistan reported the presence of blaVIM in 12% of multidrug isolates of Pseudomonas aeruginosa9,10. Algammal et al. report that on the basis of antibiogram as well as molecular analysis of the resistance genes, a
majority of tested isolates prove to be multi-drug resistant to six major classes of antimicrobials (penicillins,
tetracyclines, aminoglycosides, sulfonamides, fluoroquinolones, and lincosamides)11. It has been proposed that
with an increasing incidence of MDR Pseudomonas aeruginosa, newer treatment options such as nanoparticles
and other natural products should be explored12. Metallic nanoparticles were studied to act as promising alter-
natives to routine antibiotics for combating and defeating common resistance in pathogens that include target
site modification, promoted efflux of drugs via membranes, and enhanced expression of the efflux pumps along
with inactivation of enzyme and reduced permeability of the membranes13,14. Silver nanoparticles (AgNPs)
have been stated as potential agents that have efficacy as antibacterial agents and can help combat nosocomial
infections15. AgNPs adsorb and penetrate the bacterial cell walls, ultimately leading to the destruction of bacterial
cells through the formation of free radicals13–15. Additionally, silver nanoparticles can give rise to silver ions14. These silver ions can form bonds with crucial organelles and lead to their dysfunction14–16. According to WHO,
the point prevalence of nosocomial infections ranges from 5.7% in Low-Income and 19.1% in Middle-Income
Countries (LMICs) to 3.5–12% in developed nations. The last few years have witnessed an increased interest
in the application of Silver Nanoparticles as therapeutic regimens due to low toxicity in the environment, an
increased rate of surface capacity, and the ability to inhibit the formation of biofilm that is required for the eva-
sion of pathogens17. https://doi.org/10.1038/s41598-022-24531-9 Scientific Reports | (2022) 12:20685 | www.nature.com/scientificreports/ Table 1. Summarization of E-test® interpretive criteria of MIC for Pseudomonas aeruginosa in accordance with
CLSI 201923. a Minimun inhibitory concentration measured in µg/mL. Antibiotic
Code
Zone Diameter (nearest whole
mm)
S ≥
Zone Diameter (nearest whole
mm)
R ≤
Pseudomonas aeruginosa
ATCC 27853 MICa µg/mL
Gentamicin
GEN
8
4
1–4
Amikacin
AMK
32
16
1–4
Ciprofloxacin
CIP
2
0.5
0.25–1
Imipenem
IMP
8
2
1–4
Ceftazidime
CAZ
32
8
1–4
Aztreonam
AZT
32
38
2–8
Piperacillin/Tazobactam
TZP
128
16
1–8
Meropenem
MEM
8
2
1–4
Cefoperazone/Sulbactam
SCF
21
15 Table 1. Methodology
hi
l In accordance with the previ-
ously stated method, extraction of the template DNA was performed from isolates27. The PCR mixture included
200 µM dNTPs, 50 ng DNA templates, 0.5 U Taq Polymerase, 10 pM primers, 1.5 mM MgCl2, and giving an
eventual volume of 25 µL. Products of PCR were evaluated and construed for 30 min at 70 V. A 1.5 w/v agarose
gel plus 500 µg/100 mL ethidium bromide was used to analyze the products. Detection of MBL and ESBL genes. Confirmation of Pseudomonas aeruginosa isolates was done by singleplex
PCR. Primer sequences selected for detecting the blaIMP-1, blaTEM, blaSHV, blaOXA, and blaVIM genes have been
reported in previous studies28. Pseudomonas aeruginosa isolates for the blaVIM plus blaIMP-1 genes were screened
with the help of singleplex PCR with reported primers29,30. The existence of gene blaAmpC in Pseudomonas aer-
uginosa was evaluated through PCR amplification of 1063 bp (Table 2). The DNA amplicons were placed in a
1.5% agarose gel for 60 min at 120 V. A UV light trans-illuminator gel documentation system helped visualize
the amplified products (Thermo Fisher Scientific, US). Conditions for PCR have been enlisted in Supplementary
Table 1 (ST_1). Fungal biomass cultivation. AgNPs myogenesis was carried out employing Aspergillus flavus. Aerobic
cultivation of fungal biomass was carried out using a liquid medium that contained malt extract in a concentra-
tion of 0.3 g/100 mL, glucose 1.0 g/100 mL, yeast extract 0.3 g/100 mL, and peptone 0.5 g/100 mL. Medium’s
pH was adjusted initially to be 5.8. Growth of fungal culture was carried out at 28 °C at 150 rpm using an orbital
shaker. Extraction of the fungal mass was done through filtration employing Whatman filter paper no 1. Later,
this culture helped synthesize nanoparticles. Assay for synthesizing nanoparticles. Almost 200 mL of fungal culture without mycelia that contained
0.1 M AgNO3 as precursor salt was collected in an Erlenmeyer flask having a volume of 500 mL. This flask was
incubated on a shaker at 150 rpm in an ill-lit condition at 28 °C for a duration of 96 h. In accordance with the
methods described by Bhainsa et al. both positive control i.e. culture filtrate with the exception of silver salt along
with negative control, i.e. AgNO3 solution was run along with experimental flasks33. Characterization of AgNPs by transmission electron microscopy and X‑ray diffraction. Methodology
hi
l The potency of the antibiotic discs was studied using the
American Type Culture Collection (ATCC) standard reference strains (P. aeruginosa ATCC27853). For studying
antimicrobial susceptibility, a 0.5% McFarland turbidity standard was employed for standardizing the bacterial
inoculum suspension21. The sensitivity test results were utilized to determine the multiple antibiotic resistance
index (MAR) of Pseudomonas aeruginosa isolates. MAR helps estimate resistance trends against multiple anti-
microbial drugs and indicates the emergence of novel resistant bacterial strains. MICs (µg/mL) of meropenem
and imipenem plus cefoxitin against multidrug-resistant Pseudomonas aeruginosa were determined by E-strip
(Thermo Fisher Scientific, UK). A summarization of MIC of used antibiotics has been tabulated (Table 1). In https://doi.org/10.1038/s41598-022-24531-9 Scientific Reports | (2022) 12:20685 | www.nature.com/scientificreports/ Table 2. Sequence of primers used for detecting MBLa, ESBLb-type variants and blaAmpC. a Metallo
β-lactamase, bextended spectrum β-lactamase. c Enlisted in references. Primers
Sequence
Annealing Temperature (Tm °C)
Product of PCR
Referencesc
blaTEM
CCCCGAAGAAGTCCTTTC ATCAGCAATAGT
CCCAGC
56
500
31
blaIMP-1
AGCGCAGCATATTGATTGC ACAACCAGATGC
TGCCTTACC
54
587
28
blaSHV
AGGGCTTGACTGCCATTTTG ATTTGCGTGATT
TCATTT
55
400
31
blaAmpC
CTTCCACACTGCTGTTCGCC-TTGGCCAGGATC
ACCAGTCC
66
1063
32
blaOXA
ATATCTCGCTTGTTGCATCTCC AAACCCTTC
AGCTCATCC
55
600
31
blaVIM
ATGGTCGTTATGGCATATC TGGGCCGTGTCA
GCCAGAT
57
510
29 Table 2. Sequence of primers used for detecting MBLa, ESBLb-type variants and blaAmpC. a Metallo
β-lactamase, bextended spectrum β-lactamase. c Enlisted in references. accordance with Magiorakos et al., the tested isolates would fall in the category of MDR if the isolates expressed
resistance to at least one antimicrobial in three used or more antibiotics22. Phenotypic detection of carbapenemase and metallo‑β‑lactamases. Carbapenemases were
studied following the combined disc synergy test (CDST) and Modified Hodge test (MHT)4. Whereas, the pro-
duction of Metallo-β-lactamases was observed by the combined disc synergy test (CDST) as per the guidelines
of CLSI 2019. This test is based on using a disc of imipenem alone and a disc of IMP/EDTA disc (Oxoid, Inc. Canada) according to methodology4. A combination disc synergy test (CDST) using a solitary imipenem disc
along with an IMP/EDTA disc (Oxoid, Inc. Canada) was performed following the suggested method by Wadekar
et al.24. The modified Hodge test (MHT) was performed following the methods of Kumar et al.25. Criteria by
CLSI 2019 were used to analyze the results. IMP/EDTA E-Strips and IMP alone were used for detecting MBLs as
per the manufacturer’s instructions (Liofilchem®, Italy)26. Metallo‑β‑lactamase: molecular characterization. Molecular assays. www.nature.com/scientificreports/ had to pass the process of microcentrifugation (Microfuge® 18 Centrifuge, Beckman Coulter, USA). Eventually,
the removal of the supernatant was performed followed by overnight drying in the oven. AgNPs were finally
prepared in powdered form. Nanoparticle comprising dried sample drop-coated films on silica were eventu-
ally exposed to analysis by X-ray diffraction (XRD) working in transmission mode at 20 mA, 30 kV with Cu
Kα radiation (X’pert PRO XRD, PANalytical BV, The Netherlands). The formation of AgNPs film took place on
carbon-coated copper transmission electron microscopy (TEM) grids that were examined by TEM at an 80 kV
accelerating voltage (JEM-1010, JEOL Ltd, Tokyo, Japan). Characterization of silver nanoparticles by UV–Vis
Spectra, XRD analysis, Size distribution profile of AgNPs, and TEM Micrograph analysis has been discussed in
detail by Naqvi et al.30,34. AgNPs antibacterial activity against multidrug‑resistant Pseudomonas aeruginosa.. AgNPs
were prepared and procured from CRIMM (Centre for Research in Molecular Medicine) at the University of
Lahore. To evaluate the antibacterial action of AgNPs, the "disc diffusion method" was employed35. Individual
and combined antibacterial activities of common antibiotics plus AgNPs were explored against multidrug-resist-
ant isolates of Pseudomonas aeruginosa by the Kirby–Bauer disk-diffusion method13,14. Preparation of bacterial suspension and silver nanoparticles. LB broth (Oxoid, UK) was used to
prepare a 0.5 MacFarland suspension of MBL-producing isolates of Pseudomonas aeruginosa. A uniform suspen-
sion of AgNPs concentrated at 1000 μg/10 mL (stock solution equal to 100 μg/mL) was prepared by dissolving
AgNP powder (1 mg) in 10 mL normal saline36,37 Assays for antibacterial activity. The method of disc diffusion was employed for the analysis of the anti-
bacterial efficacy of AgNPs against multiple drug-resistant strains of Pseudomonas aeruginosa that were identi-
fied from obtained specimens. MIC and MBC values for AgNPs were also evaluated38. Nanoparticles of silver
nitrate derived from Aspergillus flavus were prepared and obtained by the microbiology lab (T-3 and 4), The
University of Lahore. Measured weight One milligram of silver nanoparticles was mixed with 10 mL of normal
saline for 15 min using a sonicator, and the prepared solution was 100 ppm (1000 µg/10 mL = 100 ppm). The
prepared silver NP solution was dropped in amounts of 10, 20, 30, 40, and 50 µL with the help of a pipette on
homemade 6 mm discs prepared from plain blotting paper and air-dried for a few seconds. www.nature.com/scientificreports/ Methodology of disc
diffusion method was employed to assess the antibacterial action of AgNPs13,39. Pseudomonas aeruginosa was
streaked on Mueller-Hinton agar for assessment of the antibacterial activity. The Petri plates were incubated at
37 °C for 24 h. Fresh cultures were then used to make the suspension. To maintain the turbidity of the bacterial
culture, 0.5% McFarland solution was employed for comparison. One millilitre of this suspension (inoculum)
was then added and spread on Mueller-Hinton Agar medium plates. Next, nanoparticles containing air-dried
sterile filter paper discs were positioned at suitable distances. Following labelling, the plates were kept at 37 °C
for 24 h. MIC and MBC of AgNPs (µg/mL). A volume of 200 µL of 0.5 McFarland bacterial suspension was pre-
pared and added to LB broth from the 1st to the 10th well. Serial dilutions from stock solutions of AgNPs
(1 mg/10 mL) were added to 96-well round-bottom microtiter plates (Thermo Fischer Scientific, UK) to final-
ize a 2 µg/mL concentration. Petri plates were incubated overnight at 37 °C following which the plates were
observed to determine the absence of growth comparing each well with positive and negative controls40. MBC
(minimum bactericidal concentration) measures the first dilution showing no growth on the agar. Wells show-
ing no visible growth in the microtiter plate were further inoculated on nutrient agar (Oxoid, UK). A schematic
illustration is shown (Fig. 1). Statistical analysis. Statistical analysis was performed for evaluating the percentage of resistance in rela-
tion to the ward and type of sample. Association between the type of sample [urine, sputum, wound, blood, pus,
and tissue] and antibiotic resistance gene were evaluated by Chi-Square test. A p-value ≤ 0.05 was considered
statistically significant. As per Pearson-correlation coefficient (r), the association between the phenotypic and
genotypic variables was determined as follows: weak correlation: |r|< 0.3, moderate correlation: 0.3 <|r|< 0.5,
strong correlation: 0.5 <|r|< 0.8541. Statistical analyses were carried out using SPSS software version 20 (IBM
Corp., Armonk, NY, USA). Methodology
hi
l Char-
acterization of myco-genized AgNPs was done by visual observations, Ultraviolet Visible (UV-Vis) Spectropho-
tometry, XRD, TEM, SEM and DLS. During the assay, 1 mL of sample volume was obtained from the reaction
mixture at time intervals of 0, 2, 4, 6, 24, 48, 72 and finally 96 h. Absorbance of sample was recorded at wave-
lengths 200–800 nm employing a UV–visible spectrophotometer (Agilent 8453 UV–Vis, Agilent Technologies,
USA). Colloid suspension from silver containing reaction mixture was eventually concentrated by centrifuga-
tion for 20 min at 12,000 rpm (centrifuge Model H-251, Kokusan Co, Ltd, Tokyo, Japan). AgNPs were obtained
by washing the silver powder thrice with sterile deionized water and pure ethanol. Thereafter, nanoparticles https://doi.org/10.1038/s41598-022-24531-9 Scientific Reports | (2022) 12:20685 | www.nature.com/scientificreports/ Results
Ph Phenotypic characteristics of the recovered Pseudomonas aeruginosa isolates. Pseudomonas
aeruginosa counts as a heterotrophic and motile, Gram-negative bacterium that is rod-shaped measuring 1–5 µm
and 0.5–1.0 µm. It has been documented as a facultative aerobe that has the ability to grow through both aerobic
and anaerobic respiration with nitrate studied to be the final electron acceptor. In accordance with Magiorakos
et al., the tested isolates would fall in the category of MDR since the isolates were resistant to a minimum of
one antimicrobial drug in three or exceeding antibiotic categories31. Pseudomonas aeruginosa has the ability to
produce elastase and protease enzymes as well as hemolysins. Antibiotic susceptibility testing. Sensitivity testing was accomplished on MH agar (Oxoid) as per rec-
ommendations of CLSI 2019 (Clinical and Laboratory Standards Institute 2019)20. This has been illustrated in
Fig. 2. The pattern of antimicrobial resistance in Pseudomonas aeruginosa in association with departments has
been tabulated in Table 3. https://doi.org/10.1038/s41598-022-24531-9 Scientific Reports | (2022) 12:20685 | www.nature.com/scientificreports/ Figure 1. Biosynthesis of AgNPs from Aspergillus flavus having antibacterial activity. (4a) Culture filtrate of
A. flavus with silver nitrate solution (0.1 M) at 0 h. (4b) Culture filtrate of A. flavus with silver nitrate solution
(0.1 M) at 96 h. (4cL) Size distribution profile of AgNPs synthesized by cultural filtrates of A. flavus {100
particles were analyzed}. (4cR) TEM micrographs of AgNPs produced by cultural filtrate of A. flavus. (4d)
UV–Vis spectra recorded for the biosynthesis of AgNPs in response to 0.1 M. silver nitrate inoculated media of
A. flavus. (4e) XRD patterns of AgNPs biosynthesized by cultural filtrates of A. flavus. (4f) Zones of inhibition
with antibiotics alone and in combination with AgNPs*.Antibiotics in combination with AgNPs (A and B) have
a wider zone of inhibition as compared to antibiotic alone showing thereby that AgNPs conjugated to antibiotics
have a stronger antibacterial potency. (4 g) In vitro activity of AgNPs* at varying volumes (10 µL, 20 µL, 30 µL,
40 µL, 50 µL). AgNPs silver nanoparticles. Figure 1. Biosynthesis of AgNPs from Aspergillus flavus having antibacterial activity. (4a) Culture filtrate of
A. flavus with silver nitrate solution (0.1 M) at 0 h. (4b) Culture filtrate of A. flavus with silver nitrate solution
(0.1 M) at 96 h. (4cL) Size distribution profile of AgNPs synthesized by cultural filtrates of A. flavus {100
particles were analyzed}. (4cR) TEM micrographs of AgNPs produced by cultural filtrate of A. flavus. Results
Ph (4d)
UV–Vis spectra recorded for the biosynthesis of AgNPs in response to 0.1 M. silver nitrate inoculated media of
A. flavus. (4e) XRD patterns of AgNPs biosynthesized by cultural filtrates of A. flavus. (4f) Zones of inhibition
with antibiotics alone and in combination with AgNPs*.Antibiotics in combination with AgNPs (A and B) have
a wider zone of inhibition as compared to antibiotic alone showing thereby that AgNPs conjugated to antibiotics
have a stronger antibacterial potency. (4 g) In vitro activity of AgNPs* at varying volumes (10 µL, 20 µL, 30 µL,
40 µL, 50 µL). AgNPs silver nanoparticles. Phenotype detection of carbapenemase activity. Of 255 total isolates, 61.5% were carbapenemase
producers, including 32 samples from urine (45%), wound swabs n = 25 (28%), sputum n = 28 (80%), blood
n = 21 (70%), tissue n = 5 (41%) and pus n = 4 (22%). Phenotypic detection of Metallo-β-lactamases was observed
by CDST as per guidelines of CLSI 2019 (Fig. 3). Antibiotic susceptibility of MBL producers is illustrated in
Fig. 4. Prevalence of MBL and ESBL producers in correlation with the department is shown in Table 4. Multiplex PCR for AMR genes (blaOXA, blaIMP, blaTEM, blaSHV, blaVIM). Multiplex PCR detected the
existence of resistance genes in 52.5% (n = 80) of the ESBL producers. Expression of blaTEM was of the order 43%
of ESBL producers (n = 34) whereby blaSHV was detected in 32% of isolates. Likewise, the expression of blaOXA
was 21%. blaVIM along with blaIMP-1 co-existed in 11.5% of MBL producers. 57.5% ESBL-positive strains exhib-
ited simultaneous existence of blaTEM, blaSHV plus blaOXA, blaTEM was concomitantly expressed with blaOXA types
in 19.5% of ESBL producers whereas blaTEM simultaneously showed presence with blaSHV in 22.5% isolates. Co-
existence of blaOXA plus blaSHV was found in 9.5% of isolates while blaSHV, blaTEM, as well as blaOXA subsisted in
7.5% of ESBL-producers. Expression of blaAmpC was positive in 15% of isolates that were cefoxitin resistant. The
correlation between phenotypic and genotypic MDR has been tabulated in Table 5. The co-expression of various
genes is depicted in Fig. 5. MIC of MBL producing Pseudomonas aeruginosa. The MIC of imipenem against 10 (13%) blaIMP-
producing Pseudomonas aeruginosa isolates inhibited five isolates at 16 μg/mL and five other isolates at 8 μg/mL. Results
Ph The MIC of imipenem for 12 (15%) blaVIM-producing Pseudomonas aeruginosa was 16 μg/mL, for three isolates https://doi.org/10.1038/s41598-022-24531-9 Scientific Reports | (2022) 12:20685 | www.nature.com/scientificreports/ 0
10
20
30
40
50
60
70
GEN MEM IMP
SCF
TZP
CIP
AZT
CAZ AMK CFX
% Resistant isolates
Gentamicin (GEN), Piperacillin-Tazobactam (TZP), Imipenem (IMP),
Meropenem (MEM), Amikacin (AMK), Cefoperazone-Sulbactam (SCF),
Cefoxitin (CFX), Ciprofloxacin (CIP), Ceftazidime (CAZ), Aztreonam (AZT)
Antimicrobial Susceptibility Pattern
% RESISTANCE
% SENSITIVITY
Figure 2. Percentage antibiotic resistance and sensitivity Pseudomonas aeruginosa isolated from patients in
Punjab, Pakistan (n = 255). 0
10
20
30
40
50
60
70
GEN MEM IMP
SCF
TZP
CIP
AZT
CAZ AMK CFX
% Resistant isolates
Gentamicin (GEN), Piperacillin-Tazobactam (TZP), Imipenem (IMP),
Meropenem (MEM), Amikacin (AMK), Cefoperazone-Sulbactam (SCF),
Cefoxitin (CFX), Ciprofloxacin (CIP), Ceftazidime (CAZ), Aztreonam (AZT)
Antimicrobial Susceptibility Pattern
% RESISTANCE
% SENSITIVITY Antimicrobial Susceptibility Pattern ntamicin (GEN), Piperacillin-Tazobactam (TZP), Imipenem (IMP) p
p
openem (MEM), Amikacin (AMK), Cefoperazone-Sulbactam (SCF p
p
Meropenem (MEM), Amikacin (AMK), Cefoperazone-Sulbactam (SCF),
Cefoxitin (CFX), Ciprofloxacin (CIP), Ceftazidime (CAZ), Aztreonam (AZT (
),
(
),
p
(
),
n (CFX), Ciprofloxacin (CIP), Ceftazidime (CAZ), Aztreonam (AZT Figure 2. Percentage antibiotic resistance and sensitivity Pseudomonas aeruginosa isolated from patients in
Punjab, Pakistan (n = 255). Figure 2. Percentage antibiotic resistance and sensitivity Pseudomonas aeruginosa isolated from patients in
Punjab, Pakistan (n = 255). Table 3. Pattern of antimicrobial resistance in Pseudomonas aeruginosa in association with departments. Vertical ranking enlists antibiotics. Horizontal ranking enlists the department of sample collection. *p
value ≤ 0.05 was considered statistically significant. Antibiotic
Wards
Surgery
Medicine
Orthopaeds
ICU
ENT
Gynaecology
Total
p-value*
Meropenem
56
42
4
14
8
6
130 (51%)
0.000
Imipenem
62
27
21
9
7
9
135 (53%)
0.005
Gentamicin
51
44
25
10
4
6
140 (55%)
0.001
Cefoxitin
43
30
10
24
2
5
114 (45%)
0.000
Ciprofloxacin
51
51
22
8
4
4
140 (55%)
0.000
Ceftazidime
65
35
16
14
10
13
153 (60%)
0.062
Aztreonam
39
32
28
18
5
6
128 (50%)
0.001
Pip-Tazo
35
25
19
16
3
4
102 (40%)
0.048
Amikacin
51
35
5
9
5
10
115 (45%)
0.001
Cefoperazone-Sulbactam
76
39
11
10
5
7
148 (58%)
0.000 Table 3. Pattern of antimicrobial resistance in Pseudomonas aeruginosa in association with departments. Vertical ranking enlists antibiotics. Horizontal ranking enlists the department of sample collection. *p
value ≤ 0.05 was considered statistically significant. Table 3. Table 3. Pattern of antimicrobial resistance in Pseudomonas aeruginosa in association with departments.
Vertical ranking enlists antibiotics. Horizontal ranking enlists the department of sample collection. *p
value ≤ 0.05 was considered statistically significant. Results
Ph Pattern of antimicrobial resistance in Pseudomonas aeruginosa in association with departments. Vertical ranking enlists antibiotics. Horizontal ranking enlists the department of sample collection. *p
value ≤ 0.05 was considered statistically significant. Figure 3. CDSTa for detection of MBL producers of Pseudomonas aeruginosa isolated from Punjab, Pakistan. This test shows using a disc of IMPb alone and IMP/EDTA disc as per methodology of Wadekar et al.24. A wider
zone of inhibition is measured with IMP/EDTA disc as compared to IMP disc alone. This identifies metallo
β-lactamase producers. aCDST Combined disc synergy disc; bImipenem. Figure 3. CDSTa for detection of MBL producers of Pseudomonas aeruginosa isolated from Punjab, Pakistan. This test shows using a disc of IMPb alone and IMP/EDTA disc as per methodology of Wadekar et al.24. A wider
zone of inhibition is measured with IMP/EDTA disc as compared to IMP disc alone. This identifies metallo
β-lactamase producers. aCDST Combined disc synergy disc; bImipenem. https://doi.org/10.1038/s41598-022-24531-9 Scientific Reports | (2022) 12:20685 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ -20
0
20
40
60
80
100
GEN MEM IMP
SCF
TZP
CIP
AZT CAZ AMK CFX
No. of isolates
Gentamicin (GEN), Meropenem (MEM), Aztreonam (AZT),
Ciprofloxacin (CIP), Imipenem (IMP), Cefoperazone-Sulbactam
(SCF), Amikacin (AMK), Cefoxitin (CFX), Piperacillin-Tazobactam
(TZP), Ceftazidime (CAZ)
% Sensitive
% Resistant
Figure 4. Antibiotic susceptibility testing of MBL producing Pseudomonas aeruginosa (n = 83). MBL metallo
β-lactamase. Gentamicin (GEN), Meropenem (MEM), Aztreonam (AZT Gentamicin (GEN), Meropenem (MEM), Aztreonam (AZT),
Ciprofloxacin (CIP), Imipenem (IMP), Cefoperazone-Sulbactam
(SCF), Amikacin (AMK), Cefoxitin (CFX), Piperacillin-Tazobactam
(TZP), Ceftazidime (CAZ) Figure 4. Antibiotic susceptibility testing of MBL producing Pseudomonas aeruginosa (n = 83). MBL metallo
β-lactamase. Figure 4. Antibiotic susceptibility testing of MBL producing Pseudomonas aeruginosa (n = 83). MBL metallo
β-lactamase. Table 4. Prevalence of MBL- and ESBL-producing Pseudomonas aeruginosa in correlation with department. Vertical ranking shows antibiotic resistance genes. Horizontal ranking shows department of sample collection
*p value ≤ 0.05 was considered statistically significant. Table 5. Correlation matrix of phenotypic and genotypic MDR isolates of Pseudomonas aeruginosa by Pearson
correlation co-efficient. *p value ≤ 0.05 was considered statistically significant. **Based on Pearson-correlation
coefficient (r), the association between the phenotypic and genotypic variables was determined as: |r|< 0.3;
weak correlation, 0.3 <|r|< 0.5; moderate correlation, 0.5 <|r|< 0.85; strong correlation. Results
Ph Isolates
Surgery
Medicine
Ortho
ICU
ENT
Gynaecology
Total
p-value*
blaIMP (n = 10)
7 (70%)
2 (20%)
0 (0%)
0 (0%)
0 (0%)
1 (10%)
10 (100%)
0.26
blaVIM (n = 12)
9 (75%)
2 (16.7%)
0 (0%)
0 (0%)
0 (0%)
1 (8.3%)
12 (100%)
0.101
blaSHV (n = 25)
19 (76%)
3 (12%)
0 (0%)
1 (4%)
1 (4%)
1 (4%)
25 (100%)
0.002
blaTEM (n = 34)
18 (52.9%)
13 (38.2%)
0 (0%)
2 (5.9%)
1 (2.9%)
0 (0%)
34 (100%)
0.012
blaOXA (n = 17)
8 (47.1%)
7 (41.1%)
0 (0%)
0 (0%)
2 (11.7%)
0 (0%)
17 (100%)
0.099
blaAmpC (n = 17)
12 (70.5%)
1 (5.9%)
3 (17.6%)
1 (5.9%)
0 (0%)
0 (0%)
17 (100%)
0.043
Total
73
28
3
4
4
3
115 Table 4. Prevalence of MBL- and ESBL-producing Pseudomonas aeruginosa in correlation with department. Vertical ranking shows antibiotic resistance genes. Horizontal ranking shows department of sample collection. *p value ≤ 0.05 was considered statistically significant. MBL producer
blaIMP
blaVIM
blaSHV
blaTEM
blaOXA
blaAmpC
ESBL producer
MBL Producer
X
0.291
0.000
0.320
0.000
− 0.06
0.339
− 0.149
0.017
− 0.085
0.176
− 0.018
0.776
0.710
0.256
Statistics {p =}*
blaIMP
0.291
0.000
X
0.909
0.000
0.001
0.983
0.04
0.529
0.027
0.668
0.027
0.668
0.038
0.550
blaVIM
0.320
0.000
0.909
0.000
X
− 0.011
0.861
0.022
0.729
0.015
0.813
0.015
0.813
0.009
0.881
blaSHV
− 0.06
0.339
0.001
0.983
− 0.011
0.861
X
0.569
0.000
0.282
0.000
0.229
0.000
0.488
0.000
blaTEM
− 0.149
0.017
0.04
0.529
0.022
0.729
0.569
0.000
X
0.589
0.000
0.126
0.044
0.555
0.000
blaOXA
− 0.085
0.176
0.027
0.668
0.015
0.813
0.282
0.000
0.589
0.000
X
− 0.008
0.894
0.395
0.000
blaAmpC
− 0.018
0.776
0.027
0.668
0.015
0.813
0.229
0.000
0.126
0.044
− 0.008
0.894
X
0.294
0.000
ESBL Producer
0.071
0.256
0.038
0.550
0.009
0.881
0.488
0.000
0.555
0.000
0.395
0.000
0.294
0.000
X
Pearson correlation coefficient (r =)** Table 5. Correlation matrix of phenotypic and genotypic MDR isolates of Pseudomonas aeruginosa by Pearson
correlation co-efficient. *p value ≤ 0.05 was considered statistically significant. **Based on Pearson-correlation
coefficient (r), the association between the phenotypic and genotypic variables was determined as: |r|< 0.3;
weak correlation, 0.3 <|r|< 0.5; moderate correlation, 0.5 <|r|< 0.85; strong correlation. Results
Ph https://doi.org/10.1038/s41598-022-24531-9 Scientific Reports | (2022) 12:20685 | www.nature.com/scientificreports/ 0
5
10
15
20
25
30
35
40
45
50
Gene Variant Percentage (%)
Gene variants
Figure 5. Co-expression of gene variants (MBL and ESBL) in isolates of Pseudomonas aeruginosa. Gene variants Figure 5. Co-expression of gene variants (MBL and ESBL) in isolates of Pseudomonas aeruginosa. was 8 μg/mL, and for the remaining three isolates measured 32 μg/mL. MIC of Imipenem for 17 (21%) blaOXA
roducers was 16 μg/mL for 12 isolates plus 8 μg/mL for five isolates. Characteristics of AgNPs. Visual observations. Changes in the color of the reaction mixture were ob-
served and images were recorded as the initial and ending stages of the experiment (Figs. 1–4a,b). UV‑spectroscopy. At the time of incubation, the reaction mixture’s UV–visible spectroscopy recorded spectra of
increased intensity having a range of 350–600 nm; crucial peaks occurred at approximately 400–470 nm (Fig. 6). Moderately increasing peak absorbance accompanied with time was most notably connected with a change in
the reaction mixture’s color exhibiting AgNPs synthesis positively (Fig. 6A). Particle size histogram has been
depicted (Fig. 6B). XRD analysis. The crystalline nature of the AgNPs was depicted using the Debye–Scherrer analysis along with
XRD of the dried sample’s drop-coated film. XRD analysis revealed four vital peaks in the total spectrum of 2θ
value that extended in the range of 20°–80°. The crystallites averaged in size from 13 to 26 nm. Debye–Scherrer
formulae were used to analyze the silver nanocrystallites ranging from 21 nm from total breadth at peak’s half
maximum. The Debye–Scherrer equation is derived from Bragg’s law that determines the diameter of crystal
samples according to the formula given below; D = k. , β cos θ, where D is the mean diameter, λ is the wavelength, k is the shape factor (0.9), θ is the Bragg angle for studied
diffraction, β is the the full width at half maximum. XRD diractograms demonstrated four vital peaks around
2θ angles at 38, 44, 64, and 77. Crystallites estimated via XRD technique revealed size in the nanometer range
(Fig. 6C). TEM of AgNPs. Bright-field image mode was employed for the analysis of samples. Ultra-sonication produced
pure ethanol based dilute suspensions of AgNPs. The suspension was spread drop-wise on 300-mesh lacy cop-
per grids that were coated with carbon followed by drying for scanning under JEOL-1010 TEM. Accelerating
high voltage was kept at 80 kV. Required adjustments and alignments were done following the selection of
apertures and sample images were scanned on screen. Micrographs were recorded at definite magnifications on
the photographic plates by focusing on the sample grids at the correct places. Standard developing procedures
were adopted for processing the exposed photographic plates. These were scanned (flatbed high-resolution scan-
ner) to finally achieve the image positives. TEM micrographs showed nanoparticles of different shapes where
predominantly the shape was spherical. AgNPs acquired a size range of 5–30 nm30,34. The majority of the AgNPs
were spread out in the micrographs with only a few places revealing larger aggregates of differing sizes (Fig. 6D). Scanning electron microscopy (SEM). Scanning electron micrographs were acquired with the JEOL 5600 fol-
lowing filtration of the samples through Millipore filters of 0.2 µm pore size to remove any contaminants that
could possibly interfere with the SEM images. Samples for analysis were prepared by overnight fixation with
2.5% glutaraldehyde at room temperature. Subsequently, dehydration of the sample was carried out with gradi-
ent alcohol (10–95%) followed by incubation for 20 min in every gradient and soaked in absolute alcohol for https://doi.org/10.1038/s41598-022-24531-9 Scientific Reports | (2022) 12:20685 | www.nature.com/scientificreports/ Figure 6. Characterization of AgNPs biosynthesized using Aspergillus flavus. (A) UV–Vis spectra recorded for
the biosynthesis of AgNPs in response to 0.1 M silver nitrate inoculated media of A. flavus. (B) Size distribution
profile of AgNPs synthesized prepared by cultural filtrates of A. flavus {A total number of 100 particles were
analyzed}. (C) XRD patterns of AgNPs biosynthesized from cultural filtrates of A. flavus. (D) TEM micrographs
of AgNPs produced by cultural filtrates of A. flavus. Figure 6. Characterization of AgNPs biosynthesized using Aspergillus flavus. β cos θ, Myco-genized AgNPs (A) Zeta potential measurements for mycogenized AgNPs. (B) SAED analy
(C) Dynamic Light Scattering (DLS) measurements (D) SEM micrographs of AgNPs. Zeta potential measurements were executed with the AgNPs obtained from stock solution along with the
resuspended employing Malvern Instruments Zeta-sizer Nano (Malvern Instruments Ltd., UK) that operated
with a variable power of (5–50 mW) together with He–Ne laser at 632 nm. Measurements were obtained in zeta
cells (DTS 1060C) at a temperature of 25 °C that were independently recorded thrice. Flow through the cell
underwent washing three times before and during measurements with ultrahigh pure water prior to the addi-
tion of the subsequent sample. AgNPs zeta potential was evaluated at pH 7.4. Zeta potential measurements for
AgNPs revealed that the maximum value was around − 19 mV (Fig. 7A). MIC and MBC of AgNPs (µg/mL). The MIC value for AgNPs was 1 µg/mL, and MBC was 2 µg/mL against
MBL-producing Pseudomonas aeruginosa. The results depict that AgNPs conjugated with antibiotics are effec-
tive in the case of multidrug-resistant Pseudomonas aeruginosa and can be employed as an alternate treatment
option following various clinical studies. The antibacterial potency of AgNPs was analyzed by recording the
diameter of zones of inhibition in millimetres. This is illustrated in Fig. 8a,b. Effect of AgNP solution volume on antibacterial action. Different volumes (10–50 μL) of 100 ppm
AgNP solution were used to assess the impact of suspension volume on inhibition zone size. In the case of each
test isolate, an increase in the inhibition zone was monitored with the corresponding rise in AgNP solution
volume as shown in Fig. 8c. Synergistic antibacterial effect of antibiotics in conjugation with AgNPs. The combined effect
of AgNPs in conjugation with multiple antibiotics was construed against multidrug-resistant Pseudomonas aer-
uginosa using the disk diffusion method. It was observed that the antibacterial potency of the antibiotics was
upgraded in the presence of AgNPs. β cos θ, (A) UV–Vis spectra recorded for
the biosynthesis of AgNPs in response to 0.1 M silver nitrate inoculated media of A. flavus. (B) Size distribution
profile of AgNPs synthesized prepared by cultural filtrates of A. flavus {A total number of 100 particles were
analyzed}. (C) XRD patterns of AgNPs biosynthesized from cultural filtrates of A. flavus. (D) TEM micrographs
of AgNPs produced by cultural filtrates of A. flavus. about 2–5 min. Approximately 25 µL of the sample was pipetted out and loaded onto a ‘stub’ supplied for SEM
analysis. The stub is approximately 1 cm in diameter has a cylindrical shape and is made of copper. Scanning of
SEM was performed at the accelerating voltage of 25 kV from a distance of 5 mm to 5 cm (Fig. 7D). Scattered area electron diffraction (SAED). The crystalline character of NPs was determined by employing the
SAED analysis supplementarily with TEM. Diffractions were specifically acquired at a distance of 80 cm. Myco-
genized AgNPs were majorly crystalline in nature, which could be perfectly listed to the Bragg reflections of the
face-centered cubic (fcc) nature of the crystalline silver (Fig. 7B). Nano‑suspensions of these myco‑genized metallic NPs. Approximately 100 ppm of NP was diffused in test tubes
that contained 100 mL of sterile deionized water to obtain nano-suspensions. Dynamic light scattering (DLS). Determination of the AgNPs size distribution was done by dynamic light
scattering measurements on Malvern Zeta Sizer Nano ZS (Malvern Instruments Ltd., UK) using disposable
clear zeta cells (DTS 1060C). The instrument allowed for an average diameter along with polydispersity index
(PDI). Recordings for zeta average diameter plus PDI reported herewith were obtained by calculating an aver-
age of three separate measurements where each measurement was recorded after ten repetitions on each sample
(Fig. 7C). Zeta potential measurement. Zeta potential analysis is for the determination of the surface charge of AgNPs in
solution form. The magnitude of zeta potential predicts the stability of the colloids. AgNPs having Zeta potential
value exceeding + 25 mV or lesser than a value of − 25 mV possess a higher probability of stability. Dispersions
having lower zeta potential value clump because of inter-particle Van Der Waal attractions. https://doi.org/10.1038/s41598-022-24531-9 Scientific Reports | (2022) 12:20685 | www.nature.com/scientificreports/ Figure 7. Myco-genized AgNPs (A) Zeta potential measurements for mycogenized AgNPs. (B) SAED analysis
(C) Dynamic Light Scattering (DLS) measurements (D) SEM micrographs of AgNPs. Figure 7. Discussionh The ability to produce Metallo-β-lactamases is the most vital mechanism by which Pseudomonas aeruginosa
gains antimicrobial resistance against several drugs. Rapidly emerging strains of Pseudomonas aeruginosa that
can produce MBLs are an urgent concern for hospitals and healthcare centers42. Hospital environment in lower
middle economic countries has certain factors that contribute to the spreading of infections by MDR bacteria. These are improper handling of medical instruments, contaminated hands of medical personnel, and various
unhygienic surfaces like floors and doorknobs42.f l
Multidrug-resistant phenotypes in Pseudomonas aeruginosa result from different mechanisms that are found
to interact with one another thus providing antimicrobial resistance43. Increased expression of the efflux pump,
reduction in the porins present in the external membrane, and modified geometry of the penicillin-binding
proteins contribute to acquired resistance43. Acquisition of dual resistance to more than a single class of anti-
microbials calls for reasoned treatment of infections owing to Pseudomonas aeruginosa39,43. Isolates of Pseu-
domonas aeruginosa resistant to ceftazidime and meropenem exhibit MBL and ESBL activity confirming that
MBL-producing genes are considered crucial to resistance against these antimicrobials25,29. p
g g
g
In this study, more isolates were obtained from samples collected from female patients (145/523) as compared
to male patients (110/636). These findings differ from a study that was carried out in Germany whereby out of a
total of 168 patients, 67.3% of males, and 32.7% of females tested positive44. The highest isolates of Pseudomonas
aeruginosa were from patients aged 40–49 years (25.4%). These results differ from another study that reported
patients aged more than 55 years having a higher prevalence of Pseudomonas aeruginosa45. This study shows a
much-increased prevalence of infections caused by Pseudomonas aeruginosa owing to poor health and sanitary
condition of patients in hospital environments, improper prescription of antimicrobial drugs, and the presence
of antimicrobial resistance in bacteria46. Almost 54.5% of the MDR strains of Pseudomonas aeruginosa were
observed by Saderi et al.47. This increase in resistance against antibiotics may be due to activated efflux pumps
in bacteria, modification of the target site of drugs, the presence of reducing enzymes, or the loss of membrane
proteins48. Strains of Pseudomonas aeruginosa isolated in this study showed higher resistance against carbapenem
drugs, i.e., imipenem (53%) and meropenem (51%). These findings are synonymous with a study conducted in
the United States that mentions 65% resistance to carbapenem drugs49. β cos θ, The reducing order of antimicrobial activity of antibiotics and AgNPs plus
their combinations was piperacillin/tazobactam + AgNPs (31 ± 1.4 mm), cefoxitin + AgNPs (30 ± 1.0 mm) > ami-
kacin + AgNPs (25 ± 1.3 mm) > aztreonam + AgNPs (23 ± 1.5 mm) > meropenem + AgNPs (22 ± 1 mm) > imipe-
nem + AgNPs (20 ± 1.5 mm) > gentamicin + AgNPs (17 ± 0.5 mm) > ciprofloxacin + AgNPs (16 ± 0.1 mm) > cefop-
erazone/sulbactam + AgNPs (14 ± 0.4 mm) ≥ ceftazidime + AgNPs (14 ± 1.2 mm). The combined effect of AgNPs https://doi.org/10.1038/s41598-022-24531-9 Scientific Reports | (2022) 12:20685 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 8. Antimicrobial effects: (a,b) AgNPs and antibiotic-AgNPs combinations, (c) AgNPs activity at varying
volumes. Figure 8. Antimicrobial effects: (a,b) AgNPs and antibiotic-AgNPs combinations, (c) AgNPs activity at varying
volumes. plus antibiotics showed a 0.15–3.51 (average, 2.09) fold-area augmentation of the antibacterial activity. The
potency of AgNPs alone and in combination with antibiotics was ascertained by measuring the inhibition zones. The inhibition zones were measured in a range of 14–31 mm. AgNPs used alone led to weaker bactericidal
activities. Combinations of standard antibiotics with AgNPs ensued in a 0.15–3.51 (average 2.09) fold-area aug-
mentation of the antibacterial activity. Typically, the highest zone of inhibition(s) was recorded by piperacillin/
tazobactam in combination with AgNPs. Isolates resistant to ceftazidime showed a zone of inhibition measuring
14 ± 1.2 mm in combination with AgNPs (Table 6). plus antibiotics showed a 0.15–3.51 (average, 2.09) fold-area augmentation of the antibacterial activity. The
potency of AgNPs alone and in combination with antibiotics was ascertained by measuring the inhibition zones. The inhibition zones were measured in a range of 14–31 mm. AgNPs used alone led to weaker bactericidal
activities. Combinations of standard antibiotics with AgNPs ensued in a 0.15–3.51 (average 2.09) fold-area aug-
mentation of the antibacterial activity. Typically, the highest zone of inhibition(s) was recorded by piperacillin/
tazobactam in combination with AgNPs. Isolates resistant to ceftazidime showed a zone of inhibition measuring
14 ± 1.2 mm in combination with AgNPs (Table 6). Discussionh The MIC values of meropenem and imi-
penem were in the range of 8 μg/mL to ≥ 32 μg/mL. These results agree with a previous study whereby the MIC
of imipenem was > 32 μg/mL50. Results of the current study have intimated a prevalence of MBL producers as https://doi.org/10.1038/s41598-022-24531-9 Scientific Reports | (2022) 12:20685 | www.nature.com/scientificreports/ ZOI (mm) of antibiotics in combination with AgNPs
Pseudomonas aeruginosa
Piperacillin/tazobactam (A) (100 µg)
18 ± 0.5
AgNP
13 ± 0.6
Piperacillin/tazobactam + AgNP (B)
31 ± 1.4
Fold increase of inhibition zone areab
1.97 (72.2%)
Cefoxitin (A) (30 µg)
18 ± 0.3
AgNP
13 ± 0.5
Cefoxitin + AgNP (B)
30 ± 1.0
Fold increase of inhibition zone areab
1.8 (66.6%)
Amikacin (A) (30 µg)
16 ± 0.7
AgNP
13 ± 0.6
Amikacin + AgNP (B)
25 ± 1.3
Fold increase of inhibition zone areab
1.4 (56.2%)
Aztreonam (A) (10 µg)
14 ± 0.4
AgNP
13 ± 0.2
Aztreonam + AgNP (B)
23 ± 1.5
Fold increase of inhibition zone areab
1.7 (64.2%)
Meropenem (A) (10 µg)
12 ± 1
AgNP
13 ± 0.5
Meropenem + AgNP (B)
22 ± 1
Fold increase of inhibition zone areab
2.3 (83.3%)
Imipenem (A) (10 µg)
10 ± 1.5
AgNP
13 ± 0.5
Imipenem + AgNP (B)
20 ± 1.5
Fold increase of inhibition zone areab
3.0 (100%)
Gentamicin (A) (10 µg)
8 ± 0.1
AgNP
13 ± 0.4
Gentamicin + AgNP (B)
17 ± 0.5
Fold increase of inhibition zone areab
3.5 (112.5%)
Ciprofloxacin (A) (5 µg)
8 ± 1.2
AgNP
13 ± 0.6
Ciprofloxacin + AgNP (B)
16 ± 0.1
Fold increase of inhibition zone areab
3.0 (100%)
Cefoperazone/sulbactam (A) (75–10 µg)
8 ± 0.5
AgNP
13 ± 1.3
Cefoperazone/sulbactam + AgNP (B)
14 ± 0.4
Fold increase of inhibition zone areab
2.1 (75%)
Ceftazidime (A) (30 µg)
0a
AgNP
13 ± 0.3
Ceftazidime + AgNP (B)
14 ± 1.2
Fold increase of inhibition zone areab
0.15 (133.3%) Table 6. Single and combined efficacy of AgNPs and antibiotics against MDR Pseudomonas aeruginosa. Rank
order is indicated by Alphabets. Vertically, the ranking is linked to combinations of antibiotics against bacterial
isolates; horizontally, the ranking is of bacterial isolates with respect to sensitivity to antibiotic amalgamation
(P, 0.05). aDisc diameter (6 mm) was employed for calculation of fold increase in absence of bacterial growth
inhibition zone34. www.nature.com/scientificreports/ combination strongly binds to the bacterial cells promoting the release of Ag(+) and resulting in an increased
concentration of Ag(+) in the vicinity of bacteria. These findings are in support of the theory that Ag(+) release
from AgNPs is the potential agent that causes toxicity52,53. g
p
g
y
Minuscule AgNPs as found in the current study having a spherical shape possessing a microscale diameter
are more sensitive to release silver because of enhanced surface area. Furthermore, AgNPs have the ability to
penetrate the cell walls of bacteria by modifying their structure due to their nanoscale size14,52. Disruption of the
cellular membranes can lead to rupture of the organelles resulting in cellular lysis13,14,39. The smaller size of nano-
particles in this study (5–30 nm) is attributed to their high efficiency in penetrating bacterial cells as compared to
previous studies where the size of nanoparticles was 65–90 nm in diameter14. The collaborative effect of AgNPs
plus antibiotics showed a 0.15–3.51 (average, 2.09) fold-area augmentation of the antibacterial activity. Several
studies have recognized the antimicrobial potency of silver nanoparticles in combating bacteria. Unfortunately,
the precise mechanism involved has not yet been ascertained44. p
y
AgNPs can be toxic due to the release of ionic Ag in combination with surface properties, shape, and size. Lesser toxicity is associated with prismatic and cubic geometries of AgNPs at a concentration of ˂ 100 µg/L54. Auclair et al. have determined that sublethal toxicity ascertained at 96 h on the basis of salient characteristic mor-
phological changes exhibits the following toxicity: ionic (2.6 µg/L), spherical (22 µg/L), and prismatic (32.5 µg/L)
AgNPs54. Auclair et al. have also concluded that the structure of nanocube was not toxic at this concentration
while nanoparticles possessing a low aspect ratio combined with high circularity as well as elongation properties
exhibit high toxicity at both sublethal and lethal levels. AgNPs shape has been studied to influence the toxic-
ity demanding further research in the field for understanding the mechanisms playing part in making AgNPs
toxic54. Furthermore, varying neurobehavioral effects have been reported by Vogt et al. for various coatings and
sizes of AgNPs while studying exposure of larvae to Ag + which suggests that AgNPs potentially act as a neu-
robehavioral disruptor55. Fu et al. have reported that exposure to AgNPs impairs social behavior and learning in
the subjects studied which indicates a strong neurotoxic effect56. Bhalodia et al. Conclusionsh The current study concludes the high prevalence of multidrug-resistant Pseudomonas aeruginosa whereby a
positive correlation has been observed between phenotypic and genotypic variants of Pseudomonas aeruginosa. Nevertheless, the antibiotic-silver nanoparticles combination has shown antibacterial potency against MDR
Pseudomonas aeruginosa. Combinations of AgNPs with conventional antibiotics can possibly be researched as
alternatives to antimicrobial agents for curing infections caused by MDR Pseudomonas aeruginosa following
multiple clinical trials. www.nature.com/scientificreports/ reported an MIC ranging from
1.406 to 5.625 µg/mL plus an MBC ranging from 2.813 to 5.625 µg/mL for Metallo-β-lactamase producers in
Pseudomonas aeruginosa35.if g
Uncoated AgNPs promote significant cytotoxic effects on PBMCs at proportionately lesser concentrations
(< 5 μg/mL) and shorter exposure times (3–12 h). PBMCs are cells of the immune system that constitute lym-
phocytes (T cells, B cells, and NK cells) plus monocytes. This suggests that the coating of AgNPs decreases the
interacting active surface area sites with the cellular components57,58. It has been reported by Kim et al. that
AgNPs exhibit genotoxic effects in BEAS-2B cells that are bronchial epithelial cell lines59. The oxidative stress
promoted by AgNPs might be a pivotal element in the genotoxic effects caused by AgNPs59. AgNPs have been
studied to cause damage in DNA while creating formation of the micronucleus in a dose-dependent approach. The specific AgNPs dose-dependent activity due to the formation of reactive oxygen radicals has been studied
to be reduced by superoxide dismutase most importantly as shown in the cytokinesis-block MN assay as well as
the comet assay. The present study demonstrates that AgNPs conjugated with antibiotics inhibited Pseudomonas
aeruginosa as the zone of inhibition significantly increased with the use of combined discs. Data availability y
All data generated or analysed during this study are included in this published article (and its Supplementary
Information file). Received: 7 July 2022; Accepted: 16 November 2022 References e e e ces
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7. Algammal, A. M. et al. Emerging MDR-Mycobacterium avium subsp. avium in house-reared domestic birds as the first report in
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7. Algammal, A. M. et al. Emerging MDR-Mycobacterium avium subsp. avium in house-reared domestic birds as the first report in
Egypt. BMC Microbial. 21, 237 (2021). gy
8. Nahid, F., Khan, A. A., Rehman, S. & Zahra, R. Prevalence of metallo-β-lactamase NDM-1-producing multidrug resistant bacteria
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8. Nahid, F., Khan, A. A., Rehman, S. & Zahra, R. Prevalence of metallo-β-lactamase NDM-1-producing multidrug resistant bacteria
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9. Qureshi, R., Shafique, M., Shahid, M., Rasool, M. H. & Muzammil, S. Molecular detection of blaVIM Metallo-β-lactamase produc-
ing clinically isolated Pseudomonas aeruginosa from tertiary care hospital, Faisalabad. Pak. J. Pharm. Sci. 31, 2673–2677 (2018). Discussionh bFold increase of inhibition zone area = (B2 − A2)/A2 As % synergism = (B −A)/A × 100. AgNP silver nanoparticles. Table 6. Single and combined efficacy of AgNPs and antibiotics against MDR Pseudomonas aeruginosa. Rank
order is indicated by Alphabets. Vertically, the ranking is linked to combinations of antibiotics against bacterial
isolates; horizontally, the ranking is of bacterial isolates with respect to sensitivity to antibiotic amalgamation
(P, 0.05). aDisc diameter (6 mm) was employed for calculation of fold increase in absence of bacterial growth
inhibition zone34. bFold increase of inhibition zone area = (B2 − A2)/A2 As % synergism = (B −A)/A × 100. AgNP silver nanoparticles. Table 6. Single and combined efficacy of AgNPs and antibiotics against MDR Pseudomonas aeruginosa. Rank
order is indicated by Alphabets. Vertically, the ranking is linked to combinations of antibiotics against bacterial
isolates; horizontally, the ranking is of bacterial isolates with respect to sensitivity to antibiotic amalgamation
(P, 0.05). aDisc diameter (6 mm) was employed for calculation of fold increase in absence of bacterial growth
inhibition zone34. bFold increase of inhibition zone area = (B2 − A2)/A2 As % synergism = (B −A)/A × 100. AgNP silver nanoparticles. 61.5% of which 11.5% expressed blaIMP-1/blaVIM while blaTEM/blaOXA were expressed in 19.5% and blaOXA/blaSHV
were concomitantly expressed in 9.5%. Tahmasebi et al. have reported the expression of 12 phenotypically
MBL-producing isolates of Pseudomonas aeruginosa, of which PCR amplification confirmed blaVIM in 33.3%,
and blaIMP in 25% of isolates41.h IMP
The antibacterial activities of silver have been discussed globally. Recently, thiol-dependent enzymes includ-
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rial sensitivity towards antibiotics by blocking the Trx system16. Concomitantly, reactive oxygen species (ROS)
generate helping bacteria become sensitive to conventional antibiotics16,51. Furthermore, the antibiotic- AgNPs Scientific Reports | (2022) 12:20685 | https://doi.org/10.1038/s41598-022-24531-9 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ 10. Algammal, A. M. et al. atpD gene sequencing, multidrug resistance traits, virulence-determinants, and antimicrobial resistance
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ing both metalloβ-lactamase VIM-2 and extended-spectrum β-lactamase IBC-1. Clin. Microbiol. Infect. 10, 757–760 (2004). Acknowledgementsh g
This work was organized by the Institute of Molecular Biology & Biotechnology (IMBB), The University of Lahore
in collaboration with Jinnah Hospital Lahore for specimen and data collection. The efforts of Dr. Mehdi at The
University of Lahore are highly acknowledged for helping in data analysis. Authors acknowledge contribution
and help from the concerned staff members at the IMBB department, The University of Lahore for all the help
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2. Deng, H. et al. Mechanistic study of the synergistic antibacterial activity of combined silver nanoparticles and common antibiotics
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59. Kim, H. R., Kim, M. J., Lee, S. Y., Oh, S. M. & Chung, K. H. Genotoxic effects of silver nanoparticles stimulated by oxidative stress
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9. Kim, H. R., Kim, M. J., Lee, S. Y., Oh, S. M. & Chung, K. H. Genotoxic effects of silver nanoparticles stimulated by oxidative stress
in human normal bronchial epithelial (BEAS-2B) cells. MRGTEM. 726, 129–135 (2011). Author contributions S.Z.H., M.M., S.Sh.-conceived the study protocol; M.M., A.R., S.M.-coordinated the study; M.M., M.A., A.B.-
participated in data collection; M.A., O.A.D. participated in data interpretation; M.M., A.R., S.M.-carried out
the literature search; M.M., M.S.B., S.S.-drafted the present manuscript; S.Z.H., S.Sh.-revised the manuscript. All the authors read and approved the final version of the manuscript and agree to be accountable for all aspects
of the work. www.nature.com/scientificreports/ Labarca, J. A., Salles, M. J., Seas, C. & Guzmán-Blanco, M. Carbapenem resistance in Pseudomonas aeruginosa and Acinetobacter
baumannii in the nosocomial setting in Latin America. Crit. Rev. Microbiol. 42, 276–292 (2016). g
0. Negi, A. et al. Assessment of doripenem, meropenem, and imipenem against respiratory isolates of Pseudomonas aeruginosa in a
tertiary care hospital of North India. Indian J. Crit. Care Med. 21, 703–706 (2017). https://doi.org/10.1038/s41598-022-24531-9 Scientific Reports | (2022) 12:20685 | © The Author(s) 2022 Funding
Th Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit
sectors. No funding was received for this study. Competing interests
The authors declare no competing interests. Competing interests h Competing interests
The authors declare no competing interests. p
g
The authors declare no competing interests. Additional information
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Different combination strategies for prophylaxis of venous thromboembolism in patients: A prospective multicenter randomized controlled study
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Different combination strategies
for prophylaxis of venous
thromboembolism in patients:
A prospective multicenter
randomized controlled study
Cui-Qin Sang1, Na Zhao1, Jian Zhang1, Shu-Zhen Wang1, Shu-Li Guo1, Shu-Hong Li1,
Ying Jiang1, Bin Li2, Jian-Liu Wang3, Lei Song4, Jian-Jun Zhai5 & Zhen-Yu Zhang1 Received: 16 February 2018
Accepted: 17 April 2018
Published: xx xx xxxx The aim was to evaluate the efficacy and safety of different combination strategies for prophylaxis of
venous thromboembolism (VTE) after gynecologic surgery in patients at different levels of risk. This
was a prospective multicenter randomized controlled study, in which 625 women who would undergo
pelvic surgery for gynecologic diseases were stratified into three risk groups and then randomized into
four groups to receive graduated compression stockings (GCS) alone (group A), GCS + low molecular
weight heparin (LMWH) (group B), GCS + intermittent pneumatic compression (IPC) (group C), and
GCS + IPC + LMWH (group C), respectively. The overall incidence of DVT was 5.1%. Group A had the
highest incidence of DVT (8.8%), followed by group C (5.2%), group B (3.8%), and group D (2.6%). There
was a significant difference in the incidence of DVT between groups A and D. The incidence of DVT was
significantly lower in LMWH-treated patients (group B + group D) than in non-LMWH-treated patients
(group A + group C). In conclusion, combination prophylaxis, especially LMWH-containing strategies,
is better than monoprophylaxis in reducing VTE after gynecologic surgery. Risk-stratified prophylactic
strategies should be implemented in patients undergoing gynecologic surgery, with LMWH-containing
strategies being recommended for high-risk and very-high-risk patients. Venous thromboembolism (VTE), manifesting mainly as deep venous thrombosis (DVT) and pulmonary embo-
lism (PE), is a common serious complication of gynecologic surgery1. Epidemiological studies from our team
and other groups show that the incidence of DVT following gynecologic surgery ranges from 6.2% to 37.9%2–4. Although the true incidence of PE in patients after gynecologic surgery is currently unknown, it has been reported
that 0.3–4.4% of patients undergoing thromboembolic prophylaxis after gynecologic oncology surgery developed
PE5,6. PE is the leading cause of mortality following gynecologic surgery, with a mortality rate as high as 40%2. Since the majority of patients with fatal PE often die within 30 min following the appearance of symptoms, there
is no chance of thrombolytic therapy or surgery. Therefore, the prevention of VTE is of particular importance. y
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Many interventions and strategies are currently available to reduce the risk of VTE, such as preoperative phar-
macologic prophylaxis and mechanical prophylaxis7. Heparin, including unfractioned heparin and low molecular
weight heparin (LMWH), is the most commonly used pharmacologic agent for the prophylaxis of VTE. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 16 February 2018
Accepted: 17 April 2018
Published: xx xx xxxx Materials and Methodsh Patients. This multicenter randomized controlled trial was sponsored by Beijing Chaoyang Hospital of
Capital Medical University and performed from June 2011 to December 2015 at five tertiary referral centers,
including Beijing Chaoyang Hospital of Capital Medical University, Beijing Anzhen Hospital of Capital Medical
University, The PLA General Hospital, Peking University People’s Hospital, and Beijing Tongren Hospital of
Capital Medical University. The study was approved by the Ethics Committee of Beijing Chao-Yang Hospital,
Capital Medical University (No. 10-Ke-42). All participants provided written informed consent before enrollment
and the study was performed in accordance with the Helsinki II declaration. Women who would undergo pelvic surgery for gynecologic diseases at the above-mentioned centers were
initially assessed for eligibility. Inclusion criteria were: (1) age >18 years; (2) carrying ≥1 risk factor for postoper-
ative VTE (see below for VTE risk stratification); (3) not taking any prophylactic measures before enrollment; and
(4) willing to sign a written informed consent form. Gynecologic diseases may be malignant or benign. Malignant
diseases included malignancies of the ovary, uterine body, uterine cervix, vulva, and other parts of the pelvis. Benign diseases included uterine myoma, uterine adenomyoma, ovarian benign tumors, pelvic floor prolapsed,
and others such as hydrosalpinx, fallopian tube abscess, encapsulated effusion, and mesosalpinx cyst. Exclusion
criteria included: (1) preoperative thrombophlebitis or PE; (2) preoperative acute lower extremity venous throm-
bosis; (3) preoperative thrombocytopenia (platelet count <100 × 109/L) or coagulation disorders; (4) usage of
anticoagulant drugs such as aspirin within 1 month; (5) bleeding tendency as revealed by coagulation indexes or
previous intracranial or gastrointestinal bleeding; (6) congestive heart failure or pulmonary edema; (7) serious leg
abnormalities (such as dermatitis, gangrene, or recent skin grafting), severe lower limb vascular atherosclerosis,
lower limb ischemic vascular disease, or severe leg deformity; and (8) imperception of dorsalis pedis artery pulse. VTE risk stratification. VTE risk stratification was performed according to the 2008 American College of
Chest Physicians guidelines on VTE prevention1 and our previous studies on risk factors for VTE4,18. Risk factors
analyzed included age ≥50 years, hypertension, previous DVT/PE, thrombophilia, previous cerebral infarction,
previous myocardial infarction, lower extremity varices, present surgery for malignancy, laparotomy surgery,
operative time ≥3 h, and postoperative bed rest time ≥48 h. www.nature.com/scientificreports/ (IPC), can avoid the risk of bleeding associated with heparin usage; however, these methods are insufficient in
reducing VTE in high-risk patients or in critically ill patients10–12. Thus, more intense prophylactic strategies are
required for high-risk patients. q
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p
Compared with either pharmacologic or mechanical prophylaxis alone, dual prophylaxis has biological plau-
sibility in further reducing postoperative VTE in high-risk patients7. A systematical review indicates that dual
prophylaxis is superior to monoprophylaxis in reducing postoperative VTE, even in high-risk patients with gyne-
cologic cancer13. The American College of Chest Physicians has recommended dual prophylaxis for patients at
high risk for postoperative VTE7,14. Consistent with these, in patients after gynecologic surgery for malignancies,
the efficacy of mechanical prophylaxis in reducing postoperative VTE can be improved significantly when a
pharmacologic prophylactic method was added11,15. Our previous study also showed that the combination of two
mechanical prophylactic methods (GCS + IPC) was better than mechanical prophylaxis alone (GCS) in prevent-
ing VTE in high-risk patients after gynecologic surgery16. Combination prophylactic strategies have been recom-
mended by some researchers for patients at very high risk for VTE after gynecologic surgery17. y
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Despite many previous studies on dual prophylaxis for postoperative VTE prevention, the data obtained from
gynecologic patients are still limited and there are currently very few large prospective randomized controlled
studies. Furthermore, previous studies rarely stratified the risk of VTE among patients to identify the optimal
strategy for patients at different levels of risk for VTE after gynecologic surgery. In this prospective multicenter
randomized controlled study, we compared the efficacy and safety of GCS alone, GCS + LMWH, GCS + IPC, and
GCS + IPC + LMWH in preventing VTE among patients who were stratified to have different levels of risk for
VTE after gynecologic surgery. Different combination strategies
for prophylaxis of venous
thromboembolism in patients:
A prospective multicenter
randomized controlled study
Cui-Qin Sang1, Na Zhao1, Jian Zhang1, Shu-Zhen Wang1, Shu-Li Guo1, Shu-Hong Li1,
Ying Jiang1, Bin Li2, Jian-Liu Wang3, Lei Song4, Jian-Jun Zhai5 & Zhen-Yu Zhang1 Many
clinical trials indicate that heparin is a reliable method to decrease VTE in postoperative patients7,8. However,
high-dose heparin is associated with a risk of perioperative bleeding, while low-dose heparin was found to be
inefficient in high-risk patients with gynecologic cancer9. Mechanical prophylaxis, including passive methods
such as graduated compression stockings (GCS) and active methods such as intermittent pneumatic compression 1Department of Obstetrics and Gynecology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing,
100020, China. 2Department of Obstetrics and Gynecology, Beijing Anzhen Hospital, Capital Medical University,
Beijing, 100029, China. 3Department of Obstetrics and Gynecology, Peking University People’s Hospital, Beijing,
100044, China. 4Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing, 100853, China. 5Department of Obstetrics and Gynecology, Beijing Tongren Hospital, Capital Medical University, Beijing, 100730,
China. Correspondence and requests for materials should be addressed to Z.-Y.Z. (email: zhangzhenyu@coga.org.cn) SCientifiC Reports | (2018) 8:8277 | DOI:10.1038/s41598-018-25274-2 1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ IPC. The Kendall Tyco SCD response compression system (USA), a 6-chamber sequential compression device,
was used. The pressure started at the ankle at 45 mmHg and moved towards to the leg at 35 mmHg and the thigh
at 30 mmHg. Each compression lasted 11 seconds, and the duration of relaxation was spontaneously adjusted
according to the venous filling velocity. The therapy started 30 min before surgery and discontinued while the
patient can ambulate1,16, generally ~24 hours after surgery. IPC. The Kendall Tyco SCD response compression system (USA), a 6-chamber sequential compression device,
was used. The pressure started at the ankle at 45 mmHg and moved towards to the leg at 35 mmHg and the thigh
at 30 mmHg. Each compression lasted 11 seconds, and the duration of relaxation was spontaneously adjusted
according to the venous filling velocity. The therapy started 30 min before surgery and discontinued while the
patient can ambulate1,16, generally ~24 hours after surgery. LMWH. Dalteparin (Fragmin Injection, 5000 IU in 0.2 ml solution; Pharmacia, Germany) was used for phar-
macologic prophylaxis22,23. Periumbilical subcutaneous injection of dalteparin 5000 IU was performed 12 h after
surgery, once a day for 5 days24,25. Measurements. Venous blood samples were collected 7 days before and 3 days after surgery to determine
hemoglobin, platelets, and coagulation indexes including prothrombin time, thrombin time, activated partial
blood coagulation time, fibrinogen, and D-dimer. Routine hematological and biochemical parameters and rou-
tine urine parameters were determined 1 day after surgery.t t
Screening of VTE was performed within 7 days before and 3–5 days after surgery. Initially, color Doppler
ultrasound imaging of lower extremities was performed by an experienced professional using an LEGIQ E9 color
Doppler system with the probe frequency set at 8.4–9 MHz26. If VTE was found or suspected, lower limb venogra-
phy was performed to confirm the presence of DVT, and computed tomography pulmonary angiography (CTPA)
was performed with the Brilliance iCT system (model# 728306, Philips Medical Systems, USA) to detect if PE was
present. Once DVT or PE was diagnosed or suspected, a joint consultation involving cardiovascular and respira-
tory clinicians was launched to decide the treatment regimen. Safety evaluation. Safety evaluation was performed as previously described27,28. All adverse events reported
by participants were recorded by investigators at various centers, and the principal investigator judged whether to
discontinue the treatment or adopt clinical interventions. www.nature.com/scientificreports/ For bleeding events, the location and amount of bleed-
ing, the amount of transfusion, and the transfused blood components were recorded. The amount of bleeding was
estimated as previously described29. p
y
Bleeding events were classified as major and minor hemorrhagic events. Major hemorrhagic events included
life-threatening bleeding, bleeding occurring in major organs or locations, non-surgical site bleeding associated
with a decrease in hemoglobin level by ≥20 g/L or requiring transfusion of two or more units of whole blood,
surgical site bleeding requiring further interventions such as a second open or endoscopic surgery and vaginal
compression therapy, and surgical site bleeding resulting in instable hemodynamics or a decrease in hemoglobin
level by ≥20 g/L. Once major bleeding was found, LMWH would be discontinued immediately.i Minor bleeding was defined as all other bleeding events that did not fall under the categories of major bleed-
ing, such as surgical field bleeding, injection site bruising, drainage port bleeding, and vaginal stump bleed-
ing. For minor superficial bleeding, such as injection site bruising and drainage port bleeding, LMWH was not
discontinued. For other minor bleeding events including deep bleeding and wound hematoma, LMWH was
discontinued. Heparin-induced thrombocytopenia was diagnosed when a platelet count ≤50 × 109/L or <50% of the base-
line platelet count occurred in patients on heparin therapy, with other causes excluded30. Once heparin-induced
thrombocytopenia occurred, LMWH would be discontinued immediately. y p
y
Lower-limb impairments included skin lesions, ulcers, and limb necrosis. Follow-up. Patients who were diagnosed with or suspected of having VTE underwent a joint consultation. Once a diagnosis of PE was made, the patient was referred to the respiratory department for treatment and given
LMWH anticoagulant therapy, which was gradually switched to oral warfarin to achieve a stable INR of ~2.0. After 2 to 3 weeks, the patient was discharged, continued to take oral warfarin for 6 months with INR maintained
at 2.0 to 3.0, and was followed for 6 months in an outpatient manner. p
If a diagnosis of DVT was made, the patient was given anticoagulant therapy under the guidance of a cardio-
vascular clinician, which was gradually switched to oral warfarin achieve a stable INR of ~2.0. Materials and Methodsh Patients with ≤2 risk factors but ≥1 risk factor were
deemed to have a moderate level of risk for DVT, those with 3 risk factors deemed to have a high level, and those
with ≥4 factors deemed to have a very high level. Randomization. Eligible patients were randomized into four groups to receive GCS alone (group A;
n = 159), GCS + LMWH (group B; n = 157), GCS + IPC (group C; n = 153), or GCS + IPC + LMWH (group D;
n = 156). A simple randomization method was adopted for this study. Based on the calculated sample size (see
below), 660 consecutive random numbers were selected from a random number table, assigned to each patient,
and then divided by four. When the remainder was 0, 1, 2, and 3, the patient was allocated to groups A, B, C, and
D, respectively. The patients were included sequentially according to the date of surgery. Interventions. GCS. Knee-length GCS (Tyco Healthcare, USA) were used as previously described19 with
some modifications. GCS were properly sized based on the maximum leg circumference as follows: small size
for patients with a leg circumference ≤30.5 cm, medium size for those with a leg circumference ≤38.1 cm but
>30.5 cm, and large size for those with a leg circumference ≤44.5 cm but >38.1 cm20. According to the literature,
patients should wear GCS from the time of arrival to the operating room until ambulation21. In this study, the
patients also wore GCS from the time of arrival to the operating room but were allowed to put GCS off before bed-
time when they can move their lower limbs (usually 2 hours after surgery), and put on again on the next morning. The patients put GCS on for ~16 hours each day, during which GCS were put off every 6–8 hours to examine if
there were ischemic manifestations or skin impairments. SCientifiC Reports | (2018) 8:8277 | DOI:10.1038/s41598-018-25274-2 2 www.nature.com/scientificreports/ After ~2 weeks, the
patient was discharged, continued to take oral warfarin for 3 months with INR maintained at 2.0 to 3.0, and was
followed for 6 months in an outpatient manner.t p
For patients without postoperative VTE, they were discharged 5 to 7 days after surgery and followed f
month in an outpatient manner or by telephone. Sample size calculation. The sample size of this study was calculated according to the findings of our
previous studies on patients at high risk for VTE following gynecologic surgery16,20, in which we found that
the incidence of VTE was 18% (17/96) in the non-prophylaxis group, 12.5% (14/112) in the GCS alone group,
6% (6/94) in the IPC alone group, 1% in the LMWH alone group, and 4.8% (5/104) in the GCS + IPC group. Based on these incidence rates, we assumed that the incidence of VTE should be <1% in the GCS + LMWH and
GCS + IPC + LMWH groups. Thus, a sample size of 150 in each group can provide sufficient statistical power. Adding 10% for dropouts, a final sample size was calculated to be 660. An intention-to-treat analysis was not done. Statistical analysis. All statistical analyses were performed using SPSS 23.0 software for Windows (SPSS
Inc, Chicago, IL, United States). Numerical data, expressed as mean ± standard deviation, were compared using
independent samples t-tests between groups. Categorical data, expressed as percentages, were compared by χ2
tests if the data followed a normal distribution, and otherwise by nonparametric rank sum tests. Fisher’s exact test
was adopted if the sample size in any cell was <40 or if the theoretical frequency was <5. To adjust the potential
confounding factors such as operative time, unconditional Logistic regression was used to screen the risk factors SCientifiC Reports | (2018) 8:8277 | DOI:10.1038/s41598-018-25274-2 3 www.nature.com/scientificreports/ Figure 1. Flow chart of patient enrollment, randomization, and completion of the study. Figure 1. Flow chart of patient enrollment, randomization, and completion of the study. for VTE. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated, and a trend test for the ORs from
the fitting model was performed. P-values < 0.05 were considered statistically significant. for VTE. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated, and a trend test for the ORs from
the fitting model was performed. P-values < 0.05 were considered statistically significant. Results Baseline characteristics. After risk stratification, 716 patients at a moderate or higher level of risk for VTE
were initially identified, of whom 91 were excluded for not meeting the inclusion/exclusion criteria (n = 75),
refusing to participate in the study (n = 5), and estimated high risk for bleeding by surgeons (n = 11). Thus, a total
of 625 patients were finally included in the study, including 411 moderate-risk patients, 133 high-risk patients,
and 81 very-high-risk-patients. Figure 1 shows the flow diagram of patient enrollment, randomization, and com-
pletion of the study.i p
y
Of the 625 patients finally included, 232 had gynecologic malignancies and 393 had benign gynecologic
diseases. They ranged in age from 18 to 86 years, with a mean age of 53.7 ± 10.3 years, and their mean body
mass index (BMI) was 24.9 ± 3.7 kg/m2. There were no significant differences among the four groups in age,
BMI, distribution of diseases including gynecologic malignancies, hypertension, diabetes, lower limb varicosity,
respiratory system diseases, cardiovascular diseases, cerebrovascular diseases, digestive system diseases, other
malignancies, and anemia, history of thrombosis, usage of anticoagulants or hormonal drugs, or preoperative
chemotherapy or radiotherapy (Table 1). Of note, the distribution of patients at different levels of risk for VTE
showed no significant difference among the four groups (P > 0.05) (Table 2), and there was also no significant
difference in the distribution of patients with malignancies among the four groups or among different risk groups
(P > 0.05) (Table 3). Results Group Risk level
A
B
C
D
Overall
χ2
Moderate
15
22
18
34
89
High
15
15
20
21
71
6.651
Very high
18
13
23
18
72
Overall
48
50
61
73
232
Table 3. Distribution of patients with malignancies in the four groups based on risk level. Note: P < 0.05, either
among the four groups or among the four risk groups. Group Risk level
A
B
C
D
Overall
χ2
Moderate
15
22
18
34
89
High
15
15
20
21
71
6.651
Very high
18
13
23
18
72
Overall
48
50
61
73
232
Table 3. Distribution of patients with malignancies in the four groups based on risk level. Note: P < 0.05, either
among the four groups or among the four risk groups. Group Risk level
A
B
C
D
Overall
χ2
Moderate
15
22
18
34
89
High
15
15
20
21
71
6.651
Very high
18
13
23
18
72
Overall
48
50
61
73
232 Table 3. Distribution of patients with malignancies in the four groups based on risk level. Note: P < 0.05, either
among the four groups or among the four risk groups. Table 3. Distribution of patients with malignancies in the four groups based on risk level. Note: P < 0.05, either
among the four groups or among the four risk groups. Table 3. Distribution of patients with malignancies in the four groups based on risk level. Note: P < 0.05, eit
among the four groups or among the four risk groups. With regard to surgical situation, there were no significant differences among the four groups in mode of sur-
gery, patient-controlled analgesia, anesthetic techniques, site of infusion, or blood transfusion (Table 1). However,
a significant difference was observed in operative time and blood loss. Operative time was significantly longer in
groups C and D than in groups A and B (P < 0.05), although there were no significant differences between groups
A and B or between groups C and D. Blood loss was significantly more in group C than in the other three groups
(P < 0.05). Results SCientifiC Reports | (2018) 8:8277 | DOI:10.1038/s41598-018-25274-2 4 www.nature.com/scientificreports/ Group A
Group B
Group C
Group D
P-value
Age (yr)
54.2 ± 9.4
54.7 ± 11.2
52.6 ± 9.9
53.3 ± 10.5
0.291
BMI (kg/m2)
24.8 ± 3.5
24.8 ± 3.6
24.8 ± 3.8
25.4 ± 4.1
0.362
History of thrombosis (%)
1.3
1.9
0.7
2.6
0.570
Hypertension (%)
30.8
29.3
30.1
25.6
0.753
Diabetes (%)
17.0
12.1
11.1
16.7
0.315
History of smoking (%)
1.9
1.9
0.0
0.0
0.115
Varicosity (%)
8.8
7.6
5.9
9.6
0.648
Preoperative
anticoagulant usage (%)
1.9
2.6
0
0
0.075
Preoperative
chemotherapy (%)
3.8
2.6
2.6
6.4
0.230
Heart disease (%)
6.9
5.7
7.2
3.8
0.585
Respiratory diseases (%)
3.8
1.9
3.3
1.3
0.469
Anemia (%)
10.7
9.6
11.8
9.0
0.856
Open surgery (%)
37.1
34.4
38.6
25.6
0.149
Operative time (min)
165.3 ± 78.7
161.7 ± 81.1
196.6 ± 93.7
198.8 ± 95.3
0.000
Blood loss (mL)
50 (20–150)
50 (20–145)
60 (30–200)
50 (20–200)
0.042
Transfusion (%)
4.4
1.3
5.9
3.2
0.196
T bl 1 B
li
h
t i ti
f th f Table 1. Baseline characteristics of the four groups. Group Risk level
A
B
C
D
Overall
χ2
Moderate
102
113
95
101
411
High
35
31
34
33
133
5.383
Very high
22
13
24
22
81
Overall
159
157
153
156
625
Table 2. Distribution of patients at different levels of risk for VTE in the four groups. Note: P < 0.05, among the
four groups. Group Risk level
A
B
C
D
Overall
χ2
Moderate
102
113
95
101
411
High
35
31
34
33
133
5.383
Very high
22
13
24
22
81
Overall
159
157
153
156
625
Table 2. Distribution of patients at different levels of risk for VTE in the four groups. Note: P < 0.05, among the
four groups. Group Risk level
A
B
C
D
Overall
χ2
Moderate
102
113
95
101
411
High
35
31
34
33
133
5.383
Very high
22
13
24
22
81
Overall
159
157
153
156
625 Table 2. Distribution of patients at different levels of risk for VTE in the four groups. Note: P < 0.05, among the
four groups. Table 2. Distribution of patients at different levels of risk for VTE in the four groups. Note: P < 0.05, among the
four groups. Results Of note, the incidence of DVT was significantly lower in LMWH-treated patients
group B + group D) than in non-LMWH-treated patients (group A + group C) (χ2 = 4.78, P < 0.05).i With regard to risk stratification, the moderate-risk patients had the lowest incidence of DVT (2.2%, 9/411),
followed by the high-risk patients (5.3%, 7/133) and very-high-risk patients (19.8%, 16/81), and there was a
significant difference among the three groups (χ2 = 42.97, P < 0.01). This finding indicates that the incidence of
DVT increased with the risk level when the same intervention was given. Of note, 71.9% (23/32) of DVT cases
occurred in the high-risk and very-high-risk groups.hf g
y
g
g
p
The incidence of DVT in moderate-risk patients in different groups ranged from 0.9% to 3.0%, and group
B had the lowest incidence (0.9%, 1/113), followed by group C (2.1%, 2/95). In high-risk patients, group D had
the lowest incidence (3.0%, 1/33), followed by group A (6.5%, 2/31). In very-high-risk patients, group D had the
lowest incidence (0%, 0/22), while group A had the highest incidence (40.9%, 9/22), and there was a significant
difference in the incidence of DVT between them (χ2 = 8.94, P < 0.01). Incidence of PE. Of 32 cases of DVT, 26 underwent CTPA, which revealed that 12 had PE and 14 did not
have; six did not undergo CTPA due to hypersensitivity to contrast medium (n = 2) or patient refusal (n = 4). Of 12 cases of PE, no involvement of pulmonary artery trunk occurred, and all affected pulmonary segmental
arteries, including 9 cases of multiple bilateral pulmonary embolisms and 3 cases of single unilateral pulmonary
embolism. No fatal PE occurred. Four (1/3) cases had apparent symptoms including chest pain (n = 2), mild chest
distress (n = 1), and postoperative syncope (n = 1), all of which disappeared with rest.hh p
p
y
p
pp
The overall incidence of PE was 1.9% (12/625). The incidence of PE in groups A-D was 4.4% (7/159), 0.64%
(1/158), 1.96% (3/153), and 0.64% (1/156), respectively (Table 6). The incidence of PE in groups B and D was
significantly lower than that in group A (P < 0.05), but there was no significant difference between group A and
group C. SCientifiC Reports | (2018) 8:8277 | DOI:10.1038/s41598-018-25274-2 Results Data shown are the number of cases/limbs. Table 4. Location of DVT. Note: Six cases developed DVT at different locations, and 26 cases developed
bilateral DVT. Data shown are the number of cases/limbs. Group Risk level
A
B
C
D
Overall
Moderate
3/102 (2.9%)
1/113 (0.9%)
2/95 (2.1%)
3/101 (3.0%)
9/411 (2.2%)#
High
2/35 (5.7%)
2/31 (6.5%)
2/34 (5.9%)
1/33 (3.0%)
7/133 (5.3%)#
Very high
9/22 (40.9%)
3/13 (23.1%)
4/24 (16.7%)
0/22 (0%)**
16/81 (19.8%)#
Overall
14/159 (8.8%)
6/157 (3.8%)
8/153 (5.2%)
4/156 (2.6%)*
32/625 (5.1%)
Table 5. Incidence of DVT in the four groups based on risk level. Note: *P < 0.05 vs. group A; #P < 0.01 among
the three risk groups; **P < 0.01 vs. group A. Table 5. Incidence of DVT in the four groups based on risk level. Note: *P < 0.05 vs. group A; #P < 0.01 among
the three risk groups; **P < 0.01 vs. group A. Group Risk level
A
B
C
D
Overall
Moderate
2/102 (2.0%)
1/113 (0.9%)
0/95 (0%)
0/101 (0%)
3/411 (0.73%)
High
1/35 (2.9%)
0/31 (0%)
1/34 (2.9%)
1/33 (3.0%)
3/133 (0.23%)#
Very high
4/22 (18.2%)
0/13 (0%)
2/24 (8.3%)
0/22 (0%)**
6/81 (7.4%)#
Overall
7/159 (4.4%)
1/157 (0.64%)*
3/153 (2.0%)
1/156 (0.64%)*
12/625 (1.9%)#
Table 6. Incidence of PE in the four groups based on risk level. Note: *P < 0.05 vs. group A; #P < 0.01 among the
three risk groups; **P < 0.05 vs. group A. Table 6. Incidence of PE in the four groups based on risk level. Note: *P < 0.05 vs. group A; #P < 0.01 among the
three risk groups; **P < 0.05 vs. group A. Table 6. Incidence of PE in the four groups based on risk level. Note: *P < 0.05 vs. group A; #P < 0.01 among the
three risk groups; **P < 0.05 vs. group A. Table 6. Incidence of PE in the four groups based on risk level. Note: *P < 0.05 vs. group A; #P < 0.01 among the
three risk groups; **P < 0.05 vs. group A. between any two other groups. Of note, the incidence of DVT was significantly lower in LMWH-treated patients
(group B + group D) than in non-LMWH-treated patients (group A + group C) (χ2 = 4.78, P < 0.05).i etween any two other groups. Results To exclude the confounding effect of operative time and blood loss, unconditional Logistic regression
analysis was performed, which revealed that the difference remained statistically significant after adjusting for
operative time (OR = 0.618, 95%CI: 0.437–0.874, P = 0.007), but not for blood loss (OR = 1.001, 95%CI: 1.003–
1.01, P = 0.189). Incidence of DVT. Of the 625 patients included, no perioperative death occurred, and 32 developed DVT,
including 14 in group A, 6 in group B, 8 in group C, and 4 in group D. Of all cases of DVT, only one (1.7%, 1/58)
was proximal, involving the popliteal vein, and the rest were distal, involving the calf muscular vein (75.9%,
44/58), peroneal vein (17.2%, 10/58), and posterior tibial vein (5.2%, 3/58) (Table 4). Of note, six cases developed
DVT at different locations, and 26 cases developed bilateral DVT. Main clinical manifestations of DVT included
limb pain, swelling and congestion, skin eczema, local tenderness and dysfunction. Symptomatic patients
accounted for only 31% (10/32) in this study.h y
y
The overall incidence of DVT was 5.1% (32/625). As shown in Table 5, group A had the highest incidence
of DVT (8.8%, 14/159), followed by group C (5.2%, 8/153), group B (3.8%, 6/157), and group D (2.6%, 4/156). There was a significant difference in the incidence of DVT between groups A and D (χ2 = 5.69, P < 0.05), but not SCientifiC Reports | (2018) 8:8277 | DOI:10.1038/s41598-018-25274-2 5 www.nature.com/scientificreports/ Group A
Group B
Group C
Group D
Overall
Calf muscular vein
12/19
6/9
7/11
4/5
29/44
Peroneal vein
3/5
1/2
2/3
0
6/10
Posterior tibial vein
1/2
0
1/1
0
2/3
Popliteal vein
1/1
0
0
0
1/1
Overall
14/27
6/11
8/15
4/5
32/58
Table 4. Location of DVT. Note: Six cases developed DVT at different locations, and 26 cases developed
bilateral DVT. Data shown are the number of cases/limbs. Group A
Group B
Group C
Group D
Overall
Calf muscular vein
12/19
6/9
7/11
4/5
29/44
Peroneal vein
3/5
1/2
2/3
0
6/10
Posterior tibial vein
1/2
0
1/1
0
2/3
Popliteal vein
1/1
0
0
0
1/1
Overall
14/27
6/11
8/15
4/5
32/58
Table 4. Location of DVT. Note: Six cases developed DVT at different locations, and 26 cases developed
bilateral DVT. Data shown are the number of cases/limbs. Table 4. Location of DVT. Note: Six cases developed DVT at different locations, and 26 cases developed
bilateral DVT. ncidence (18.2%, 4/22), and there was a significant difference in the incidence of DVT between them (χ2 = 4.40
< 0.05). Rate of PE in patients with DVT. The rate of PE in patients with DVT showed no significant difference
either among the four groups or among the different risk groups (P > 0.05) (Table 7), suggesting that the inci-
dence of PE had a similar trend to that of DVT. Overall, the number of cases with DVT and/or PE was lower in
groups B and C for moderate-risk patients, in groups B and D for high-risk patients, and in group D for very
high-risk patients. Adverse events. Table 8 lists the bleeding events observed within 7 days after surgery in the four groups. No major bleeding or heparin-induced thrombocytopenia occurred in this study. Compared with groups A and
C, the number of mild bleeding events significantly increased in groups B and D (P < 0.01). These mild bleeding
events mostly occurred following 2–3 injections of LMWH. After LMWH was discontinued, bleeding stopped
and surgical hemostasis was not required. A total of 27 patients discontinued LMWH, but transfusion or hemo-
stasis was not required. q
During the follow-up period, four cases sought treatment for minor vaginal bleeding, including 1 case in
group A, 1 case in group C, and 2 cases in group D. Since the two cases in group D did not develop vaginal bleed-
ing within 7 days after surgery, their bleeding may not be caused by LMWH usage, but may be associated with
poor wound healing secondary to local inflammation or suture loosening. After anti-inflammatory treatment,
bleeding stopped. In addition, vaginal stump bleeding with an amount like that of menstrual bleeding occurred
in a patient with PE 12 days after surgery and a patient with DVT 14 days after surgery, both of whom belonged
to group A. Warfarin was discontinued in both cases until bleeding stopped, and then LMWH+ warfarin was
restarted. No lower-limb impairments occurred throughout the study period. Results incidence (18.2%, 4/22), and there was a significant difference in the incidence of DVT between them (χ2 = 4.40,
P < 0.05). Results Of note, the incidence of PE was significantly lower in LMWH-treated patients (group B + group D)
than in non-LMWH-treated patients (group A + group C) (P < 0.05).i With regard to risk stratification, the moderate-risk patients had the lowest incidence of PE (0.73%, 3/411),
followed by the high-risk patients (2.3%, 3/133) and very-high-risk patients (7.4%, 6/81), and there was a signif-
icant difference among the three groups (χ2 = 16.12, P < 0.01). This finding indicates that the incidence of PE
increased with the risk grade when the same intervention was given. Of note, 75% (9/12) of PE cases occurred in
the high-risk and very-high-risk groups.hf The incidence of PE in moderate-risk patients in different groups ranged from 0.0% to 2.0%, and groups C and
D had the lowest incidence (0.0% for both). In high-risk patients, group B had the lowest incidence (0.0%, 0/31). In very-high-risk patients, groups B and D had the lowest incidence (0% for both), while group A had the highest SCientifiC Reports | (2018) 8:8277 | DOI:10.1038/s41598-018-25274-2 6 www.nature.com/scientificreports/ Group Risk level
A
B
C
D
Overall
Moderate
2/3
1/1
0/2
0/3
3/9
High
1/2
0/2
1/2
1/1
3/7
Very high
4/9
0/3
2/4
0/0
6/16#5
Overall
7/14
1/6
3/8
1/4
12/32
Table 7. Incidence of DVT with PE in the four groups based on risk level. Group Risk level
A
B
C
D
Overall
Moderate
2/3
1/1
0/2
0/3
3/9
High
1/2
0/2
1/2
1/1
3/7
Very high
4/9
0/3
2/4
0/0
6/16#5
Overall
7/14
1/6
3/8
1/4
12/32
Table 7. Incidence of DVT with PE in the four groups based on risk level. Table 7. Incidence of DVT with PE in the four groups based on risk level. Table 7. Incidence of DVT with PE in the four groups based on risk level. Group A
Group B
Group C
Group D
Injection site bruising
13
13
Wound hematoma
0
8
0
6
Drainage port bleeding
0
0
2
3
Vaginal bleeding
0
1
0
5
Pelvic bleeding
2
4
1
0
Hematuria
0
1
0
1
Bleeding of unknown
cause
0
1
0
0
Overall
2
28
3
28
Table 8. Bleeding events observed within 7 days after surgery in the four groups. Table 8. Bleeding events observed within 7 days after surgery in the four groups. Table 8. Bleeding events observed within 7 days after surgery in the four groups. Discussionh 2–6 h postoperatively)
at a lower dose (5000 U, once daily) in the present study compared to conventional administrations24,25. These
modifications may explain why no major bleeding events occurred in this study. Combination prophylaxis, especially LMWH-containing strategies, is better than monoprophy-
laxis. Many randomized trials performed in other fields such as urology and general surgery have demon-
strated that combination prophylactic strategy is superior to either pharmacologic or mechanical prophylaxis
alone in reducing the incidence of postoperative VTE7. Despite few trials on gynecologic patients, combination
prophylaxis strategy has also been recommended by some researchers for this group of patients, especially those
who have a high risk for VTE, such as gynecologic oncology patients after a major surgery17. In agreement with
previous findings, this study indicated that combination strategies including GCS + IPC (5.2%), GCS + LMWH
(3.8%), and GCS + IPC + LMWH (2.6%) were better than GCS alone (8.8%) in reducing the incidence of VTE in
patients after gynecologic surgery. Thus, our study provides several additional options for combination prophy-
laxis of postoperative VTE in gynecologic patients. p
p
gy
g
p
A remarkable finding of this study is that LMWH-containing strategies are significantly better than
non-LMWH-containing ones in the prophylaxis of postoperative VTE in gynecologic patients. Compared to
the combination of two mechanical methods (GCS + IPC), the strategy combining both mechanical and phar-
macologic prophylaxis (GCS + LMWH or GCS + IPC + LMWH) exhibited better efficacy. LMWH has been
demonstrated to be reliable in decreasing VTE in postoperative patients7,8; however, high-dose LMWH may cause
perioperative bleeding while low-dose LMWH has insufficient efficacy in high-risk patients9. The combination
of mechanical prophylaxis with LMWH optimized for dosage and timing (e.g., 12 h postoperatively in this study)
may overcome these problems. In fact, although LMWH-containing strategies were associated with significantly
more bleeding events, all of them were mild and manageable. Different prophylactic strategies should be implemented in patients at different levels of risk
for VTE. Many risk factors for VTE have currently been identified4,18. Based on these risk factors, individual
risk assessment can be performed to guide the degree of prophylaxis. This study showed that the incidence of both
DVT and PE increased with the risk level even in patients receiving the same intervention, further suggesting
the importance of risk stratification to guide the administration of different interventions to patients at different
levels of risk. Discussionh Three primary factors predisposing to thrombosis (Virchow’s triad) are: (1) endothelial injury; (2) stasis or tur-
bulence of blood flow; and (3) hypercoagulability of blood31. Directing against these predisposing factors, many
prophylactic strategies have been developed to prevent postoperative VTE7. Since GCS, IPC, and LMWH are rela-
tively commonly used strategies, the efficacy and safety of their combination in three different ways in preventing
VTE after gynecologic surgery were evaluated in the present study. Technical details for prophylactic strategies used. Both GCS and IPC are mechanical prophylactic
methods, and they can increase venous return, reduce venous distention, and thus prevent venous stasis that con-
tributes to an increased risk of VTE7. In this study, IPC was performed using a commercial device as previously
described1,16, while some modifications were made to the use of GCS. Since knee-length GCS are as effective as
thigh-length ones, more comfortable to wear, and easier to apply19, the former was used in this study. Although
the use of GCS for the entire day was advocated in many previous studies1,21, this is associated with an increased
risk of pressure ulcer and rarely limb necrosis19. Furthermore, improperly fitted stockings may act as a tourni-
quet to result in venous stasis and increase the risk of VTE2. Particularly, the folding and distortion of stockings
that easily occur when patients are sleeping can result in the tourniquet effect. For these reasons, we allowed our
patients to put GCS off before bedtime once they can move their lower limbs (usually 2 hours after surgery), and
put on again on the next morning. This greatly increased patients’ comfort and improved their compliance. As SCientifiC Reports | (2018) 8:8277 | DOI:10.1038/s41598-018-25274-2 7 www.nature.com/scientificreports/ a consequence, the incidence of VTE (8.8%) in the GCS alone group was lower than that reported previously
(12.5%)16, and no pressure ulcer or limb necrosis occurred throughout the study period. In this study, we used dalteparin as a pharmacological agent. As an LMWH, dalteparin has the advantages
of simplicity, good anticoagulation effects, and low risk of heparin-induced thrombocytopenia22,23. Compared
to enoxaparin and nadroparin, dalteparin has a shorter plasma elimination half-life (2.8–3.8 h)32,33. Thus, once
bleeding occurs, discontinuation of dalteparin will result in a more rapid recovery of coagulation function. Due
to the combined use of dalteparin with GCS and/or IPC, dalteparin was given later (12 h vs. Discussionh Since the majority of DVT and PE cases were present in the high-risk and very-high-risk groups, the
emphasis of prophylaxis should be placed on this group of patients. p
p
p y
p
g
p
p
An ideal prophylaxis regimen should be tailed to the patient’s thromboembolic risk, thus allowing clinicians to
maximize the benefits and minimize the harms7. Although all four strategies were effective in the moderate-risk
patients, their efficacy still differed. Based on their efficacy, GCS + LMWH and GCS + IPC are recommended
for this group of patients although all four strategies are acceptable. When patients are not suitable for the use of
anticoagulants, GCS + IPC is recommended to avoid the risk of increased bleeding. For high-risk patients, both
LMWH-containing strategies resulted in an acceptable incidence of DVT. From a health-economics point of view,
GCS + LMWH is recommended. GCS + IPC + LMWH was associated with the lowest incidence of DVT in the
very-high-risk group. Therefore, this strategy is recommended for very-high-risk patients. Of note, GCS alone
are not recommended for both high-risk and very-high-risk patients due to its poor preventive effects, which is
consistent with the previous finding34. Limitations. Despite some promising results, there are several limitations to our study. First, the relatively
small number of high-risk and very-high-risk patients was underpowered to show statistical differences between
groups. In fact, although GCS + LMWH and GCS + IPC exhibited better efficacy than GCS alone in high-risk
and very-high-risk patients, no significant difference was observed. Second, 6 of 32 patients with DVT did not
undergo CTPA, and this might have resulted in the underestimation of the incidence of PE. Finally, the patients
without DVT were followed for 4 weeks in this study; however, it has been reported that DVT may develop 7–12
weeks after surgery35. Future studies with larger sample size and longer follow-up periods are needed to carefully
address these issues. Conclusions To conclude, this study shows that combination prophylaxis, especially LMWH-containing strategies, is superior
to monoprophylaxis in reducing the incidence of VTE after gynecologic surgery. Given the incidence of both
DVT and PE increases with the risk level even in patients receiving interventions, risk-stratified prophylactic
strategies should be implemented in patients undergoing gynecologic surgery, with LMWH-containing strategies
being recommended for high-risk and very-high-risk patients. 1. Geerts, W. H. et al. Prevention of venous thromboembolism: American College of Chest Physicians Evidence-Based Clinical
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26. Schellong, S. M. et al. References 1. Geerts, W. H. et al. Prevention of venous thromboembolism: American College of Chest Physicians Evidence-Based Clinica
Practice Guidelines (8th Edition). Chest 133, 381S–453S, https://doi.org/10.1378/chest.08-0656 (2008). SCientifiC Reports | (2018) 8:8277 | DOI:10.1038/s41598-018-25274-2 8 Author Contributions C.W.S. and Z.Y.Z. contributed to the conception and design of the study; N.Z., J.Z., S.Z.W. and S.L.G. contributed
to the acquisition of data; S.H.L., Y.J., B.L. and J.L.W. contributed to the analysis of data; L.S. and J.J.Z. made the
tables and figures; C.Q.S. wrote the manuscript; N.Z. revised the manuscript; All authors reviewed and approved
the final version of the manuscript. Acknowledgementsf g
We thank the staff at Departments of Gynecology, Ultrasound, and Imaging, Beijing Chaoyang Hospital of Capital
Medical University for their technical assistance. This study was supported by The Capital Health Research and SCientifiC Reports | (2018) 8:8277 | DOI:10.1038/s41598-018-25274-2 9 www.nature.com/scientificreports/ Development of Special Project (No. 2011-2003-03). This study was registered at the Chinese Clinical Trial
Registry, and the registration number is ChiCTR-IPR-15007324, data is Oct-30-2015. The study was approved by
the Ethics Committee of Beijing Chao-Yang Hospital, Capital Medical University (No. 10-Ke-42). SCientifiC Reports | (2018) 8:8277 | DOI:10.1038/s41598-018-25274-2 © The Author(s) 2018 Additional Informationh Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
ative Commons license, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 SCientifiC Reports | (2018) 8:8277 | DOI:10.1038/s41598-018-25274-2 10
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English
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Partial monosomy14q involving FOXG1 and NOVA1 in an infant with microcephaly, seizures and severe developmental delay
|
Molecular cytogenetics
| 2,016
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cc-by
| 4,003
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© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: manolakos@atg-labs.gr
3“Access to Genome” Clinical Laboratory Genetics, Athens Thessaloniki, Greece
8Department of Medical Genetics, Binaghi Hospital, University of Cagliari,
Cagliari, Italy
Full list of author information is available at the end of the article Background Since 2008, when mutations in the FOXG1 gene were ini-
tially described in two patients with Rett-like symptoms,
more than 90 patients have been reported to have muta-
tions involving the FOXG1 gene [1–6]. Rett syndrome is
typically an X-linked neurodegenerative condition [6] that
was initially described by Andreas Rett in 1966. Common
clinical features of Rett syndrome include postnatal micro-
cephaly, autism, seizures, breathing abnormalities, growth
retardation and gait apraxia [7, 8]. The majority of patients
with typical Rett syndrome carry mutations in the gene en-
coding Methyl-CpG-binding protein 2 (MECP2) located at
Xq28. Variant forms of Rett syndrome have been revised * Correspondence: manolakos@atg-labs.gr
3“Access to Genome” Clinical Laboratory Genetics, Athens Thessaloniki, Greece
8Department of Medical Genetics, Binaghi Hospital, University of Cagliari,
Cagliari, Italy
Full list of author information is available at the end of the article Partial monosomy14q involving FOXG1 and
NOVA1 in an infant with microcephaly,
seizures and severe developmental delay H. Fryssira1, E. Tsoutsou1, S. Psoni1, S. Amenta2, T. Liehr6, E. Anastasakis7, Ch Skentou5, A. Ntouflia3, I. Papoulidis3,
E. Manolakos3,8* and N. Chaliasos4 Abstract Background: FOXG1 gene mutations have been associated with the congenital variant of Rett syndrome (RTT) since
the initial description of two patients in 2008. The on-going accumulation of clinical data suggests that the FOXG1-
variant of RTT forms a distinguishable phenotype, consisting mainly of postnatal microcephaly, seizures, hypotonia,
developmental delay and corpus callosum agenesis. Case presentation: We report a 6-month-old female infant, born at 38 weeks of gestation after in vitro fertilization,
who presented with feeding difficulties, irritability and developmental delay from the first months of life. Microcephaly
with bitemporal narrowing, dyspraxia, poor eye contact and strabismus were also noted. At 10 months, the proband
exhibited focal seizures and required valproic acid treatment. Array-Comparative Genomic Hybridization revealed a 4. 09 Mb deletion in 14q12 region, encompassing the FOXG1 and NOVA1 genes. The proband presented similar feature
with patients with 14q12 deletions except for dysgenesis of corpus callosum. Disruption of the NOVA1 gene which
promotes the motor neurons apoptosis has not yet been linked to any human phenotypes and it is uncertain if it
affects our patient’s phenotype. Conclusions: Since our patient is the first reported case with deletion of both genes (FOXG1-NOVA1), thorough clinical
follow up would further delineate the Congenital Rett-Variant phenotypes. Keywords: FOXG1 syndrome, Rett syndrome, NOVA1, Array-CGH, Postnatal microcephaly, Seizures in 2010 by Neul et al [7]. He classified them in to three
main variant categories: 1. Preserved speech, 2. early sei-
zures and, 3. the congenital variant. Mutations in the
cyclin-dependent kinase-like 5 (CDKL5) gene located in
Xp22 are found in most cases of early-onset seizure vari-
ants, while the congenital variant of the syndrome has re-
cently been found to be associated with heterozygous
mutations or deletions in the forkhead box protein G1
(FOXG1) gene, located in the 14q12 chromosomal region
[7, 9–11]. In this study, we report a new case of atypical
Rett Syndrome with a 14q12 deletion, 4.09 Mb in size,
encompassing only two genes, the FOXG1 and the NOVA1
and we discuss the role of the NOVA1 haploinsufficiency. Fryssira et al. Molecular Cytogenetics (2016) 9:55
DOI 10.1186/s13039-016-0269-1 Fryssira et al. Molecular Cytogenetics (2016) 9:55
DOI 10.1186/s13039-016-0269-1 Open Access * Correspondence: manolakos@atg-labs.gr
3 Case presentation Our patient was a 6-month- old female infant and the
only child of non-consanguineous parents. She was born Page 2 of 7 Page 2 of 7 Fryssira et al. Molecular Cytogenetics (2016) 9:55 by caesarean section at 38 weeks of gestation. Her birth
weight was 2.990 gr (25th -50th centile), her length was
49 cm (25th -50th centile) and her head circumference
was 33 cm (10–25th centile). with bitemporal narrowing, strabismus, broad base to
nose and long philtrum. Her weight was 6.18 kg (25th
centile) and her height was 62 cm (25th centile). Due to gastroesophageal reflux she was treated with
ranitidine. It should be noted that the embryo was the product of
in vitro fertilization (IVF) by intracytoplasmic sperm in-
jection (ICSI) due to the husband’s low sperm count. The mother underwent treatment with Follitropin Beta
(Puregon), human Chorionic Gonadotropin, hCG (Preg-
nyl), and Cetrorelix Acetate (Cetrotide). Haematological, biochemical, metabolic, thyroid, am-
monia and blood gasses were all normal. The ultra-
sounds of the heart and abdomen were also normal. At 10 months the proband exhibited focal seizures and
required valproic acid treatment. Sleep deprived EEG
examination revealed slower focal activity for her age
and sleep spindles which are indicative signs of focal
cerebral dysfunction. Brain magnetic resonance imaging
(MRI) showed no serious findings except for a septum
pellucidum cyst and brain asymmetry—the left hemi-
sphere was larger than the right one. The patient as a neonate was hypotonic. She was re-
ferred for clinical evaluation at the age of 6 months due
to irritability and feeding difficulties. The examinations
revealed peripheral hypertonia, psychomotor retardation
(limited facial expression—no smile, poor eye contact),
borderline microcephaly (head circumference of 39 cm,
at 3rd centile) (Fig. 1a), periodical stereotypic movements
and minor dysmorphic features such as a small forehead At the age of 12 months, the head circumference was
40.7 cm (<< 3rd centile) (Fig. 1a) while the height and Fig. 1 Growth curve ¨Head circumference—for—age GIRLS. First Paediatric department, National and Kapodistrian University of Athens. Professor
G.Chroussos. The standard curves are used by the Greek National Health System Fig. 1 Growth curve ¨Head circumference—for—age GIRLS. First Paediatric department, National and Kapodistrian University of Athens. Professor
G.Chroussos. The standard curves are used by the Greek National Health System Fig. 1 Growth curve ¨Head circumference—for—age GIRLS. First Paediatric department, National and Kapodistrian University of Athens. Professor
G.Chroussos. Case presentation The standard curves are used by the Greek National Health System Fryssira et al. Molecular Cytogenetics (2016) 9:55 Fryssira et al. Molecular Cytogenetics (2016) 9:55 Page 3 of 7 Page 3 of 7 Page 3 of 7 Chromosome and array-based analysis Chromosome and array-based analysis weight remained between 10th and 25th centile. Severe
neurodevelopmental delay was obvious. Metaphase chromosomes were obtained from phyto-
hemagglutinin
(PHA)-stimulated
peripheral
blood
lymphocytes and high resolution (550–650 bands) thy-
midine treatment G-banding karyotype analysis was
performed, using standard procedures. Twenty meta-
phase spreads were analysed, and the result was a nor-
mal female karyotype (46, XX). The molecular analysis
of the MECP2 gene was normal also. Re-examination at the age of 19- months and 2-
years, revealed head circumference of 42 cm (<< 3rd
centile) (Fig. 1a), height of 75.5 cm (slightly below the
3rd centile) and weight of 9 Kg (slightly below the 3rd
centile). The proband exhibited severe psychomotor
retardation as she could neither sit independently nor
speak at all. Fig. 2 Array-CGH analysis detected a 4.09 Mb loss of the copy numbers in the spanning region 14q12. b Chromosome 14, region 25,843,560-29,938,629. Represents the 4,095,070 bp deletion described in our case report. The region includes both FOXG1 and NOVA1 genes. Figure adapted from
http://genome.ucsc.edu/ Accessed at 18/10/2015 Fig. 2 Array-CGH analysis detected a 4.09 Mb loss of the copy numbers in the spanning region 14q12. b Chromosome 14, region 25,843,560-29,938,629
Represents the 4,095,070 bp deletion described in our case report. The region includes both FOXG1 and NOVA1 genes. Figure adapted from
http://genome ucsc edu/ Accessed at 18/10/2015 Fig. 2 Array-CGH analysis detected a 4.09 Mb loss of the copy numbers in the spanning region 14q12. b Chromosome 14, region 25,843,560-29,938,629. Represents the 4,095,070 bp deletion described in our case report. The region includes both FOXG1 and NOVA1 genes. Figure adapted from
http://genome.ucsc.edu/ Accessed at 18/10/2015 Fryssira et al. Molecular Cytogenetics (2016) 9:55 Page 4 of 7 Page 4 of 7 Fryssira et al. Molecular Cytogenetics (2016) 9:55 Fryssira et al. Molecular Cytogenetics (2016) 9:55 Page 4 of 7 Further investigation with array-Comparative Genomic
Hybridization was performed by hybridizing the sample
against a male human reference commercial DNA sam-
ple (Promega biotech) using an array-CGH platform that
includes 60,000 oligonucleotides distributed across the
entire genome (Agilent Technologies). The statistical
test used as parameter to estimate the number of copies
was ADAM-2 (provided by the DNA analytics software,
Agilent Techn.) with a window of 0.5 Mb, A = 6. Fryssira et al. Molecular Cytogenetics (2016) 9:55 Only
those copy number changes that affect at least 5 consecu-
tive probes with identically oriented change were consid-
ered as Copy Number Variations (CNV). For the majority
of the genome, the average genomic power of resolution
of this analysis was 200 kilobases. Array-CGH analysis
detected a 4.09 Mb loss in the 14q12 region (Fig. 2a). The deleted segment was mapped at chr14:25,843,560-
29,938,629 region. The genomic coordinates are listed
according to genomic build GRCh37/hg19, and in-
cludes FOXG1 and NOVA1 genes (Fig. 2b). Discussion 2015 case 3 has a deletion beyond 14q12 and is drawn with a discontinued line Table 1 Clinical presentation of patients with FOXG1 mutations and 14q12 deletions Table 1 Clinical presentation of patients with FOXG1 mutations and 14q12 deletions
Kumakura
et al. 2014
Byun et al. 2015
Patients (n = 8) Cellini et al. 2015
Patients (n = 7)
Allou et al. 2012
Patients (n = 2)
Philippe et al. 2011
Jacob et al. 2009
Yeung et
al. 2009
Patients (n = 2)
Ariani et al. 2008
Papa et al. Discussion Recently, the FOXG1 congenital variant of Rett syndrome
has been described as a clinically identifiable phenotype,
called the “FOXG1 syndrome” [13]. This is an epileptic-
dyskinetic developmental encephalopathy with features of
classic Rett syndrome, but earlier onset from the first
months of life. The main phenotype comprises postnatal
microcephaly, severe developmental delay and lack of
speech, hypotonia, dyskinesia and corpus callosum hypo-
plasia. Other symptoms include strabismus, feeding diffi-
culties, bruxism and seizures [8, 12–16]. In the present study, a novel FOXG1 and a NOVA1
deletion is described. According to the diagnostic criteria
for typical and atypical Rett syndrome [7], our patient
possesses only some of the diagnostic criteria for the
atypical Rett syndrome. She has slight facial dysmorph-
ism, feeding difficulties, severe psychomotor retardation,
postnatal
microcephaly,
seizures
and
focal
cerebral
dysfunction, strabismus and hypertonia. A deletion of
4.09 Mb on chromosome 14q12 including the FOXG1
and NOVA1 genes was identified by array-CGH tech-
nique. The clinical phenotypes vary between the re-
ported cases that have mutations or deletions involving
the FOXG1 gene and other genes (Table 1, Fig. 3). Our
case differs as only two genes, FOXG1 and NOVA1, are
involved. These genes were found to be highly expressed
in the human brain throughout ontogeny, and double
strand breaks in 14q12 region are implicated in the neu-
rodegenerative disease: ataxia telangiectasia [17]. The result of array-CGH was confirmed with FISH. Ten metaphases were analysed from phytohemagglutinin
(PHA)-stimulated peripheral blood lymphocytes of the
patient with RP11-120I18 in 14q12 and wcp 14 probes. The rearrangement was molecular cytogenetically unbal-
anced (data not shown), therefore the karyotype is: 46,XX,
del[12](q12q12).ish14q12(RP11-120I18-). arr[hg19]14q12
(25,843,560-29,938,629)x1. Blood samples from both par-
ents were examined with FISH in order to exclude any
chromosome rearrangement like inversion. Ten meta-
phases were analysed from each subject with RP11-332 N6
in 14q11.2, RP11-120I18 in 14q12 and wcp 14 probes, and
no rearrangement was detected (data not shown). Different clinical features have also been described in
patients with FOXG1 point mutations. The severity or
lack of the symptoms among cases could depend Fig. 3 Ideogram of the deletions described in Table 1. The upper thin line indicates the 14q12 region (hg38), and the bold lines indicate the
region which is deleted in each case. Cellini et al. Competing interests
Th
h
d
l
h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Funding The whole study was funded by the private company Access to Genome,
Clinical Laboratory Genetics, 33A Ethn. Antistaseos str, 55134 Thessaloniki,
Greece. Consent for publication Written informed consent was obtained from the patient for publication of
this case report and any accompanying images. A copy of the written
consent is available for review by the Editor-in-Chief of this journal. Abbreviations CGH, comparative genomic hybridization; EEG, electroencephalogram; ICSI,
intracytoplasmic sperm injection; IVF, in vitro fertilization; MRI, magnetic
resonance imaging; PHA, phytohemagglutinin; RTT, Rett syndrome Authors’ contributions
d
h AN and CS wrote the manuscript; HF, ET, SP and SA coordinated the clinical
analysis of the patient; EM performed the cytogenetic analysis; IP signed out
the molecular cytogenetic results; EM, IP and NC coordinated the study; All
authors have read and approved the manuscript. Author details
1 1Medical genetics, School of Medicine, National and Kapodistrian University
of Athens, “Aghia Sophia” Children’s Hospital, Athens, Greece. 2“Mitera”
Maternity Hospital, Athens, Greece. 3“Access to Genome” Clinical Laboratory
Genetics, Athens Thessaloniki, Greece. 4Child Health Department, University
Hospital of Ioannina (UHI), Ioannina, Greece. 5“Mitera kai emvrio” Medical centre,
Larisa, Greece. 6Institute of Human Genetics, Jena University Hospital, Friedrich
Schiller University, Jena, Germany. 7Hellenic Navy Hospital, Deinokratous 70,
Athens 11521, Greece. 8Department of Medical Genetics, Binaghi Hospital,
University of Cagliari, Cagliari, Italy. Neuro-Oncological Ventral Antigen 1 (NOVA1) gene
(OMIM*602157)
encodes
a
neuron-specific
RNA-
binding protein that is inhibited by paraneoplastic anti-
bodies [18]. Recently, Storchel H et al., 2015 [19] found
that NOVA1 protein converges on Ago proteins and
controls miRNA-induced silencing complex (miRISC),
possibly resulting on the regulation of neuronal develop-
ment and synaptic plasticity. Despite the significant role
of the NOVA1 gene, it has not been linked to any human
phenotype yet. In the case of our patient with FOXG1
syndrome, we are unable to estimate the impact of the
NOVA1 haploinsufficiency. NOVA1 haploinsufficiency
may act synergistically with the FOXG1 gene or it
may cause no difference in the final clinical outcome. Also, we cannot define whether the IVF by ICSI plays
a role in the microdeletion of chromosome 14q12 re-
gion. Further research should investigate if ICSI could
be associated with haploinsufficiency as was the case
in our patient. University of Cagliari, Cagliari, Italy. Discussion Molecular Cytogenetics (2016) 9:55 Page 6 of 7 Page 6 of 7 Fryssira et al. Molecular Cytogenetics (2016) 9:55 Page 6 of 7 Conclusion additionally on genetic and/or environmental factors
[13]. Jacob F. et al. in 2009 [9] reported a case of a
2.594 Mb deletion involving the FOXG1 and C23orf14
(Table 1). As in our case, patient had a normal cor-
pus callosum but showed all the typical characteristics
found in patients with FOXG1 alterations. Table 1 in-
dicates that almost all cases suffer from microcephaly,
dyskinesia, lack of speech, seizures and developmental
delay. On the other hand, hypotonia and corpus callo-
sum hypoplasia are not apparent in every case. Pro-
gressive hypertonia was obvious in our patient and in
at least 10 other reported cases with FOXG1 point
mutations [8]. additionally on genetic and/or environmental factors
[13]. Jacob F. et al. in 2009 [9] reported a case of a
2.594 Mb deletion involving the FOXG1 and C23orf14
(Table 1). As in our case, patient had a normal cor-
pus callosum but showed all the typical characteristics
found in patients with FOXG1 alterations. Table 1 in-
dicates that almost all cases suffer from microcephaly,
dyskinesia, lack of speech, seizures and developmental
delay. On the other hand, hypotonia and corpus callo-
sum hypoplasia are not apparent in every case. Pro-
gressive hypertonia was obvious in our patient and in
at least 10 other reported cases with FOXG1 point
mutations [8]. Since our patient is the only one with a deletion encom-
passing only FOXG1 and NOVA1 genes, in the future
the thorough clinical follow-up of her neurological sta-
tus will help for further delineation of the role of these
genes. Acknowledgments The authors would like to thank the members of the family of the patient for
their collaboration. Forkhead BOX G1 (FOXG1) gene (OMIM*164874)
encodes a developmental transcription factor with re-
pressor activity, important for the development of the
ventral telencephalon in the embryonic forebrain and
crucial for the regulation of neurogenesis and neurite
outgrowth. It is also expressed in neurogenetic regions
of the postnatal brain [15]. Postnatal microcephaly is
another common feature of FOXG1 syndrome. In
cases with a duplication of the FOXG1 gene, micro-
cephaly is not found (Table 1), indicating that the
phenotype is dependent on FOXG1 dosage. Further-
more, other reported cases with deletions close but not
disturbing the FOXG1 gene have a similar phenotype
as patients with ¨FOXG1 syndrome¨, suggesting that a
position effect causing altered expression of FOXG1 gene
may be the cause. The protein Kinase D1 (PRKD1) gene
(OMIM*605435), located close to FOXG1 gene has been
reported to be implicated in transcription and expression
of FOXG1 gene [14]. Availability of data and materials The datasets during and/or analysed during the current study are available
from the corresponding author on reasonable request. Received: 28 April 2016 Accepted: 25 July 2016 Received: 28 April 2016 Accepted: 25 July 2016 Discussion 2008
Present
case
1
2
3
4
4 cases with
point mutations
in FOXG1
Age (Y,M)
8.0
8.0
9.0
3.6
2.0
10.0
2.6–17.0
N/A
22.0 and 10.0 3.0
9.0
22.0 and 7.0
7.0
0.6
Sex
M
F
F
M
F
F
3 F, 1 M
4 F, 3 M
F
F
F
F
F
F
FOXG1
mutation on
14q12 CNV
(Mb)
0.54 Mb
Del
2.5 Mb
Del
2.5 Mb
Del
2.8 Mb Del
9.1 Mb Del
7.3 Mb
Dup (de
novo)
p.Gln100Serfs*92,
p.Q46X,
p.Glu154Glyfs*301,
p.Gln86Argfs*106
(de novo)
0.4–4.1 Mb
p.Trp308X
and
p.Tyr400X
2.594 Mb Del
4.45 Mb
Dup
p.W255X and
p.S323fsX325
3 Mb Del
4,09 Del
Genes involved
in mutation or
point mutations
FOXG1,
C14orf23
(de novo)
FOXG1,
C14orf23,
PRKD1 (de
novo)
FOXG1,
C14orf23,
PRKD1
FOXG1,
C14orf23,
PRKD1, SCFD1,
G2E3, COCH,
STRN3 (de
novo)
STXBP6, NOVA1,
FOXG1,
C14orf23,
PRKD1, SCFD1,
G2E3, COCH,
STRN3, AP4S1,
HECTD1, DTD2,
NUBPL,
ARHGAP5, AKAP,
NPAS3
(de novo)
FOXG1
and 50
additional
genes (de
novo)
FOXG1
Deletions in 5
cases, 3 of
them distal to
FOXG1 and 2
cases of point
mutations
involving
FOXG1
FOXG1
FOXG1,
C14orf23
FOXG1,
NOVA1,
c14orf23,
PRKD1,
G2E3,
SDFD,
COCH
FOXG1
FOXG1,
PRKD1,
SCFD1,
G2E3,
COCH,
STRN3
FOXG1,
NOVA1
Postnatal
microcephaly
yes
yes
yes
yes
yes
no
yes
yes
yes
yes
no
yes
yes
yes
Psychomotor
retardation
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
Hypotonia
yes
no
yes
yes
yes
yes
2/4 cases
hypotonic
All except 1
case
1/2 cases
yes
yes
yes
yes
Hypertonia
Diskinesia
yes
yes
yes
yes
yes
yes
3/4 cases
diskinetic
3/7 cases
yes
yes
yes
yes
yes
yes
Speech
no
no
no
no
no
no
no
no
no
no
no
no
no
no
Hand
stereotypies
no
other
sterotypies
yes
no
yes
yes
yes
4/7 cases
yes
yes
hand
flapping
only
yes
yes
yes
Corpus
callosum
hypogenesis
yes
no
yes
yes
yes
no
3/4 cases with
hypoplasia
brain
abnormalities
in all cases
1/2 cases
no,
brachycephaly
observed
no
yes
no
no
Seizures
yes
yes
yes
yes
yes
yes
yes
5/7 cases
1/2 cases
yes
yes
yes
yes
yes
Developmental
delay
yes
yes
yes
yes
yes
yes
yes
5/7 cases
1/2 cases
yes
yes
yes
yes
yes
F female M male Y years M months Del deletion Dup duplication
Fryssira et al. Molecular Cytogenetics (2016) 9:55
Pa Fryssira et al. Fryssira et al. Molecular Cytogenetics (2016) 9:55 Fryssira et al. Molecular Cytogenetics (2016) 9:55 Fryssira et al. Molecular Cytogenetics (2016) 9:55 clinical and molecular genetics, diagnositic testing and personalized
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criteria and nomenclature. Ann Neurol. 2010;68:944–50. criteria and nomenclature. Ann Neurol. 2010;68:944–50. 8. Byun CK, Lee JS, Lim BC, Kim KJ, Hwang YS and Chae J. FOXG1 Mutation is
a Low-Incidence Genetic Cause in Atypical Rett Syndrome. Child Neurology
Open. 2015;8:1-5 9. Jacob F, Ramaswamy V, Andersen J, Bolduc F. Atypical Rett syndrome with
selective FOXG1 deletion detected by comparative genomic hybridization:
case report and review of literature. Eur J Hum Genet. 2009;17:1577–81. 10. Papa FT, Mencarelli MA, Caselli R, et al. A 3 Mb Deletion in 14q12 Causes
Severe Mental Retardation, Mild Facial Dysmorphisms and Rett-like Features. Am J Med Genet. 2008;146:1994–8. 11. Pantaleón FG, Juvier RT. Molecular basis of Rett syndrome: A current look. Rev Chil Pediatr. 2015;86:142–51. 12. Allou L, Lambert L, Amsallem D, Bieth E, Edery P, Destree A, et al. 14q12
and severe Rett-like phenotypes: new clinical insights and physical mapping
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et al. The core FOXG1 syndrome phenotype consists of postnatal
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corpus callosum hypogenesis. J Med Genet. 2011;48:396–406. 14. Pratt DW, Warner JV, Williams MG. Genotyping FOXG1 mutations in patients
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large-scale functional screen identifies Nova1 and Ncoa3 as regulators of
neuronal miRNA function. EMBO J. 2015;34:2237–54. Submit your next manuscript to BioMed Central
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English
| null |
A new icriodontid conodont cluster with specific mesowear supports an alternative apparatus motion model for Icriodontidae
|
Journal of systematic palaeontology
| 2,017
|
cc-by
| 11,047
|
*Corresponding author. Email: thomas.suttner@uni-graz.at
2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
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 unre-
stricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (Received 28 November 2016; accepted 25 June 2017; published online 15 August 2017) Increasing numbers of conodont discoveries with soft tissue preservation, natural assemblages and fused clusters of the
hard tissue have strengthened the hypothesis regarding the function and mechanism of the conodont feeding apparatus. Exceptional fossil preservation serves as a solid basis for modern reconstructions of the conodont apparatus illustrating the
complex interplay of the single apparatus elements. Reliable published models concern the ozarkodinid apparatus of
Pennsylvanian and Early Triassic conodonts. Recognition of microwear and mammal-like occlusion, especially of
platform elements belonging to individuals of the genus Idiognathodus, allows rotational closure to be interpreted as the
crushing mechanism of ozarkodinid platform (P1) elements. Here we describe a new icriodontid conodont cluster of
Caudicriodus woschmidti that consists of one pair of icriodontan (I) and 10 pairs of coniform (C1–5) elements, with I
elements being preserved in interlocking position. The special kind of element arrangement within the fused cluster
provides new insights into icriodontid apparatus reconstruction and notation of elements. However, orientation of coniform
elements is limited to a certain degree by possible preservational bias. Four possible apparatus models are introduced and
discussed. Recognition of specific wear on denticle tips of one of the icriodontan elements forms the basis for an
alternative hypothesis of apparatus motion. Analysis of tip wear suggests a horizontal, slightly elliptical motion of
opposed, antagonistically operating I elements. This is supported by similar tip wear from much better preserved, but
isolated, elements of Middle Devonian icriodontids. More detailed interpretation of the masticatory movement will allow
enhanced understanding of anatomical specifications, diet and palaeobiology of different euconodont groups. Keywords: Caudicriodus woschmidti; conodont cluster; apparatus reconstruction; mesowear; mastication mode 1971) or trimembrate (Nicoll 1977) into a septimembrate
icriodontid apparatus model (Nicoll 1982). Since then, the
notation and number of elements included within the indi-
vidual apparatus has changed, especially among Early
Devonian icriodontids. However, these models of the
apparatus have been reconstructed on statistical analysis
of isolated elements, which are supposed to belong to
Caudicriodus woschmidti (Serpagli 1983) and Cypricrio-
dus hesperius (Simpson 1998; Murphy et al. 2016). Serpagli (1983) introduced an icriodontid apparatus
model that includes a set of ramiform elements. Journal of Systematic Palaeontology, 2018
Vol. 16, No. 11, 909–926, https://doi.org/10.1080/14772019.2017.1354090 Journal of Systematic Palaeontology, 2018
Vol. 16, No. 11, 909–926, https://doi.org/10.1080/14772019.2017.1354090 A new icriodontid conodont cluster with specific mesowear supports an alternative
apparatus motion model for Icriodontidae
Thomas J. Suttner
a,b*, Erika Kido
a and Antonino Briguglio
c
aUniversity of Graz, Institute for Earth Sciences, Heinrichstrasse 26, 8010 Graz, Austria; bGeological-Palaeontological Department,
Natural History Museum Vienna, Burgring 7, 1010 Vienna, Austria; cFaculty of Science, Universiti Brunei Darussalam, Jalan Tungku
Link, Gadong BE1410, Brunei Darussalam Systematic palaeontology General remarks. Although we use the term Conodonta
of Eichenberg (1930), the subdivisions of order (Paraco-
nodontida excluded) and family as introduced by Sweet &
Donoghue (2001, fig. 6) are followed here. A slightly
modified notation of Nicoll (1982) for coniform elements
of the Caudicriodus apparatus is used. This Early Devo-
nian species did not possess coniform elements with
prominent shoulder spurs (Ce element of Nicoll 1982) and
therefore the apparatus reconstruction of Nicoll (1982)
cannot be applied entirely, although all other element
types are recognized. In order to keep the relation to Late
Devonian icriodontids, we retain the notation of ‘C’ for
‘coniform’ but use numbers instead of letters for subdivi-
sion of elements. Our revised notation based on Nicoll
(1982) is as follows: Ca D C3; Cb D C5; Cc D C1; Cd D
C4; Ce D not observed; Cf D C2a–f; I D I. Scanning electron microscope (SEM) images of cono-
donts were produced using a Zeiss DSM 982 Gemini elec-
tron microscope (Institute for Earth Sciences, University
of Graz). Conodonts were coated with gold/palladium
alloy for 10 minutes. For three-dimensional reconstruction and discrimination
of single apparatus elements, computer microtomography
(Micro-CT) was used for the icriodontid conodont cluster. The specimen was scanned for c. 5 hours at 80 kV (source
voltage) and 100 uA (source current) with an image rota-
tion step of 0.2600 degrees using a SkyScan 1173 (Depart-
ment of Palaeontology, University of Vienna). The material figured herein was collected from two
localities. The conodont cluster of Caudicriodus wosch-
midti was obtained from bed Ki/4/2a (hesperius Biozone,
Lochkovian) of the ‘Kottwitz’ quarry near the village of
Kirchfidisch,
southern
Burgenland,
Austria
(Suttner
2009a). Two isolated icriodontan elements of Icriodus aff. michiganus and Icriodus sp. came from sample BL-12-
29c (kockelianus Biozone, Eifelian), Blankenheim Syn-
cline, Eifel, Germany (K€onigshof et al. 2016). Because in vivo orientation of icriodontid apparatus ele-
ments is unknown, we apply conventional terminology
and orientation here for description of elements and indi-
cation of relative disposition in proposed models. Terms
used follow the suggestions of Purnell et al. (2000,
pp. 117, 119, table 2). Introduction Fused clusters of icriodontid conodonts have been known
since the late 1960s. The first publication discussed the
apparatus of the Late Devonian species Icriodus alterna-
tus (Lange 1968). This paper concluded that about 30
coniform elements belonging to the form taxon Acodina,
together with one pair of icriodontan elements, could rep-
resent the apparatus of one individual. Concerns regarding
the absolute number of acodinan elements were raised by
Lange (1968) because the conodont elements were clus-
tered within a coprolite. In the early 1980s, more than 850
conodont clusters of Icriodus expansus from Late Devo-
nian deposits of the Canning Basin in Western Australia
were analysed and described by Nicoll (1982). He con-
cluded that the apparatus consisted of one pair of icrio-
dontan (I) elements and more than 140 cone elements. Nicoll discriminated seven element types and changed the
common opinion of a bimembrate (Klapper & Philip The conodont cluster of Caudicriodus woschmidti
described here provides new insights into apparatus com-
position and notation of elements. The architecture of the
cluster is very similar to the apparatus composition
described by Nicoll (1982), except that coniform ele-
ments, which have prominent shoulder spurs, are lacking. It differs from the reconstructions of Serpagli (1983) in
having no ramiform or denticulate elements preserved. 910 T. J. Suttner et al. Therefore, the notation of coniform elements by Nicoll
(1982) is modified here. Material and methods Extraction of conodonts followed the standard chemical
methods for phosphatic microfossils (Jeppsson & Anehus
1995). For dissolution of conodont-bearing rocks (marl
and limestone), we used 5% formic acid. Sodium poly-
tungstate (density 2.79 g/cm3) was used for heavy liquid
separation (Mitchell & Heckert 2010). Geological setting The icriodontid conodont cluster was found in Unit 4 of the
‘Kottwitz’ quarry near Kirchfidisch (Fig. 1), which repre-
sents one of few localities in southern Burgenland (Austria)
where Silurian to Devonian rocks are exposed. Other out-
crops are found near the villages of Hannersdorf, Punitz
and Sulz (Pollak 1962; Sch€onlaub 1984, 1994, 2000). Additionally, the sequence is documented from subsurface
drilling cores in the Styrian Basin near the villages of
Arnwiesen and Blumau (Ebner 1988; Fl€ugel 1988). Although clusters of icriodontan elements in interlock-
ing position were found prior to our discovery, distinctive
tip wear was not recognized. Weddige (1990) attempted
to classify wear of isolated conodont specimens, resulting
in the identification of Occlusio, Depressio and Duplicatio
pathologies for the genera Polygnathus and Icriodus. Based on pathologies seen on the oral surface of conodont
elements, Weddige (1990) hypothesized a permanent see-
saw movement of antagonistically working platform
element pairs. Alternative models of the motion of the
icriodontid apparatus do not exist. Thus, recognition of
denticle tip wear on one of the icriodontan elements of the
Caudicriodus cluster provides a unique opportunity to
reconsider the icriodontid apparatus motion model. Anal-
ysis of mesowear (Purnell & Jones 2012) on the fused
cluster is supported by measurements of denticle tip wear
on isolated, but much better preserved, Middle Devonian
icriodontan elements. At the ‘Kottwitz’ quarry, the latest Silurian (Pridolian)
and Early Devonian (Lochkovian) sequence is about
40 metres thick and consists of phyllitic shale, calcareous
marls, laminated limestone, dolomitic limestone and dolo-
stone (Suttner & Lukeneder 2004; Suttner 2009a). The
interval yielding the fused cluster of Caudicriodus com-
prises well-bedded, laminated argillaceous and silty lime-
stones. Beds are 7 to 25 cm thick and yield a low-diversity
invertebrate fauna including brachiopods, ostracods and
crinoid ossicles. Other specimens of fused coniform ele-
ments (icriodontids) and ramiform elements (ozarkodinids)
were found in beds Ki/4/1c and Ki/4/2a (Suttner 2009b, c). Systematic palaeontology Phylum Chordata Bateson, 1886
Class Conodonta Eichenberg, 1930 sensu Sweet &
Donoghue, 2001
Order Prioniodontida Dzik, 1976
Family Icriodontidae M€uller & M€uller, 1957
Genus Caudicriodus Bultynck, 1976 The conodont cluster of Caudicriodus woschmidti is
stored in the micropalaeontological collection of the Geo-
logical-Palaeontological
Department,
Natural
History
Museum Vienna (Austria) under the repository number
NHMW 2011/0374/0001. A new icriodontid conodont cluster 911 Figure 1. Locality map and section log from the ‘Kottwitz’ quarry (southern Burgenland, Austria), where the Caudicriodus woschmidti
conodont cluster was found. Figure 1. Locality map and section log from the ‘Kottwitz’ quarry (southern Burgenland, Austria), where the Caudicriodus woschmidti
conodont cluster was found. Type
species. Icriodus
woschmidti
Ziegler,
1960;
lower Lochkovian; Untenr€uden, Rhenish Slate Mountains,
Germany. Type
species. Icriodus
woschmidti
Ziegler,
1960;
lower Lochkovian; Untenr€uden, Rhenish Slate Mountains,
Germany. specific angle, and a spur developed between the cusp
and ‘posterior’-most transverse denticle row on the
‘inner’ side of the element. Bultynck (1976) included the following species within
Caudicriodus: Caudicriodus woschmidti, C. postwosch-
midti, C. angustoides, C. curvicauda, C. celtibericus and
C. sigmoidalis. Later, Drygant (2010) included four addi-
tional species, Caudicriodus hesperius, C. ruthmawsonae,
C. transiens and C. serus, and Drygant & Szaniawski
(2012)
described
Caudicriodus
schoenlaubi. More
recently, Murphy et al. (2016) excluded Caudicriodus
hesperius, placing it in their new genus Cypricriodus. Remarks. The
generic
diagnosis
follows
Bultynck
(1976). The outline of the widely opened basal cavity
(termed
platform
in
earlier
publications,
but
not
homologous to the platform in polygnathids, for exam-
ple) is identical in shape to that of other species of
Icriodus. Features which discriminate Caudicriodus
from Icriodus are the lateral process (denticulate or
adenticulate) that extends posterior of the cusp at a 912 T. J. Suttner et al. Caudicriodus woschmidti (Ziegler, 1960)
(Figs 2, 4) 1980 Icriodus woschmidti woschmidti Ziegler; Jaeger &
Sch€onlaub: pl. 4, figs 4–5/16, 6/16. Icriodontan elements are preserved with the oral side
opposing each other in an interlocking position. Lateral
walls of the basal cavity, especially in the ‘posterior’ part
of either element, are adpressed and show strong fractures. Therefore, the lower margin of the basal cavity is very
irregular, not reflecting the original outline. However, the
basal cavity of this species is widest below the cusp. The
initial part of the ‘anterior’ portion of both I elements is
broken off. Additionally, the ‘posterior’-most portion of
the lateral process is broken too. Four transverse denticle
rows are bar-like (denticles are connected by high ridges;
Fig. 4B) with deep interspacing on the rather low spindle. Some of the lateral row denticles show strong fractures. No surface ornamentation is observed. 1980 Icriodus woschmidti woschmidti Ziegler; Pickett: 70,
fig 3B–D. 1980
Icriodus
woschmidti
Ziegler;
Serpagli
&
Mastandrea: 39, figs 2–4. 1981 Caudicriodus woschmidti woschmidti (Ziegler);
Norris & Uyeno: pl. 5, figs 10–17. 1981 Icriodus woschmidti Ziegler; Wang: 77, pl. 1, figs
22–25. 1983
Icriodus
woschmidti
Ziegler;
Broadhead
&
McComb: 153, figs 2E, 3H–J. 1983 Icriodus woschmidti woschmidti Ziegler; Serpagli:
155, figs 2, 5–7. 1986 Caudicriodus woschmidti woschmidti (Ziegler);
Borremans & Bultynck: 52, pl. 1, figs 1–9. Coniform elements are clustered in bidirectional orien-
tation around the ‘posterior’ part of the icriodontan ele-
ments (‘inner’ side). Few elements are found on the
‘outer’ side of the sinistral I element which indicate post-
mortem distortion of the original orientation of the coni-
form assemblage. Basically, two sets of different-shaped
pairs of small (C1, C2a, C2b, C2c, C2d, C4) and large
(C2e, C2f, C3, C5) coniform elements are observed. All
of these are adenticulate. 1988 Icriodus woschmidti woschmidti Ziegler; Denkler &
Harris: B8, pl. 1, figs A, B. 1990 Icriodus woschmidti woschmidti Ziegler; Olivieri &
Serpagli: 63, pl. 1, figs 12–14. g
g
1990 aff. Icriodus cf. postwoschmidti Mashkova; Weyant
& Morzadec: 752, pl. 1, figs 1, 3–5. , p
, g
,
1994
Icriodus
woschmidti
woschmidti
Ziegler;
Valenzuela-Rıos: 87, pl. 8, figs 14, 15, 28. C1 elements are small, gracile coniform elements with
a recurved cusp and striate surface ornamentation. The
cusp and basal outline are elliptical in cross section
(‘posterior’ margin more convex than ‘anterior’ margin)
with sharp margins that represent costae. 1995 Icriodus woschmidti woschmidti Ziegler; Luppold:
pl. 2, fig. 11. Caudicriodus woschmidti (Ziegler, 1960)
(Figs 2, 4) 2005 Icriodus woschmidti woschmidti Ziegler; Corradini
et al.: fig. 5e. 2005 Icriodus woschmidti woschmidti Ziegler; Corradini
et al.: fig. 5e. 2009a Icriodus woschmidti woschmidti Ziegler; Suttner:
77, pl. 1, figs 1–6. 1960 Icriodus woschmidti Ziegler: 185, pl. 15, figs 16–18,
20–22. 2010 Caudicriodus woschmidti (Ziegler); Drygant: 57,
pl. 2, figs 3, 6–13. 2010 Caudicriodus woschmidti (Ziegler); Drygant: 57,
pl. 2, figs 3, 6–13. 2012 Caudicriodus woschmidti (Ziegler); Drygant &
Szaniawski: 846, figs 9B, 10C, D. 1962 Icriodus woschmidti Ziegler; Jentzsch: 967, pl. 1,
figs 17–23. 2012 Caudicriodus woschmidti (Ziegler); Drygant &
Szaniawski: 846, figs 9B, 10C, D. g
1964 Icriodus woschmidti Ziegler; Walliser: 38, pl. 9, fig. g
p
g
22, pl. 11, figs 14–22. Material. NHMW
2011/0374/0001,
single
conodont
cluster including 10 pairs of coniform and both I elements. Additional icriodontan elements from the same locality
were described by Suttner (2009a). , p
, g
1969 Icriodus woschmidti transiens Carls & Gandl: 174,
pl. 15, figs 1–7. 1969 Icriodus woschmidti Ziegler; Klapper: 10, pl. 2,
figs 3–5. Description. The icriodontid conodont cluster consists of
crown tissue only and includes one pair of I elements and
20 coniform elements, which can be distinguished in 10
pairs (C1–C5). No basal plate is preserved. Although the
cluster shows numerous micro-fractures on the surface of
I elements with some coniform elements being broken in
two or more pieces (still attached in the cluster) or having
lost their tips, it can be reconstructed based on SEM and
micro-CT analysis (Fig. 2). 1975 Icriodus woschmidti woschmidti Ziegler; Carls: 410,
pl. 2, figs 19–21. Description. The icriodontid conodont cluster consists of
crown tissue only and includes one pair of I elements and
20 coniform elements, which can be distinguished in 10
pairs (C1–C5). No basal plate is preserved. Although the
cluster shows numerous micro-fractures on the surface of
I elements with some coniform elements being broken in
two or more pieces (still attached in the cluster) or having
lost their tips, it can be reconstructed based on SEM and
micro-CT analysis (Fig. 2). 1976 Caudicriodus woschmidti (Ziegler); Bultynck: 21,
figs 1, 3–4 [cum syn.]. 1977 Caudicriodus woschmidti (Ziegler); Bultynck: pl. 39, fig. 10, pl. 40, fig. 24. 39, fig. 10, pl. 40, fig. 24. 1977 Icriodus woschmidti woschmidti Ziegler; Chatterton
& Perry: 793, pl. 3, figs 18–22. 1977 Icriodus woschmidti woschmidti Ziegler; Chatterton
& Perry: 793, pl. 3, figs 18–22. Caudicriodus woschmidti (Ziegler, 1960)
(Figs 2, 4) However, the large number
of coniform elements counted in single clusters led to the
conclusion that these were arranged serially within the
apparatus of one individual. Like Icriodus expansus, the
fused cluster of Caudicriodus woschmidti did not preserve
ramiform elements. The latter elements are part of the
apparatus reconstruction suggested by Serpagli (1983). His analysis of disarticulated elements of Caudicriodus
woschmidti from the Early Devonian of southern Sardinia
(Italy) resulted in an apparatus that included ramiform
(a, b and c), coniform (e and f) and icriodiform (g) ele-
ments. These formed two transitional series, each consist-
ing of three morphotypes (a, b, c and e, f, g). This
hypothesis followed the analysis of Cypricriodus hesper-
ius from the Silurian to Devonian of north Queensland,
Australia, by Simpson (1998), who proposed an apparatus
that contained variably ornamented coniform elements
(Sa, Sb1, Sb2 and Sc elements), M elements, Pb elements
and Pa elements. In his model, S elements represent a
symmetry transition series. Originally introduced for the
skeletal apparatus of Oulodus by Sweet & Sch€onlaub
(1975), this notation scheme was used by Simpson (1998)
for documenting the analogous relationship regarding the
position occupied by elements in different euconodont
apparatuses. The most recent study of Cypricriodus hes-
perius by Murphy et al. (2016) suggested a new apparatus
structure followed by introduction of a new element nota-
tion based on statistical analysis of isolated elements. These authors discriminated five elements, including three
flared elements: one with plication (Fp), a second with the
‘inner’ wall of the base straighter than the ‘outer’ wall
(Fi), and a third with the ‘outer’ wall straighter than the
‘inner’ wall (Fo). The fourth coniform element is denticu-
late (D) and the fifth is represented by the icriodontan ele-
ment (I). However, apparatus architecture and notation
schemes for Early Devonian icriodontids are based exclu-
sively on statistical analysis of isolated elements, which is
expected to suffer a higher bias error compared with anal-
ysis of fused conodont clusters (see discussion of ‘bias
and biology’ by Purnell & Donoghue 2005). Therefore,
these are not applied here. The largest pair of coniform elements is identified as
C3. Both cones have a widely excavated base with an
irregular, flared outline. Elements have a keel extending
from the base of the cone to the base of the cusp. Caudicriodus woschmidti (Ziegler, 1960)
(Figs 2, 4) 1998
Icriodus
woschmidti
woschmidti
Ziegler;
¸Capkinoglu & Bekta¸s: 167, pl. 5, figs 10, 11. 1999 Caudicriodus woschmidti (Ziegler); Benfrika: 318,
pl. 1, fig. 10. C2 elements (C2a–C2f) differ in size but all possess a
circular outline of the basal margin. All are erect or
slightly recurved and show a striate surface ornament
where preserved. Generally, neither costae nor keels are
developed. Some of elements have the base fractured and 2002 Icriodus woschmidti woschmidti Ziegler; Garcıa-
Lopez et al.: pl. 1, figs 5–7. 2003 Caudicriodus woschmidti woschmidti (Ziegler);
Bultynck: pl. 1, figs 1–3. A new icriodontid conodont cluster 913 igure 2. Conodont cluster of Caudicriodus woschmidti, Early Devonian, southern Burgenland, Austria; Ki/4/2a-1, NHMW 2011/0374
001. A, SEM scan of the conodont cluster. B, detailed view of the coniform elements (C1–C5) close to the dextral I element. C, D, com
uter microtomography-based three-dimensional reconstruction with identification of all elements. E, hypothetical arrangement of a
ements preserved within the fused conodont cluster. A new icriodontid conodont cluster
91 Figure 2. Conodont cluster of Caudicriodus woschmidti, Early Devonian, southern Burgenland, Austria; Ki/4/2a-1, NHMW 2011/0374/
0001. A, SEM scan of the conodont cluster. B, detailed view of the coniform elements (C1–C5) close to the dextral I element. C, D, com-
puter microtomography-based three-dimensional reconstruction with identification of all elements. E, hypothetical arrangement of all
elements preserved within the fused conodont cluster. T. J. Suttner et al. 914 therefore the basal margin appears elliptically com-
pressed. C2a elements are broken into two parts: base
with major part of cusp and tip of cusp preserved close to
each other. The tip of the cusp seems elliptical in cross
section (‘posterior’ margin more convex than ‘anterior’
margin) with rather sharp margins. This differs somewhat
from other C2 elements which have a cusp with a rather
round cross section. C2e and C2f elements are larger than
other C2 elements, comparable in size to C3 and C5 ele-
ments. However, C2 elements can be discriminated easily
by having a circular basal outline and a more slender
shape in general. Because of recrystallization, surface
ornamentation of C2e and C2f is difficult to ascertain. and notation of icriodontid conodonts. His reconstruction
includes one pair of opposed platform elements (I ele-
ments) and other associated coniform elements (Ca, Cb,
Cc, Cd, Ce and Cf elements). No ramiform elements are
included within this apparatus. Caudicriodus woschmidti (Ziegler, 1960)
(Figs 2, 4) The
angle
between
the
‘posterior’
lower
part
and
the
‘posterior’ margin of the cusp is about 97. The angle
between the lower and ‘anterior’ margin of the element is
about 55, slightly curved in the lower one-fifth, continu-
ing rather straight towards the tip of the cusp. No surface
ornament is observed. C4 elements are erect and seem symmetrical with an
oval outline of the base. Although it is rather small, one
element of the C4 pair is preserved with the same orienta-
tion between two large coniform elements, close to the
‘anterior’ margins of C3 and C5. C5 elements are about half the size of C3 elements,
with an erect cusp and a wide, probably oval to circular
basal margin. Although the base appears rather conical,
the original outline and shape is unknown because of
post-mortem deformation. Neither costae nor keels are
observed. Remarks. A chronological summary of the icriodontid
element notation (Fig. 3) shows that a bimembrate nature
of the apparatus was suggested by Lange (1968) based on
the first finding of clusters of Icriodus alternatus. A few
years later, coniform elements were termed S2 (acodinan)
elements by Klapper & Philip (1971). Although previ-
ously speculated upon by Klapper & Ziegler (1975),
Nicoll (1977) was the first to propose a trimembrate appa-
ratus by including an additional type of coniform element
(M2 element). Further evidence to support this model
came from statistical analysis of the apparatus reconstruc-
tion of Icriodus trojani by Johnson & Klapper (1981). In
the same year, Norris & Uyeno (1981) introduced three
coniform types (S2a, S2b and S2c) for the apparatus of
Icriodus subterminus, of which their S2a element equates
with the classically known S2 element, and their S2b ele-
ment with the M2 element of Nicoll (1977). Nicoll (1982)
set a milestone with his publication on the analysis of hun-
dreds of fused clusters of Icriodus expansus from the Can-
ning Basin in which he revised the apparatus architecture Caudicriodus woschmidti (Ziegler, 1960)
(Figs 2, 4) However, the large number
of coniform elements counted in single clusters led to the
conclusion that these were arranged serially within the
apparatus of one individual. Like Icriodus expansus, the
fused cluster of Caudicriodus woschmidti did not preserve
ramiform elements. The latter elements are part of the
apparatus reconstruction suggested by Serpagli (1983). His analysis of disarticulated elements of Caudicriodus
woschmidti from the Early Devonian of southern Sardinia
(Italy) resulted in an apparatus that included ramiform
(a, b and c), coniform (e and f) and icriodiform (g) ele-
ments. These formed two transitional series, each consist-
ing of three morphotypes (a, b, c and e, f, g). This
hypothesis followed the analysis of Cypricriodus hesper-
ius from the Silurian to Devonian of north Queensland,
Australia, by Simpson (1998), who proposed an apparatus
that contained variably ornamented coniform elements
(Sa, Sb1, Sb2 and Sc elements), M elements, Pb elements
and Pa elements. In his model, S elements represent a
symmetry transition series. Originally introduced for the
skeletal apparatus of Oulodus by Sweet & Sch€onlaub
(1975), this notation scheme was used by Simpson (1998)
for documenting the analogous relationship regarding the
position occupied by elements in different euconodont
apparatuses. The most recent study of Cypricriodus hes-
perius by Murphy et al. (2016) suggested a new apparatus
structure followed by introduction of a new element nota-
tion based on statistical analysis of isolated elements. These authors discriminated five elements, including three
flared elements: one with plication (Fp), a second with the
‘inner’ wall of the base straighter than the ‘outer’ wall
(Fi), and a third with the ‘outer’ wall straighter than the
‘inner’ wall (Fo). The fourth coniform element is denticu-
late (D) and the fifth is represented by the icriodontan ele-
ment (I). However, apparatus architecture and notation
schemes for Early Devonian icriodontids are based exclu-
sively on statistical analysis of isolated elements, which is
expected to suffer a higher bias error compared with anal-
ysis of fused conodont clusters (see discussion of ‘bias
and biology’ by Purnell & Donoghue 2005). Therefore, and notation of icriodontid conodonts. His reconstruction
includes one pair of opposed platform elements (I ele-
ments) and other associated coniform elements (Ca, Cb,
Cc, Cd, Ce and Cf elements). No ramiform elements are
included within this apparatus. Element notation The skeletal apparatus of Caudicriodus woschmidti con-
sists of 10 pairs of coniform (C1, C2a–f, C3, C4 and C5)
and one pair of icriodontan (I) elements. Because of the
specific arrangement of elements within the cluster, ele-
ments are considered to belong to one individual only. A new icriodontid conodont cluster 915 Figure 3. Chronological listing of notation history for icriodontid apparatus elements. Morphologically similar coniform element types
and the icriodontan element evaluated for this study are highlighted in different colours or shades. Figure 3. Chronological listing of notation history for icriodontid apparatus elements. Morphologically similar coniform element types
and the icriodontan element evaluated for this study are highlighted in different colours or shades. The in vivo orientation and position occupied by coniform
elements and their relative position to the I element pair
remain uncertain. post-depositional breakage, a search has been made for
additional collections of much better preserved, but iso-
lated icriodontan elements. We found several late Eifelian icriodontan elements
(Eifel area, Germany) possessing similar wear. Tip wear
of two elements is analysed in detail and illustrated in Fig-
ures 5 and 6. The first icriodontan element of Icriodus aff. michiganus (Figs 5A–C, 6A; Supplemental Fig. 1) shows
that tip wear of three median row denticles is located on
the ‘inner’ –‘posterior’ denticle quarter and inclined in an
‘anterior’ direction. Tip wear of lateral row denticles is
documented
in
opposite
denticle
quarters
(‘outer’–
‘posterior’ vs ‘inner’–‘anterior’) with an inverted inclina-
tion angle. The largest denticle on the oral surface
(D cusp), shows wear along the ‘inner’ side, which is
inclined in an ‘outer’ direction nearly perpendicular to
the direction of wear measured from other denticles Recognition of meso- and microwear Occlusion of icriodontid elements resulted in specifically
directed mesowear of denticle tips. Within the Caudicrio-
dus woschmidti cluster, mesowear is documented only
from denticles 1 and 2 of the ‘inner’ lateral denticle row
(dextral I element). Smoothly polished facets of rather
elliptical outline are inclined in a more or less ‘anterior’
direction (Fig. 4A, B, indicated by arrows). Because of
poor preservation, no further meso- or microwear could
be observed with certainty. In order to verify whether tip
wear recognized on the dextral I element of the Caudi-
criodus cluster is due to occlusal stress and not merely 916 T. J. Suttner et al. T. J. Suttner et al. Figure 4. A, denticle tip wear of the dextral I element of Caudicriodus woschmidti; Early Devonian, southern Burgenland, Austria; Ki/
4/2a-1, NHMW 2011/0374/0001. B, detailed view of oral surface of the dextral I element with extent and orientation of tip wear indi-
cated by dotted line and arrow head. Figure 4. A, denticle tip wear of the dextral I element of Caudicriodus woschmidti; Early Devonian, southern Burgenland, Austria; Ki/
4/2a-1, NHMW 2011/0374/0001. B, detailed view of oral surface of the dextral I element with extent and orientation of tip wear indi-
cated by dotted line and arrow head. side of denticles 2 and 3, respectively. Although a
major part of the ‘inner’ lateral row denticle 2 is bro-
ken (Fig. 5D–F), direction of wear can be recon-
structed
based
on
the
remaining
damage
of
the
transverse ridge extending from the lateral row den-
ticles towards the base of middle row denticles. The
‘posterior’-most three ‘inner’ lateral row denticles pos-
sess facets which are inclined ‘posteriorly’. Those of
the ‘outer’ lateral row denticles and the entire fourth
denticle row show opposite inclination (the process of
facet formation is illustrated for the fourth transverse
denticle row in Supplemental Video 1). Some of the
transverse ridges also show spalling (‘inner’ lateral
row denticle 1 and ‘outer’ lateral row denticle 2 on the
‘posterior’ side of the transverse ridge, and ‘outer’ lat-
eral row denticle 3 on either side). Similar to the other
specimen, wear of the cusp (and here the pre-cusp too)
is nearly perpendicular to tip wear of the other den-
ticles. Spalling
is
observed
within
the
‘posterior’
–‘outer’ quarter of cusp and pre-cusp. Although both (Fig. 6A). There is a range of variation of tip wear
observed within one element. Recognition of meso- and microwear Some denticles are damaged
more strongly compared to others possessing steeply
inclined, rather ovate to elliptical outlined wear facets that
reach deeply down to the denticle base (Fig. 5A–C:
‘inner’ lateral row denticle 2 and median row denticle 2). Other tips show a less inclined crescent-shaped facet. Three
denticles
clearly
have
layered
microstructure
exposed on the somewhat blunted tips (Fig. 5A–C: ‘inner’
lateral row denticle 3 and ‘outer’ lateral row denticles 2
and 3, counted from the ‘posterior’ towards the ‘anterior’). (Fig. 6A). There is a range of variation of tip wear
observed within one element. Some denticles are damaged
more strongly compared to others possessing steeply
inclined, rather ovate to elliptical outlined wear facets that
reach deeply down to the denticle base (Fig. 5A–C:
‘inner’ lateral row denticle 2 and median row denticle 2). Other tips show a less inclined crescent-shaped facet. Three
denticles
clearly
have
layered
microstructure
exposed on the somewhat blunted tips (Fig. 5A–C: ‘inner’
lateral row denticle 3 and ‘outer’ lateral row denticles 2
and 3, counted from the ‘posterior’ towards the ‘anterior’). For comparison, the crown tissue of a second icrio-
dontan specimen from the same locality is illustrated
in Figures 5D–F and 6B. It shows a more strongly and
rather roughly damaged oral surface. Tips of median
row denticles possess planar wear facets without a spe-
cific inclination direction (Figs 5D–F, 6B). However,
spalling occurs on median row denticles. It is docu-
mented on the ‘inner’, ‘posterior’ and ‘outer’ side of
denticle 1, and the ‘inner’–‘posterior’ and ‘posterior’ (Fig. 6A). There is a range of variation of tip wear
observed within one element. Some denticles are damaged
more strongly compared to others possessing steeply
inclined, rather ovate to elliptical outlined wear facets that
reach deeply down to the denticle base (Fig. 5A–C:
‘inner’ lateral row denticle 2 and median row denticle 2). Other tips show a less inclined crescent-shaped facet. Three
denticles
clearly
have
layered
microstructure
exposed on the somewhat blunted tips (Fig. 5A–C: ‘inner’
lateral row denticle 3 and ‘outer’ lateral row denticles 2
and 3, counted from the ‘posterior’ towards the ‘anterior’). For comparison, the crown tissue of a second icrio-
dontan specimen from the same locality is illustrated
in Figures 5D–F and 6B. It shows a more strongly and
rather roughly damaged oral surface. Recognition of meso- and microwear Tips of median
row denticles possess planar wear facets without a spe-
cific inclination direction (Figs 5D–F, 6B). However,
spalling occurs on median row denticles. It is docu-
mented on the ‘inner’, ‘posterior’ and ‘outer’ side of
denticle 1, and the ‘inner’–‘posterior’ and ‘posterior’ For comparison, the crown tissue of a second icrio-
dontan specimen from the same locality is illustrated
in Figures 5D–F and 6B. It shows a more strongly and
rather roughly damaged oral surface. Tips of median
row denticles possess planar wear facets without a spe-
cific inclination direction (Figs 5D–F, 6B). However,
spalling occurs on median row denticles. It is docu-
mented on the ‘inner’, ‘posterior’ and ‘outer’ side of
denticle 1, and the ‘inner’–‘posterior’ and ‘posterior’ A new icriodontid conodont cluster 917 Figure 5. Denticle tip wear of icriodontid I elements. A–C, Icriodus aff. michiganus, dextral I element, lateral and oral view; Middle
Devonian, Eifel, Germany; sample BL-12-29c-9. D–F, Icriodus sp., dextral I element, lateral and oral view; Middle Devonian, Eifel,
Germany; sample BL-12-29c-3. Extent and orientation of tip wear are indicated by dotted lines and arrowheads. Figure 5. Denticle tip wear of icriodontid I elements. A–C, Icriodus aff. michiganus, dextral I element, lateral and oral view; Middle
Devonian, Eifel, Germany; sample BL-12-29c-9. D–F, Icriodus sp., dextral I element, lateral and oral view; Middle Devonian, Eifel,
Germany; sample BL-12-29c-3. Extent and orientation of tip wear are indicated by dotted lines and arrowheads. Figure 5. Denticle tip wear of icriodontid I elements. A–C, Icriodus aff. michiganus, dextral I element, lateral and oral view; Middle
Devonian, Eifel, Germany; sample BL-12-29c-9. D–F, Icriodus sp., dextral I element, lateral and oral view; Middle Devonian, Eifel,
Germany; sample BL-12-29c-3. Extent and orientation of tip wear are indicated by dotted lines and arrowheads. Discussion specimens are dextral elements, the inclination of wear
of the cusp runs in the opposite direction. A significant difference exists between the Caudicriodus
cluster and those published by Nicoll (1982). Most coni-
form elements of the specimen shown here are attached
nearly perpendicularly to the I element pair, possessing
a
more
or
less
bidirectional
orientation
(Fig. 2). Microwear is observed only on a few isolated late
Eifelian coniform elements from the conodont collec-
tion of the Eifel area (Germany). Well-preserved speci-
mens show a smoothly polished tip of the otherwise
striate cusp. T. J. Suttner et al. 918 Figure 6. Diagrams illustrating orientation and direction of denticle tip wear. A, Icriodus aff. michiganus; left-side illustration shows
the orientation of the inclined facet plane, right-side illustration the direction of vertically inclined facet; Middle Devonian, Eifel, Ger-
many; sample BL-12-29c-9. B, Icriodus sp. left-side illustration shows the orientation of the inclined facet plane, middle the direction of
the vertically inclined facet, and right the orientation and direction of the facet plane of median row denticles; Middle Devonian, Eifel,
Germany; sample BL-12-29c-3). Figure 6. Diagrams illustrating orientation and direction of denticle tip wear. A, Icriodus aff. michiganus; left-side illustration shows
the orientation of the inclined facet plane, right-side illustration the direction of vertically inclined facet; Middle Devonian, Eifel, Ger-
many; sample BL-12-29c-9. B, Icriodus sp. left-side illustration shows the orientation of the inclined facet plane, middle the direction of
the vertically inclined facet, and right the orientation and direction of the facet plane of median row denticles; Middle Devonian, Eifel,
Germany; sample BL-12-29c-3). However, a discrepancy can be recognized regarding
the absolute orientation of the elements when comparing
the in vivo orientation proposed for the apparatuses of
ozarkodinids (Aldridge et al. 1987, 1993; Purnell 1993,
1995; Purnell & Donoghue 1997; Purnell et al. 2000) and
prioniodontids (Gabbott et al. 1995; Freedman 1999;
Purnell et al. 2000). All of these have the denticle tips of
ramiform elements oriented in a dorsal direction with the
‘posterior’ part of the platform elements oriented in the
same direction (originally based on the apparatus model
deduced from individuals of Clydagnathus by Aldridge
et al. 1987). In the Caudicriodus cluster, almost all denti-
cle tips of coniform elements point in an opposite direc-
tion relative to the orientation of the ‘posterior’ part of the
icriodontan element. This kind of preservational bias
allows only hypothetical reconstructions. Discussion Based on the
arrangement and orientation of elements and without hav-
ing evidence of in vivo orientation of icriodontids based
on natural assemblages with soft tissue preservation, four
possible models of the icriodontid apparatus architecture
are introduced and discussed below. Compared to this, coniform elements of the Icriodus
expansus clusters are oriented either chaotically around
the icriodontan elements (Nicoll 1982, fig. 4.Aa) or show
a rather parallel unidirectional arrangement alongside the
faecal pellet (Nicoll 1982, fig. 11). Such specific orienta-
tion and the quantity of 10 element-pairs (it is unclear
whether additional elements are missing) suggest that the
conodont cluster represents skeletal remnants of one indi-
vidual only. Furthermore, the presence of coniform ele-
ments in pairs suggests bilaterally symmetrical apparatus
architecture. This is supported by microwear of some
coniform elements published by Nicoll (1982, fig. 10.Ic,
Mc), where sharp lateral margins of the cones’ cusps are
produced by polishing away the striate micro-ornament
on the lateral edges (of either one or both sides) of the
‘anterior’ and/or ‘posterior’ surface. Other cusps of coni-
form elements show removed micro-ornament only in the
tip-region of the ‘posterior’ surface (Nicoll 1982, fig. 10. Rc). Such microwear is considered to be the result of
‘tooth-to-tooth’ contact of opposing interdigitating coni-
form elements. 919 A new icriodontid conodont cluster gure 7. Hypothetical apparatus reconstruction deduced from the element arrangement within the Caudicriodus woschmidti conodont
uster. A, Model 1 with tips of coniform elements pointing dorsally and ‘posterior’ part of icriodontan elements oriented ventrally. B,
odel 2 with tips of coniform elements and ‘posterior’ part of icriodontan elements oriented ventrally. C, Model 3 with tips of coniform
ements pointing ventrally and ‘posterior’ part of icriodontan elements oriented dorsally. D, Model 4 with tips of coniform elements
nd ‘posterior’ part of icriodontan elements oriented ventrally. Coniform elements are arranged in multiple rows. A new icriodontid conodont cluster
919 Figure 7. Hypothetical apparatus reconstruction deduced from the element arrangement within the Caudicriodus woschmidti conodont
cluster. A, Model 1 with tips of coniform elements pointing dorsally and ‘posterior’ part of icriodontan elements oriented ventrally. B,
Model 2 with tips of coniform elements and ‘posterior’ part of icriodontan elements oriented ventrally. C, Model 3 with tips of coniform
elements pointing ventrally and ‘posterior’ part of icriodontan elements oriented dorsally. D, Model 4 with tips of coniform elements
and ‘posterior’ part of icriodontan elements oriented ventrally. Discussion The type model for ozarkodinid apparatuses (Purnell &
Donoghue 1997, 1998) is based on the analysis of wear
and surface damage of articulated platform element pairs
from natural assemblages of Idiognathodus. It concludes
that opposing platform elements were located close to
each other in a slightly offset position, performing a rota-
tional movement, with the pivot point on the ventral part
of the element where the platform joins the free blade
(Fig. 8A). This movement resulted in a complex interlock-
ing occlusion of the oral surface of P1 elements
(Donoghue & Purnell 1999b). In the second model (Fig. 7B), conventional orientation
of icriodontan elements is retained because no proof of
opposite orientation is known for icriodontid conodonts. Here, the coniform part is reoriented with the tip of the
cusp pointing ventrally. The model is based on the
assumption that collapse of the head was followed by
post-mortem displacement of the coniform elements – at
that time still connected. Such displacement could have
resulted in rotation and partial disintegration of the coni-
form apparatus part. In this case, small elements are
located in front of the large coniform elements. A similar kind of platform element motion was sug-
gested for Early Triassic individuals of the genus Novispa-
thodus (Fig. 8B). In this reconstruction, P elements
persistently operate in synchronous rotational movement
by shearing the lateral blade surfaces against each other,
while ramiform elements (M and S types) act indepen-
dently to grasp hard tissue (Goudemand et al. 2011). Coordination of such apparatus motion requires a complex
arrangement of muscle tissue. In the third model (Fig. 7C) orientation of the icriodon-
tan element pair follows the in vivo orientation of platform
elements suggested for ozarkodinids (sensu Purnell et al. 2000). Here, coniform element tips would point ventrally
and large elements would cover a position in front of
small elements. Jones et al. (2012) suggested that in addition to rota-
tional occlusion, elements must have separated during the
occlusal cycle (Fig. 8C), because of smoothly polished
wear on either side of lateral surfaces of the elements
(occlusal and non-occlusal sides). Based on sharpness
analysis they concluded that the sharper dorsal edges of
cusp and denticles indicate a dorsal rotation direction of
the primary power stroke of the platform elements. Discussion Coniform elements are arranged in multiple rows. In the first model (Fig. 7A), the original post-mortem
orientation of the majority of coniform elements relative to
the icriodontan pair is illustrated. Except for four coniform
elements (C2a, C2c, C2d and C3), all are clustering near
the ‘posterior’ part of the interlocked I element pair. Most
of the coniform elements are oriented with the ‘posterior’
margin of the cusp pointing ‘posteriorly’. A few elements
are oriented in the opposite direction (with the ‘anterior’
margin of the cusp pointing ‘posteriorly’: each of C2a, C2c and C3), or with one of lateral margins, base or tip
‘posteriorly’ (each of C1, C2a, C2b and C4). Apparently,
this is due to post-mortem displacement. In the apparatus
reconstruction, these elements are reoriented such that the
‘posterior’ margin points in a ‘posterior’ direction with the
tip of the cusp dorsal, a reconstruction resembling the in
vivo orientation in the sense of Aldridge et al. (1987) for
ozarkodinids. The apparatus would have most of the small
coniform elements (C1–C2d, except for C4) in a position T. J. Suttner et al. 920 Briggs et al. 1983; Aldridge et al. 1987, 1993, 1995;
Sweet 1988; Gabbott et al. 1995; Purnell 1995, 2001;
Donoghue & Purnell 1999a; Donoghue et al. 2000, 2008;
Purnell et al. 2000; Sweet & Donoghue 2001; Zhuravlev
2007; Jones et al. 2012; Purnell & Jones 2012; Martınez-
Perez et al. 2014a, b, 2016). Briggs et al. 1983; Aldridge et al. 1987, 1993, 1995;
Sweet 1988; Gabbott et al. 1995; Purnell 1995, 2001;
Donoghue & Purnell 1999a; Donoghue et al. 2000, 2008;
Purnell et al. 2000; Sweet & Donoghue 2001; Zhuravlev
2007; Jones et al. 2012; Purnell & Jones 2012; Martınez-
Perez et al. 2014a, b, 2016). close to the icriodontan element pair. Large coniform ele-
ments (C2e–C3 and C5) would cover a position in front of
the small coniform group. Although orientation of coni-
form elements in this model (with the tip of the cusp dor-
sal) accords with the in vivo orientation suggested for
ramiform elements of ozarkodinids, the orientation of
icriodontan elements (‘posterior’ part in a ventral direc-
tion) contradicts the published in vivo orientation of most
models since Aldridge et al. (1987). Surprisingly, it fol-
lows the conventional orientation suggested for isolated
icriodontan elements (Branson & Mehl 1938). Discussion The
authors pointed out that separation of platform elements
during the occlusal cycle also allowed food to move
between elements, which would otherwise be difficult. Studies on functional morphology and element kinematics
by Jones et al. (2012) were performed on natural assemb-
lages of the Silurian species Wurmiella excavata. The fourth model (Fig. 7D) hypothesizes an absolutely
different apparatus architecture consisting of multiple
rows of coniform elements. Generally, orientation of coni-
form and icriodontan elements follows the second model
(Fig. 7B), but here the coniform part of the apparatus is
divided into two rows of alternating elements. The lower
row consists of small and the upper row of large elements. The model is based on the evidence that one of the C4 ele-
ments is preserved between C3 and C5. Additionally,
most of the small elements (C1–C2d) are preserved close
to each other within the cluster and are located between
the icriodontan pair and the group of large coniform ele-
ments (C2e–C5). Such specific sorting of coniform ele-
ments points to a neighbouring position covered by the
groups of small and large coniform elements in the origi-
nal apparatus configuration. Martınez-Perez et al. (2014a) provided a slightly differ-
ent model for platform element motion after studying two
fused clusters from Slovenia (Krivic & Stojanovic 1978)
and additional disarticulated platform elements from
Spain (Plasencia 2009) of the Middle–Late Triassic cono-
dont Pseudofurnishius murcianus. Similar to the model
for Wurmiella excavata of Jones et al. (2012), elements
are considered to separate completely during each occlu-
sal cycle, moving more or less orthogonally to the oral
surface against each other for the next power stroke. Occlusion was refined by interdigitation of platform den-
ticles. In this model, rotational occlusion is not a major
part of the occlusal cycle (Fig. 8D). However, it is consid-
ered possible when P1 elements are interlocked. Smooth
polishing, chipping and spalling are observed on either Existing apparatus motion models Earlier models of the euconodont apparatus largely
resulted in the reconstruction of commonly known disar-
ticulated elements. The discovery of conodont clusters
and natural assemblages with soft tissue preservation and
the observation of meso- and microwear on the surfaces
of the crown tissue opened the path to new directions in
conodont research towards a better understanding of cono-
dont palaeobiology (Nicoll 1982, 1984, 1987, 1995; 921 A new icriodontid conodont cluster A new icriodontid conodont cluster A new icriodontid conodont cluster . Motion of P1 elements of ozarkodinid apparatuses summarized from the literature. A, Idiognathodus (Pennsylvanian); B,
hodus (Early Triassic); C, Wurmiella excavata (Silurian); D, Pseudofurnishius murcianus (Middle–Late Triassic); E, Polygna-
s xylus (Middle Devonian). Grey dots mark the pivot point; black arrows indicate the direction of occlusion and interlocking of
nts, grey arrows its reversal. Figure 8. Motion of P1 elements of ozarkodinid apparatuses summarized from the literature. A, Idiognathodus (Pennsylvanian); B,
Novispathodus (Early Triassic); C, Wurmiella excavata (Silurian); D, Pseudofurnishius murcianus (Middle–Late Triassic); E, Polygna-
thus xylus xylus (Middle Devonian). Grey dots mark the pivot point; black arrows indicate the direction of occlusion and interlocking of
P1 elements, grey arrows its reversal. Figure 8. Motion of P1 elements of ozarkodinid apparatuses summarized from the literature. A, Idiognathodus (Pennsylvanian); B,
Novispathodus (Early Triassic); C, Wurmiella excavata (Silurian); D, Pseudofurnishius murcianus (Middle–Late Triassic); E, Polygna-
thus xylus xylus (Middle Devonian). Grey dots mark the pivot point; black arrows indicate the direction of occlusion and interlocking of
P1 elements, grey arrows its reversal. T. J. Suttner et al. 922 2012; Martınez-Perez et al. 2014a, 2016), supported by
analogous pathodynamic mechanisms of tooth wear in
dental sciences (e.g. Grippo et al. 2004; Fondriest &
Ralgrodski 2012), results in a slightly different model of
apparatus motion for icriodontid conodonts. Compared to
other models for ozarkodinid P1 elements (Jones et al. 2012; Martınez-Perez et al. 2014a, 2016), oral surfaces of
opposed icriodontid I elements approach each other with
a slightly elliptical rather than directly orthogonal motion
(Fig. 9; Supplemental Video 2). We agree with Jones
et al. (2012) that occlusion was not always precise
depending on the morphology of the oral surface and
related to denticle guidance during the element interlock-
ing process. However, rotational movement as suggested
for ozarkodinids is not part of the occlusal cycle in icrio-
dontids. In this respect, no specific meso- and/or micro-
wear is observed. Existing apparatus motion models lateral surface of the cusp and denticles (more weakly
developed on the non-occlusal side), demonstrating that
occlusion was not always precise (Martınez-Perez et al. 2014a). )
Another apparatus motion model based on the study of
clusters of Polygnathus xylus xylus (Fig. 8E) was introduced
by Martınez-Perez et al. (2016). Originally the material was
published by Nicoll (1984), who illustrated an apparatus
model with ‘anterior’–‘posterior’ axis of P1 and P2 ele-
ments in rostro-caudal orientation. Martınez-Perez et al. (2016) revised the orientation of platform elements accord-
ing to the suggestions of Purnell et al. (2000) and proposed
a new motion model for P1 elements of Polygnathus. In this
model, opposing P1 elements are brought together bilater-
ally with the blade of the left element behind the right ele-
ment. Blades act as guides, aligning elements while
platforms approach each other. Once elements are in inter-
locking position, a short rotational movement follows from
ventral to dorsal along the elements’ curvature. Because of
imprecise occlusion of the carina and the platform troughs
in the dorsal region of the platform, rotational occlusion is
considered to stop in the middle part of the platform, not
contacting dorsal-most regions of the element pair. Because of poor preservation, tip wear of coniform ele-
ments is difficult to ascertain from the Caudicriodus clus-
ter. However, some coniform elements possess narrow
‘posterior’ keels or costae on either lateral side, which
implies coniform guidance. Additional hints on apparatus
function and motion can be inferred from isolated coni-
form elements from the collection containing icriodontan
elements of the genus Icriodus (Fig. 5). Specific occlusion
patterns such as smoothing of striate micro-ornament on
the ‘posterior’ surface of the tip of the cusp or the lateral
margin of either the ‘posterior’ and/or ‘anterior’ surface Alternative model for icriodontid apparatus motion
Direction of breakage and stress on elements (Purnell
1995; Zhuravlev 2007; Jones et al. 2012; Purnell & Jones Figure 9. Model of masticatory motion of icriodontid I elements. A, oblique lateral view; B, ‘anterior’ view. Figure 9. Model of masticatory motion of icriodontid I elements. A, oblique lateral view; B, ‘anterior’ view. Figure 9. Model of masticatory motion of icriodontid I elements. A, oblique lateral view; B, ‘anterior’ view. A new icriodontid conodont cluster 923 elements based on microwear is less well understood. Conclusions For financial support the Austrian Academy of Sciences
(Project: NAP0001 and ESS subproject to IGCP 596) and
the Austrian Science Fund (Projects: FWF P23775-B17
and FWF P23459-B17) are thanked. Dipl.-Ing. Alexander
Kottwitz-Erd€odi is kindly thanked for providing access
and sampling permission for the ‘Kottwitz’ quarry near
Kirchfidisch (southern Burgenland, Austria). Johnny A. Waters (Appalachian State University, USA) is thanked
for improving the English of the manuscript. Two anony-
mous
reviewers
and
the
Editor-in-chief
are
kindly
acknowledged for their constructive comments and useful
suggestions regarding the structure of the manuscript. This is a contribution to IGCP 596 and IGCP 652. The fused conodont cluster of Caudicriodus woschmidti
provides new insights into the apparatus structure of Early
Devonian icriodontid conodonts. The apparatus consists
of 11 element pairs (10 pairs of coniform elements and
one pair of icriodontan elements). Specific post-mortem
arrangement of most coniform elements suggests that all
elements are skeletal remnants of one individual only. The icriodontid element notation provided by Nicoll
(1982) for Icriodus expansus can be adopted in part. One
of the coniform element types of Nicoll (1982, Ce ele-
ment) was not observed. Contrary to earlier reconstruc-
tions of Caudicriodus woschmidti (Serpagli 1983), no
ramiform elements are preserved within the described
cluster. However, a preservational bias is evident which
restricts our conclusions regarding the absolute number of
coniform elements and its orientation relative to the I ele-
ment pair. With this in mind, we discuss four hypothetical
models on the apparatus architecture of Caudicriodus
woschmidti. Existing apparatus motion models Wear on the cusp is located in specific areas of different
element types and related to removed striate micro-orna-
ment. Two types of microwear are observed: (1) polished
tip of the cusp (‘anterior’ and/or ‘posterior’ side), and (2)
polished and sharpened lateral margins (left and/or right
margin of ‘anterior’ and/or ‘posterior’ side). can be documented. Such wear is characteristic for ele-
ment-element attrition (see Jones et al. 2012). Distinc-
tively different element types show specific attrition
patterns; thus, it is assumed that coniform elements oper-
ated – probably arranged in multiple rows – against each
other, organized in a bilaterally symmetrical disposition
during the occlusal cycle (compare tip wear published by
Nicoll 1982, fig. 10.Ic, Mc, Rc). However, architecture
and function of the coniform part of the icriodontid appa-
ratus are not understood well enough to provide a model
of apparatus motion illustrating how coniform and icrio-
dontan elements worked together within one individual
apparatus. Such studies will be the topic of future
research. The many unsolved questions regarding the orientation
and position of coniform elements preclude complete
reconstruction of the icriodontid apparatus motion model. Illustration of the interaction of coniform and icriodontan
elements during the occlusal cycle will be issue for future
study based on additional conodont clusters and natural
assemblages with soft tissue preservation. Supplemental data Supplemental material for this article can be accessed at:
https://doi.org/10.1080/14772019.2017.1354090 Distinctive tip wear is observed on one of the icriodon-
tan elements. Together with more significant results from
meso- and microwear analyses of additional Middle
Devonian icriodontan conodont material from the Eifel
area (Germany), a new model for icriodontid apparatus
motion is suggested. The occlusal cycle consists of a
slightly elliptical rather than straight orthogonal motion of
opposed I elements when approaching each other for the
interlocking phase. Rotational movement in interlocking
position is not considered for this euconodont group. We
recognize that neither does element-element attrition
always affect all denticles on the oral side, nor is tip wear
present within the same area of each denticle. The investi-
gated material shows that density, inclination and orienta-
tion of tip wear are related mainly to individual denticle
size, growth form and the relative position of denticles on
oral sides of opposed elements during the interlocking
phase. Compared to this, the occlusal cycle of coniform ORCID Thomas J. Suttner
http://orcid.org/0000-0002-8803-
7611
Erika Kido
http://orcid.org/0000-0001-5130-8804
Antonino Briguglio
http://orcid.org/0000-0001-5799-
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Nonlinear and delayed impacts of climate on dengue risk in Barbados: A modelling study
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RESEARCH ARTICLE * rachel.lowe@lshtm.ac.uk Background Academic Editor: Madeleine Thomson, Africa
Program, UNITED STATES Over the last 5 years (2013–2017), the Caribbean region has faced an unprecedented crisis
of co-occurring epidemics of febrile illness due to arboviruses transmitted by the Aedes sp. mosquito (dengue, chikungunya, and Zika). Since 2013, the Caribbean island of Barbados
has experienced 3 dengue outbreaks, 1 chikungunya outbreak, and 1 Zika fever outbreak. Prior studies have demonstrated that climate variability influences arbovirus transmission
and vector population dynamics in the region, indicating the potential to develop public
health interventions using climate information. The aim of this study is to quantify the nonlin-
ear and delayed effects of climate indicators, such as drought and extreme rainfall, on den-
gue risk in Barbados from 1999 to 2016. Received: February 11, 2018
Accepted: June 15, 2018
Published: July 17, 2018
Copyright: © 2018 Lowe 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. Received: February 11, 2018
Accepted: June 15, 2018
Published: July 17, 2018 Copyright: © 2018 Lowe 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. OPEN ACCESS Citation: Lowe R, Gasparrini A, Van Meerbeeck CJ,
Lippi CA, Mahon R, Trotman AR, et al. (2018)
Nonlinear and delayed impacts of climate on
dengue risk in Barbados: A modelling study. PLoS
Med 15(7): e1002613. https://doi.org/10.1371/
journal.pmed.1002613 Nonlinear and delayed impacts of climate on
dengue risk in Barbados: A modelling study Rachel Lowe1,2,3*, Antonio Gasparrini4,5, Ce´dric J. Van Meerbeeck6, Catherine A. Lippi7,
Roche´ Mahon6, Adrian R. Trotman6, Leslie Rollock8, Avery Q. J. Hinds9, Sadie J. Ryan7,10,
Anna M. Stewart-Ibarra11,12 1 Department of Infectious Disease Epidemiology, London School of Hygiene & Tropical Medicine, London,
United Kingdom, 2 Centre for the Mathematical Modelling of Infectious Diseases, London School of Hygiene
& Tropical Medicine, London, United Kingdom, 3 Barcelona Institute for Global Health (ISGLOBAL),
Barcelona, Spain, 4 Department of Public Health, Environments and Society, London School of Hygiene &
Tropical Medicine, London, United Kingdom, 5 Centre for Statistical Methodology, London School of Hygiene
& Tropical Medicine, London, United Kingdom, 6 Caribbean Institute for Meteorology and Hydrology,
St. James, Barbados, 7 Quantitative Disease Ecology and Conservation Lab Group, Department of
Geography and Emerging Pathogens Institute, University of Florida, Gainesville, Florida, United States of
America, 8 Ministry of Health, St. Michael, Barbados, 9 Caribbean Public Health Agency, Port of Spain,
Trinidad and Tobago, 10 School of Life Sciences, University of KwaZulu-Natal, Durban, South Africa,
11 Institute for Global Health and Translational Science, SUNY Upstate Medical University, Syracuse, New
York, United States of America, 12 Department of Medicine and Department of Public Health and
Preventative Medicine, SUNY Upstate Medical University, Syracuse, New York, United States of America a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 Why was this study done? • Changes in local climate conditions (i.e., rainfall, temperature) can affect the risk of out-
breaks of diseases transmitted by mosquitoes, such as dengue fever, chikungunya, and
Zika. • Climate information can be used to develop forecasts of disease outbreaks. RL was supported by a Royal Society
Dorothy Hodgkin Fellowship. AG was supported by
a grant from the Medical Research Council UK
(Grant ID: MR/M022625/1). 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. Abbreviations: AUC, area under the ROC curve;
CariCOF, Caribbean Climate Outlook Forum;
CARPHA, Caribbean Public Health Agency;
CDPMN, Caribbean Drought and Precipitation
Monitoring Network; CIMH, Caribbean Institute for
Meteorology and Hydrology; CV, cross-validated;
DENV, dengue virus; DIC, deviance information
criterion; DLNM, distributed lag nonlinear model;
ENSO, El Niño Southern Oscillation; EWISACTs,
Early Warning Information Systems Across Climate
Timescales; GAIA, Grantley Adams International
Airport; INLA, Integrated Nested Laplace
Approximation; NS1, nonstructural protein 1; RCC,
Regional Climate Centre; ROC, relative (receiver)
operating characteristic; SIDS, Small Island
Developing States; SPI, Standardised Precipitation
Index; SPI-6, SPI averaged over a 6-month period;
Tmin, minimum temperature. Climate and dengue risk in Barbados 5 months, while excess rainfall increased the risk at shorter lead times between 1 and 2
months. The SPI averaged over a 6-month period (SPI-6), designed to monitor drought and
extreme rainfall, better explained variations in dengue risk than monthly precipitation data
measured in millimetres. Tmin was found to be a better predictor than mean and maximum
temperature. Furthermore, including bidimensional exposure–lag–response functions of
these indicators—rather than linear effects for individual lags—more appropriately
described the climate–disease associations than traditional modelling approaches. In pre-
diction mode, the model was successfully able to distinguish outbreaks from nonoutbreaks
for most years, with an overall proportion of correct predictions (hits and correct rejections)
of 86% (81%:91%) compared with 64% (58%:71%) for the baseline model. The ability of the
model to predict dengue outbreaks in recent years was complicated by the lack of data on
the emergence of new arboviruses, including chikungunya and Zika. 5 months, while excess rainfall increased the risk at shorter lead times between 1 and 2
months. The SPI averaged over a 6-month period (SPI-6), designed to monitor drought and
extreme rainfall, better explained variations in dengue risk than monthly precipitation data
measured in millimetres. Tmin was found to be a better predictor than mean and maximum
temperature. Furthermore, including bidimensional exposure–lag–response functions of
these indicators—rather than linear effects for individual lags—more appropriately
described the climate–disease associations than traditional modelling approaches. In pre-
diction mode, the model was successfully able to distinguish outbreaks from nonoutbreaks
for most years, with an overall proportion of correct predictions (hits and correct rejections)
of 86% (81%:91%) compared with 64% (58%:71%) for the baseline model. The ability of the
model to predict dengue outbreaks in recent years was complicated by the lack of data on
the emergence of new arboviruses, including chikungunya and Zika. 00001) Programme for Building Regional Climate
Capacity in the Caribbean (BRCCC Programme:
rcc.cimh.edu.bb/brccc) with funding made
possible by the generous support of the American
people. RL was supported by a Royal Society
Dorothy Hodgkin Fellowship. AG was supported by
a grant from the Medical Research Council UK
(Grant ID: MR/M022625/1). The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript. 00001) Programme for Building Regional Climate
Capacity in the Caribbean (BRCCC Programme:
rcc.cimh.edu.bb/brccc) with funding made
possible by the generous support of the American
people. Methods and findings Data Availability Statement: All meteorological
data used in this study are available from the
Caribbean Institute for Meteorology and Hydrology
via Mr. Wayne Depradine (wdepradine@cimh.edu. bb). Health data can be requested from the
Barbados Ministry of Health (EPI.Unit@health.gov. bb). Distributed lag nonlinear models (DLNMs) coupled with a hierarchal mixed-model frame-
work were used to understand the exposure–lag–response association between dengue rel-
ative risk and key climate indicators, including the standardised precipitation index (SPI)
and minimum temperature (Tmin). The model parameters were estimated in a Bayesian
framework to produce probabilistic predictions of exceeding an island-specific outbreak
threshold. The ability of the model to successfully detect outbreaks was assessed and com-
pared to a baseline model, representative of standard dengue surveillance practice. Drought
conditions were found to positively influence dengue relative risk at long lead times of up to Funding: This study was solicited by the Caribbean
Institute for Meteorology and Hydrology (CIMH)
through the United States Agency for International
Development’s (USAID, Grant ID: AID-538-10-14- 1 / 24 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Conclusion We present a modelling approach to infer the risk of dengue outbreaks given the cumulative
effect of climate variations in the months leading up to an outbreak. By combining the den-
gue prediction model with climate indicators, which are routinely monitored and forecasted
by the Regional Climate Centre (RCC) at the Caribbean Institute for Meteorology and
Hydrology (CIMH), probabilistic dengue outlooks could be included in the Caribbean Health-
Climatic Bulletin, issued on a quarterly basis to provide climate-smart decision-making guid-
ance for Caribbean health practitioners. This flexible modelling approach could be extended
to model the risk of dengue and other arboviruses in the Caribbean region. Author summary What did the researchers do and find? • In this study, we devised a statistical model to test whether dengue outbreaks in the
Caribbean island of Barbados could be predicted using weather station data for temper-
ature and a precipitation index—used to monitor drought and extreme rainfall—as
model inputs from June 1999 to May 2016. • The model was able to successfully predict months with dengue outbreaks versus non-
outbreaks in most years. • The risk of dengue outbreaks increased with increasing minimum temperature (Tmin;
up to 25˚C). Disease outbreaks were more likely to occur 4 to 5 months after periods of
drought and 1 month after periods of excess rainfall. 2 / 24 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Climate and dengue risk in Barbados • The modelling results suggest that a drought period followed by intense rainfall 4 to 5
months later could provide optimum conditions for an imminent dengue outbreak. • The modelling results suggest that a drought period followed by intense rainfall 4 to 5
months later could provide optimum conditions for an imminent dengue outbreak. What do these findings mean? • In practice, the Regional Climate Centre (RCC) could use this dengue model to generate
disease forecasts using their seasonal climate forecast products. • In practice, the Regional Climate Centre (RCC) could use this dengue model to generate
disease forecasts using their seasonal climate forecast products. • Public health decision-makers can use the dengue forecasts as an early warning tool to
plan interventions to reduce the risk of dengue and other mosquito-borne diseases. • The ability of the model to predict dengue outbreaks is complicated by the emergence of
new diseases with similar symptoms that are transmitted by the same mosquito vector,
including chikungunya and Zika. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Climate and dengue risk in Barbados determine the type and abundance of larval habitat in the environment [14]. Rainfall can
increase mosquito population densities by increasing the availability of larval habitat in aban-
doned rain-filled containers in the patio around the home. Drought conditions that result in
household water scarcity can also potentially increase larval habitat by increasing the number
of water storage containers around the home [15]. However, few studies have examined the
effects of prolonged drought on dengue transmission. Prior studies in Barbados documented the seasonal linkages between climate and dengue
(from 1980 to 2000), finding that epidemics occurred in the latter part of the year [16]. The
study also found that there was a trend in increasing temperatures and declining rainfall in
recent decades. Other studies found that dengue was significantly associated with lagged cli-
mate variables, including rainfall at a 7-week lag and minimum temperature (Tmin) at a
12-week lag [10]. In a different study, investigators analysed a time series of dengue from 2004
to 2013 using generalised linear models, which were able to predict dengue cases with limited
success using monthly rainfall, monthly number of rain days, and average temperature, after
pre-adjusting for unmeasured cyclical and long-term trends [17]. Most recently, investigators
analysed monthly dengue cases from 2006 to 2015 and found a positive correlation between
rainfall and relative humidity of the same month. However, they did not assess lagged relation-
ships [5]. The El Niño Southern Oscillation (ENSO) is a major driver of regional year-to-year climate
variability in the Caribbean [18,19]. ENSO is associated with a gradient in sea surface tempera-
ture anomalies between the eastern Tropical Pacific Ocean and the Tropical North Atlantic
Ocean and Caribbean Sea. During the early and mature stages of El Niño events, this gradient
is often positive (i.e., the surface waters of the Tropical Pacific are anomalously warmer than
the Tropical North Atlantic and Caribbean Sea). Most periods of regional drought, weaker
hurricane season activity, and warmer temperatures are associated with the El Niño–induced
positive gradient. By contrast, wet episodes and more active hurricane seasons coincide with
La Niña events [19], i.e., when the ocean gradient tends to be negative. Recent examples of El
Niño–associated regional drought occurred in 1997–1998, 2009–2010, and 2014–2016,
whereas La Niña events in 2010–2011 and 2011–2012 coincided with very wet conditions in
large parts of the Caribbean [20]. Introduction Small Island Developing States (SIDS) in the Caribbean are among the most vulnerable coun-
tries to extreme climate events (e.g., droughts and tropical storms), which are becoming more
frequent and severe due to climate change [1]. Climate events have a major impact on human
health in the Caribbean, including impacts on arboviral disease transmission, heat-induced
morbidity, water-borne diseases, injuries, respiratory complications, and mental health [2]. The social and economic cost of these adverse health outcomes is a major burden on SIDS,
and the burden is projected to increase with the changing climate [3]. In recent years, the Caribbean region has experienced an unprecedented crisis of co-
occurring epidemics of febrile illness due to dengue, chikungunya, and Zika viruses. These
diseases are transmitted principally by the female Aedes aegypti, which is a mostly domestic,
urban mosquito that lays its eggs in water-bearing containers in and around the home. Over
a 5-year period (2012 to 2016), Barbados reported a total of 7,298 dengue cases [4], with
most cases reported in children up to 15 years of age [5]. Three outbreaks of dengue fever
were reported during that period (2013, 2014, and 2016). Recent studies reported an upward
trend in dengue transmission in Barbados from 2006 to 2015, with all 4 dengue serotypes
(dengue virus [DENV]-1 through DENV-4) cocirculating in the population [5]. Illness due
to dengue virus is estimated to cost $321.4 million USD per year in the Caribbean (excluding
the costs of vector control and other prevention programmes) [6]. With the recent emer-
gence of chikungunya and Zika in the Caribbean, the cost of Aedes-transmitted diseases has
increased dramatically. In a region with low economic growth and high public debt [7,8], the
burden of arboviruses is unsustainable, and new tools are urgently needed to support the
public health sector. Prior studies have shown that climate variability influences dengue transmission and A. aegypti population dynamics in the Caribbean [9–12]. Ambient temperatures impact disease
transmission by affecting mosquito development rates, reproduction, survival, biting rates,
and viral replication in the mosquito [13]. Warmer temperatures increase the risk of disease
transmission up to an optimum temperature range of 26 to 29˚C [13]. The effect of rainfall on
dengue risk is more complex and depends on the local socioecological conditions that PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 3 / 24 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Study area This study was carried out for the Caribbean island of Barbados (13˚ N 59˚ W) in the Lesser
Antilles. Barbados is relatively small in land area (425 km2), with a resident population of over
277,000 people [29]. Due to a thriving tourism-based economy, over 1 million people visit Bar-
bados each year [30]. The island is administratively divided into 11 parishes, the most densely
populated being the urban parish of St. Michael, which contains the capital city of Bridgetown
(see S1 Fig). Dengue is one of the primary febrile illnesses in Barbados, especially during the
wet season, when the mosquito vector is most abundant. A. aegypti is the only known vector
on the island. Health data The surveillance unit of the Environmental Health Department in the Ministry of Health of
Barbados is responsible for maintaining records on the incidence of dengue and other arbo-
viral disease cases. As of January 2018, the surveillance unit liaises with the Leptospirosis lab
(national reference lab) to record laboratory-confirmed cases of dengue virus, chikungunya
virus, and Zika virus. Specimens are tested for the dengue virus nonstructural protein 1 (NS1)
antigen, dengue virus IgM/IgG, and chikungunya virus IgM/IgG using commercial ELISA
kits. Since September 2016, the lab has also conducted real-time PCR using the CDC Trioplex
assay for dengue virus, chikungunya virus, and Zika virus, and they conduct serotyping of
positive dengue virus samples. National population estimates, obtained from the Barbados
Population and Housing Census (1990, 2000, and 2010) [29], were used to estimate yearly pop-
ulation during the study period by linear interpolation. Population data were used to calculate
monthly incidence rates of laboratory-confirmed dengue cases in Barbados from June 1999 to
May 2016. Climate and dengue risk in Barbados This study aims to quantify the nonlinear and delayed effects of climate impacts, such as
drought, extreme rainfall, and temperature variations, on dengue risk in the eastern Caribbean
island of Barbados by coupling DLNMs with a Bayesian model estimation framework. The
model is then used to produce out-of-sample predicted probabilities of exceeding island-spe-
cific outbreak thresholds. Barbados is an ideal case study due to high-quality historical climate
and health data, a relatively high burden of disease, and prior experience in building projects
on climate and health, paving the way for a sustainable collaboration to develop integrated
early warning information to predict the risk of dengue and other mosquito-transmitted dis-
eases in the Caribbean. In Barbados, we hypothesise that the most important
extreme climate events with respect to arbovirus risk are droughts, which extend into the dry
season when water availability is reduced [20], and periods of excessive rainfall during the wet
season. Before an effective disease forecast can be developed, it is important to identify the key cli-
mate drivers, lag periods, and appropriate model formulation that reflects the local disease
transmission ecology and climatology. It takes time for anomalies in the climate to manifest
and contribute to disease risk. The various components of the time lag include the period for
mosquito larval habitat to increase, the development period of the mosquito, the time before
the first blood meal in which the mosquito transmits the virus to a human host, and the time
before the appearance of clinical manifestations of dengue [10]. Several studies have employed
distributed lag nonlinear models (DLNMs) to explore associations between meteorological
variables and dengue incidence in several countries—primarily in Asia—that experience den-
gue epidemics, including Bangladesh [21], China [22], Indonesia [23], Malaysia [24], Singa-
pore [25], Sri Lanka [26], and Taiwan [27]. A major advantage to using DLNM methodology
is the possibility of describing the lag structure of either linear or nonlinear exposure–response
relationships, through the choice of 2 functions that define the association along the dimen-
sions of the predictor (e.g., climate variables) and lags [28]. The analysis of the temporal evolu-
tion of disease risk associated with protracted time-varying climatic exposures has
implications for the development of climate-based disease early warning systems. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 4 / 24 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Standardised Precipitation Index The Standardised Precipitation Index (SPI) is a drought index first developed by McKee et al. [35]. The SPI is a representation of rainfall totals in units of standard deviation following a
gamma-fitted distribution. Positive values indicate greater than average rainfall totals or excess
rainfall, while negative values indicate less than average rainfall or rainfall deficits—otherwise
called meteorological drought. The SPI can best be interpreted as the severity of meteorological
drought or excessive rainfall by the end of the period of interest and is based on monthly pre-
cipitation totals alone—the most commonly available climate variable from rainfall stations
around the world. As such, wet or dry conditions can be monitored by the SPI at a variety of
time horizons, from subseasonal to multi-annual time scales. SPI at different time scales are representative of different types of drought. Generally, the
longer the meteorological drought persists and the more negative the SPI value, the greater
the societal impact. The 1-month SPI (SPI-1) allows for early detection of meteorological
droughts. Operationally, with the aim to enable drought early warning systems, the Caribbean
Drought and Precipitation Monitoring Network (CDPMN)—coordinated by the CIMH—
tracks drought using SPIs at 1 month, 3 months (SPI-3), 6 months (SPI-6), 12 months (SPI-
12), and 24 months (SPI-24) [31] to cover the range of drought durations experienced in its cli-
mate [20]. For example, rainfall deficits over 1 month may be enough to dry the topsoil, espe-
cially during the latter part of the dry season. However, in the Caribbean, agricultural drought
(related to shortage of surface or soil moisture for optimal crop growth) manifests more signif-
icantly after 3 months [20]. Therefore, SPI-3 and SPI-6 can be used to track agricultural
drought. Soil moisture and surface water reservoir depletion typically take at least 6 months to
substantially impact water availability at the national level. Aquifer depletion and repletion
typically occur at time scales of 9 to 12 months, affecting large groundwater reservoirs. There-
fore, hydrological drought can be tracked with SPI-6 and SPI-12. With drought being a slow-onset climate hazard, early warning (e.g., based on SPIs) with
ample lead time is a realistic and effective option. This is especially the case when monitoring
is combined with forecasting. Meteorological data Monthly rainfall totals and average minimum, mean, and maximum temperatures were
extracted from the 2 synoptic weather stations on the island—at the Grantley Adams Interna-
tional Airport (GAIA), at an elevation of 56 m in the southern parish of Christ Church, and at
the Caribbean Institute for Meteorology and Hydrology (CIMH), at an elevation of 112 m in
the western parish of St. James (see S1 Fig and S3 Fig). Monthly minimum (maximum) tem-
perature is defined as the arithmetic average of the daily minimum (maximum) dry-bulb tem-
perature, measured under standard Stevenson screen conditions. Climate and dengue risk in Barbados lying areas to around 2,000-mm per annum above 300-m elevation [31]. S2 Fig shows the
1981–2010 average rainfall seasonality in terms of rainfall chance and rainfall intensity on wet
days for the synoptic station located at the CIMH. The combination of relatively little rainfall,
high population density, and a mostly urban population makes Barbados a particularly water-
scarce country [34]. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Climate of Barbados The climatology of Barbados fits well within that of the Lesser Antilles. The wet season lasts
from June until November and coincides with the Atlantic Hurricane Season [31]. The dry sea-
son occurs from December to May. More than 70% of annual rainfall totals are recorded on
average during the wet season [32], which is further characterised by elevated relative humid-
ity, increased rainfall frequency and intensity, and the irregular occurrence of spells of very
heavy rainfall [31]. The wet season typically features 2 to 3˚C higher dry-bulb temperatures
than the relatively cool period of December to February, with heat waves becoming more fre-
quent in recent decades [31,33]. Given its low topography, Barbados records significantly less rainfall, on average, than
neighbouring mountainous islands, from less than 1,200-mm rainfall per annum in some low- PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 5 / 24 Climate and dengue risk in Barbados Fig 1. Annual cycle of dengue incidence rates, SPI-6, and Tmin June 1999–May 2016. Annual cycle of (a) dengue incidence rates (per 100,000 population) in Barbados,
(b) SPI-6, and (c) Tmin (˚C) averaged over CIMH and GAIA weather stations at the monthly time scale from June 1999 to May 2016. CIMH, Caribbean Institute for
Meteorology and Hydrology; GAIA, Grantley Adams International Airport; SPI-6, 6-month Standardised Precipitation Index. https://doi.org/10.1371/journal.pmed.1002613.g001 Fig 1. Annual cycle of dengue incidence rates, SPI-6, and Tmin June 1999–May 2016. Annual cycle of (a) dengue incidence rates (per 100,000 population) in Barbados,
(b) SPI-6, and (c) Tmin (˚C) averaged over CIMH and GAIA weather stations at the monthly time scale from June 1999 to May 2016. CIMH, Caribbean Institute for
Meteorology and Hydrology; GAIA, Grantley Adams International Airport; SPI-6, 6-month Standardised Precipitation Index. Fig 1. Annual cycle of dengue incidence rates, SPI-6, and Tmin June 1999–May 2016. Annual cycle of (a) dengue incidence rates (per 100,000 population) in Barbados,
(b) SPI-6, and (c) Tmin (˚C) averaged over CIMH and GAIA weather stations at the monthly time scale from June 1999 to May 2016. CIMH, Caribbean Institute for
Meteorology and Hydrology; GAIA, Grantley Adams International Airport; SPI-6, 6-month Standardised Precipitation Index. https://doi.org/10.1371/journal.pmed.1002613.g001 https://doi.org/10.1371/journal.pmed.1002613.g001 original severity classes are shown in S1 Table. Impacts experienced (particularly in agricul-
ture) during the Caribbean drought of 2009–2010 led to a redefinition of the categories (see S2
Table), which has been used for decision-making since 2011. For this study, all SPI values were calculated based on the records of daily rainfall totals at
the CIMH and the GAIA stations in Barbados, which start in January of 1981 and 1971,
respectively. The historical climatological reference was calculated using the period 1981–2010
to conform with World Meteorological Organization regulations. Fig 1 shows the annual cycle of dengue incidence rates (per 100,000 population), the SPI-6,
and Tmin in Barbados from June 1999 to May 2016. Dengue outbreaks occurred in the dengue
seasons 2003–2004, 2007–2008, 2010–2011, 2013–2014, 2014–2015 and 2015–2016. Severe
droughts (negative SPI-6 values) occurred in 2002–2003, 2009–2010, and 2015–2016, which
coincided with El Niño events (see S4 Fig). During wetter than average rainy seasons, dengue
outbreaks tended to peak earlier in the dengue season, in September (see S5 Fig). Standardised Precipitation Index Operationally, the Caribbean Climate Outlook Forum (Cari-
COF) provides drought forecasts using predictions of SPI-6 with a lead time of 3 months (i.e.,
3 months in advance) and SPI-12 with lead times up to 6 months [20]. In contrast, while SPIs
are used by the CDPMN to track periods of excess precipitation, flooding potential—the
hydrometeorological hazard component of flood risk—is a compounded factor of both long-
term rainfall accumulations (i.e., soil moisture saturation) and short-term wet spells. The PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 6 / 24 Exposure–lag–response model DLNMs [28] were used to explore and understand possible nonlinear and delayed associations
between dengue incidence rates and a variety of climate impact indicators. This modelling
approach is based on the definition of a cross-basis, obtained by the combination of 2 functions
to flexibly model linear or nonlinear exposure–responses and the lag structure of the relation-
ship [28]. A standard exposure–response function f(x) is defined to express the potentially non-
linear exposure–response curve along the dimension of the climate predictors. An additional
lag–response function w(l) expresses instead the temporal dimension. Their combination in a
bidimensional exposure–lag–response function f.w(x,l) through a cross-basis is defined to flexi-
bly model both intensity and timing of past exposures. Meteorological variables—including
monthly precipitation, minimum, mean, and maximum temperatures as well as the SPI at dif-
ferent time scales (1-, 3-, 6-, and 12-month averages)—were tested in the model, in turn. Note
that an El Niño index was not included in the model because it is highly correlated with the SPI
in Barbados (see S4 Fig), and including both would have resulted in overfitting. Each variable
was included as a linear term at individual lags and as a nonlinear exposure–lag–response func-
tion. The delayed effects of the climate indicators were assessed for up to 5 months, which was
determined on the basis of exploratory analysis and previous studies [27,38]. Natural cubic
splines were selected for both the exposure and the lag dimensions to allow enough flexibility to
capture potentially complex associations between climate indicators and their delayed impact
on dengue risk. Climate and dengue risk in Barbados First, a baseline model was formulated by including a first-order random walk latent model
for month βt’(t), where t’(t) = 1, . . ., 12 and β1 represents the parameter estimate for the month
of June. This term helps to capture the seasonality in dengue, which is assumed to be stationary
each year (monthly random effect). The first-order random walk prior allows dengue inci-
dence rates in 1 month to depend on the previous month, to reflect both seasonality and the
infectious nature of the disease. Next, exchangeable random effects for each year γT’(t) (where
T’(t) = 1, . . ., 17 and γ1 represents the dengue year June 1999–May 2000) was included in the
model to account for interannual variation in dengue over time (yearly random effect). This
term potentially allows for changes in population immunity between outbreak years, lapses in
vector control, and other slowly changing factors, such as changes in mosquito-control inten-
sity and the introduction of new dengue virus serotypes or other viruses, which could be mis-
taken for dengue. Note that chikungunya and Zika viruses were introduced to Barbados in
2014 and at the end of 2015, respectively [5,37]. Model framework As dengue incidence tends to peak between September and February (see S3 Fig panel a), we
defined a ‘dengue’ year as running from June to May. A hierarchical mixed model was formu-
lated using counts of laboratory-confirmed dengue cases per month over 17 years—from June
1999 to May 2016—in Barbados as the response variable. Counts of dengue cases, yt (t = 1, . . .,
204), were assumed to follow a negative binomial distribution, ytjmt NegBinðmt ¼ pT0ðtÞrt; kÞ logðmtÞ ¼ logðpT0ðtÞÞ þ logðrtÞ
ð1Þ ð1Þ where μt is the corresponding distribution mean, which is equal to the population per 100,000
pT’(t) multiplied by the unknown dengue incidence rate estimate ρt for time t. κ is the scale (or
overdispersion) parameter [36]. Population effects were accounted for by including log popu-
lation per 100,000 in the model as an offset at the linear predictor scale (which is assigned a
coefficient of 1). The model equation can then be rearranged such that ρt is equivalent to the
dengue incidence rate per 100,000 population, (yt / pT’(t)). Then, the most suitable estimate for
ρt was sought via a combination of climate covariates (included as both linear terms at individ-
ual lags and as distributed lagged nonlinear terms) and random effects to account for seasonal-
ity and unmeasured and unknown interannual variability. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 7 / 24 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Climate and dengue risk in Barbados comparing models of increasing complexity, in terms of input variables and model structure, to
the baseline model (including only the monthly random effect to account for seasonality). Posterior predictive distributions and out-of-sample predictions To test the predictive ability of the model, we refitted the model 17 times, removing 1 year at a
time to produce out-of-sample predictions (i.e., 17-fold cross-validation). Note that the annual
random-effect term was included in the model-fitting stage to better quantify the association
between climate factors and variation in dengue incidence rates. However, when producing
out-of-sample predictions, no effect was estimated for the year in which the prediction was
valid, hence the term does not contribute to incidence rate estimates in prediction mode. Therefore, model predictions are based solely on exposure–lag–response functions of key cli-
mate variables and the seasonality term, meaning the model can be used to predict any year
not included in the model-fitting process. To evaluate the output from the selected Bayesian hierarchical model, posterior predictive
distributions of the response variable were simulated using samples from the posterior distri-
bution of the parameters and hyperparameters in the model [44]. The posterior predictive dis-
tribution of dengue cases, yt, for each month (June 1999–May 2016) was estimated by drawing
1,000 random values from a negative binomial distribution with mean corresponding to the
elements of μt and scale parameter corresponding to the elements of the overdispersion
parameter κ, estimated from the models, estimated in 17-fold cross-validation mode. This pro-
cedure allows for uncertainty in the response variable, given the model parameters. We there-
fore generated 204 (17 × 12) out-of-sample posterior predictive distributions and use these to
evaluate the ability of the model to produce probabilistic dengue outbreak predictions given
predefined epidemic thresholds. Model selection and estimation Model parameters were estimated in a Bayesian framework using Integrated Nested Laplace
Approximation (INLA; www.r-inla.org) [39] (see S1 Text for specification of prior and hyper-
prior distributions). The model was estimated in ‘leave one out’ cross-validation mode from
June 1999 to May 2016, i.e., by excluding the month for which the prediction is valid when
estimating model parameters [40]. Exploratory analysis and model selection criteria were used
to select the final model, including the cross-validated (CV) log score [41] and the deviance
information criterion (DIC) [42], for which smaller values indicate better fitting models. An
R2
LR statistic for mixed-effects models based on a likelihood ratio test between the candidate
model, and an intercept-only model was also formulated [43], where 0 R2
LR 1, with R2
LR =
1 corresponding to a perfect fit and R2
LR 0 for any reasonable model specification. R2
LR is
useful as a measure of goodness-of-fit and provides an intuitive measure of the ability of the
model to account for the variation in the dependent variable. The final model was selected by PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 8 / 24 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Model selection As part of the model selection procedure, a range of climate variables were tested, first as linear
terms for individual lags from 0 to 5 months and second as exposure–lag functions for individ-
ual climate indicators (see S3 Table, S4 Table and S5 Table). Key variables were tested with and
without the interannual term to understand the relative contribution of each term to dengue
incidence rate variations. The best estimate of log(ρt) comprised an intercept, α, monthly, βt’(t),
and yearly, γT’(t), random effects (to account for seasonality and unmeasured interannual vari-
ability) and nonlinear exposure–lag functions f.w(xit, l) of the SPI-6, x1t, and Tmin, x2t, with
lags, l, from 0 to 5 months. ð2Þ logðrtÞ ¼ a þ bt0ðtÞ þ gT0ðtÞ þ f:wðx1t; lÞ þ f:wðx2t; lÞ
ð2Þ Table 1 shows model adequacy statistics for a series of models of increasing complexity. Firstly,
a baseline model was fitted including a monthly random effect (Model 1), which accounted for
23% of the variation in dengue relative risk. Next, a yearly random effect was included (Model
2), which explained an additional 31% of the variation in addition to the annual cycle term. Exposure–lag–response functions for the selected climate factors, SPI-6, and Tmin were then
added to the baseline model (individually in Model 3 and 4 and together in Model 5). This
showed that 24% of the interannual variation could be explained by the key selected climate vari-
ables. The final selected model included monthly and yearly random effects and exposure–lag–
response functions for both the SPI-6 and Tmin (Model 6), explaining—overall—68% of the var-
iation in dengue incidence rates. Therefore, in addition to climate factors, yearly random effects
accounted for an extra 21% of the variation in dengue incidence rates. Climate and dengue risk in Barbados threshold as the point on the curve closest to the point of perfect discrimination (0, 1). Perfor-
mance measures, including the hit rate (probability of detection), false alarm rate, and proportion
of correct predictions, were then calculated for both the final model and the baseline model. threshold as the point on the curve closest to the point of perfect discrimination (0, 1). Perfor-
mance measures, including the hit rate (probability of detection), false alarm rate, and proportion
of correct predictions, were then calculated for both the final model and the baseline model. Model evaluation Dengue control programmes often monitor new cases against historical case data, which
define a typical dengue transmission season [45]. While various methods for calculating epi-
demic thresholds are available, we chose the monthly moving thresholds of the upper quartile
(75th percentile) of the distribution of observed dengue incidence. This is in line with the epi-
demic threshold methods described by the World Health Organization [45]. Prediction mod-
els that can detect outbreaks are crucial because they provide information to public health
decision-makers who can advocate for increased disease control interventions when necessary
[46]. To assess the performance of the final model against current practice, we considered the
ability of both the final model and the baseline model (a submodel of the final model based
only on the seasonality term) to detect outbreaks in Barbados. An outbreak threshold was cal-
culated as the 75th percentile of the distribution of dengue cases per month between June 1999
and May 2016, excluding the ‘dengue’ year for which the prediction was valid. The probability
of exceeding the moving outbreak threshold was mapped and verified with actual exceedance
data. To determine an optimum decision trigger threshold for issuing outbreak alerts, we cal-
culated a relative (or receiver) operating characteristic (ROC) curve for the binary events of
exceeding the moving outbreak threshold. The ROC is a graph of the hit rate against the false
alarm rate for varying decision trigger thresholds [47]. The hit rate represents the proportion
of events that occurred (i.e., outbreaks) that were correctly predicted (also known as the true
positive rate or sensitivity). The false alarm rate represents the proportion of events that were
predicted to occur but did not occur (also known as the false positive rate or 1-specificity). We
calculated the curve using probabilistic out-of-sample predictions of exceeding the moving
outbreak threshold compared with the observed binary outcome. We defined a trigger alert PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 9 / 24 Climate and dengue risk in Barbados i ti
f
diff
t
i
h
i Fi 3 Th
l ti
i k i di
l
d
Fig 2. Relative risk of dengue given climatic exposures and time lags. Three-dimensional (upper panel) and contour (lower panel) plots of the exposure–lag–response
association between (left) SPI-6 relative to normal conditions (SPI-6 = 0) and (right) mean temperature anomalies relative to Tmin of 20˚C, at lags between 0 and 5
months. SPI-6, 6-month Standardised Precipitation Index; Tmin, minimum temperature. https://doi.org/10.1371/journal.pmed.1002613.g002 Fig 2. Relative risk of dengue given climatic exposures and time lags. Three-dimensional (upper panel) and contour (lower panel) plots of the exposure–lag–response
association between (left) SPI-6 relative to normal conditions (SPI-6 = 0) and (right) mean temperature anomalies relative to Tmin of 20˚C, at lags between 0 and 5
months. SPI-6, 6-month Standardised Precipitation Index; Tmin, minimum temperature. https://doi org/10 1371/journal pmed 1002613 g002 Fig 2. Relative risk of dengue given climatic exposures and time lags. Three-dimensional (upper panel) and contour (lower panel) plots of the exposure–lag–response
association between (left) SPI-6 relative to normal conditions (SPI-6 = 0) and (right) mean temperature anomalies relative to Tmin of 20˚C, at lags between 0 and 5
months. SPI-6, 6-month Standardised Precipitation Index; Tmin, minimum temperature. https://doi.org/10.1371/journal.pmed.1002613.g002 Fig 2. Relative risk of dengue given climatic exposures and time lags. Three-dimensional (upper panel) and contour (lower panel) plots of the exposure–lag–response
association between (left) SPI-6 relative to normal conditions (SPI-6 = 0) and (right) mean temperature anomalies relative to Tmin of 20˚C, at lags between 0 and 5
months. SPI-6, 6-month Standardised Precipitation Index; Tmin, minimum temperature. Fig 2. Relative risk of dengue given climatic exposures and time lags. Three-dimensional (upper panel) and contour (lower panel) plots of the exposure–lag–response
association between (left) SPI-6 relative to normal conditions (SPI-6 = 0) and (right) mean temperature anomalies relative to Tmin of 20˚C, at lags between 0 and 5
months. SPI-6, 6-month Standardised Precipitation Index; Tmin, minimum temperature. https://doi.org/10.1371/journal.pmed.1002613.g002 https://doi.org/10.1371/journal.pmed.1002613.g002 response associations for different scenarios are shown in Fig 3. The relative risk is displayed
as differences from the relative risk when SPI-6 = 0 (i.e., normal category, see S2 Table) and
Tmin = 20˚C (e.g., the lowest Tmin observed over the time period). The relative risk of dengue
is greatest at short lead times (between 1 and 2 months) for exceptionally wet conditions (Fig 2
and Fig 3A). Climate–dengue associations Associations between the selected climate variables (SPI-6 and Tmin) and dengue relative risk
are presented as three-dimensional graphs and two-dimensional contour plots in Fig 2. Lag– Table 1. Model adequacy results for models of increasing complexity. The CV mean logarithmic score, the DIC, and the likelihood ratio RLR
2 statistic for models of
increasing complexity. Model
log(ρt)
CV log score
DIC
RLR
2
1
α + βt’(t)
Baseline (seasonal random effect)
4.46
1,801.36
0.23
2
α + βt’(t) + γT’(t)
Seasonal + interannual random effect
4.23
1,719.46
0.54
3
α + βt’(t) + f.w(x1t, l)
Seasonal + function of SPI-6
4.32
1,759.07
0.41
4
α + βt’(t) + f.w(x2t, l)
Seasonal + function of Tmin
4.34
1,770.52
0.34
5
α + βt’(t) + f.w(x1t, l) + f.w(x2t, l)
Seasonal + functions of SPI-6 + Tmin
4.28
1,742.98
0.47
6
α + βt’(t) + γT’(t) + f.w(x1t, l) + f.w(x2t, l)
Final (seasonal + interannual + climate functions)
4.09
1,664.94
0.68
Abbreviations: CV cross validated; DIC deviance information criterion; SPI 6 6 month Standardised Precipitation Index; Tmin minimum temperature ncreasing complexity. The CV mean logarithmic score, the DIC, and the likelihood ratio RLR
2 statistic for models of PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 10 / 24 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Climate and dengue risk in Barbados Fig 3. Lag–response for extreme climatic scenarios. Lag–response association for scenarios of (a) SPI-6: exceptionally dry (SPI-6 = −2.5) and exceptionally wet (SPI-
6 = 2.5) conditions relative to the baseline (SPI-6 = 0) and (b) Tmin: Tmin = 21.5˚C and Tmin = 25.5˚C relative to the baseline (Tmin = 20˚C), at lags between 0 and 5
months. SPI-6, 6-month Standardised Precipitation Index; Tmin, minimum temperature. https://doi.org/10.1371/journal.pmed.1002613.g003 Fig 3. Lag–response for extreme climatic scenarios. Lag–response association for scenarios of (a) SPI-6: exceptionally dry (SPI-6 = −2.5) and exceptionally wet (SPI-
6 = 2.5) conditions relative to the baseline (SPI-6 = 0) and (b) Tmin: Tmin = 21.5˚C and Tmin = 25.5˚C relative to the baseline (Tmin = 20˚C), at lags between 0 and 5
months. SPI-6, 6-month Standardised Precipitation Index; Tmin, minimum temperature. Fig 3. Lag–response for extreme climatic scenarios. Lag–response association for scenarios of (a) SPI-6: exceptionally dry (SPI-6 = −2.5) and exceptionally wet (SPI-
6 = 2.5) conditions relative to the baseline (SPI-6 = 0) and (b) Tmin: Tmin = 21.5˚C and Tmin = 25.5˚C relative to the baseline (Tmin = 20˚C), at lags between 0 and 5
months. SPI-6, 6-month Standardised Precipitation Index; Tmin, minimum temperature. https://doi.org/10.1371/journal.pmed.1002613.g003 https://doi.org/10.1371/journal.pmed.1002613.g003 However, exceptionally dry conditions are associated with increased dengue rela-
tive risk 5 months later. Dengue relative risk is greatest 2 to 3 months after higher Tmin values
(i.e., 25.5˚C) (Fig 2 and Fig 3B). The cumulative exposure–response association across all lags
for SPI-6 and Tmin show that, overall, moderately dry and warmer conditions contribute
more to the relative risk (see S6 Fig). response associations for different scenarios are shown in Fig 3. The relative risk is displayed
as differences from the relative risk when SPI-6 = 0 (i.e., normal category, see S2 Table) and
Tmin = 20˚C (e.g., the lowest Tmin observed over the time period). The relative risk of dengue
is greatest at short lead times (between 1 and 2 months) for exceptionally wet conditions (Fig 2
and Fig 3A). However, exceptionally dry conditions are associated with increased dengue rela-
tive risk 5 months later. Dengue relative risk is greatest 2 to 3 months after higher Tmin values
(i.e., 25.5˚C) (Fig 2 and Fig 3B). The cumulative exposure–response association across all lags
for SPI-6 and Tmin show that, overall, moderately dry and warmer conditions contribute
more to the relative risk (see S6 Fig). PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 11 / 24 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Detecting outbreaks Moving outbreak thresholds were calculated as the 75th percentile of the distribution of den-
gue cases per month between June 1999 and May 2016, excluding the ‘dengue’ year for which
the prediction was valid. We applied this moving threshold to produce out-of-sample probabi-
listic predictions of exceeding the outbreak threshold for all 17 years (June 1999–May 2016) as
a demonstration. Fig 4 shows time series of observed and out-of-sample predicted dengue incidence rates
(per 100,000 population) from May 1999 to June 2016. Posterior predictive mean and the
upper 95% prediction (credible) interval from the final model (fitted 17 times in cross-valida-
tion mode, excluding 1 dengue year at a time), and the moving outbreak threshold (75th per-
centile of observed dengue incidence rates per month, excluding the year for which the
prediction is valid) are also included (note, a scatter plot of observed versus posterior predic-
tive mean dengue incidence rates from the final model is shown in S7 Fig; for comparison,
posterior predictive mean dengue incidence rates from the baseline model are also shown). Fig
5 shows the probability of exceeding the moving outbreak threshold. For each time step, the
proportion of the 1,000 samples simulated from the final model that exceeded the moving out-
break threshold was calculated. The deeper the colour, the greater the predicted probability of
exceeding the outbreak threshold. Observed exceedance events are marked with a cross. The
model was able to correctly forecast peak months and distinguish between outbreak and non-
outbreak years, except in the last 2 dengue seasons (2014–2015, 2015–2016). The model simu-
lated a high probability (expressed as deeper colours) of exceeding the threshold in 2007–2008
and 2010–2011, and also in 2013 (Fig 5) when peaks occurred either earlier or later than
expected (Fig 4). For nonoutbreak years (e.g., 2004–2005 and 2005–2006), the model correctly
predicted a very low probability of outbreaks (depicted by pale colours). PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 12 / 24 Climate and dengue risk in Barbados Fig 4. Predicted versus observed dengue incidence rates. Posterior predicted mean (dashed purple curve) and 95%
prediction interval (shaded area) for dengue incidence rates (per 100,000 population) in Barbados from June 1999 to
May 2016, simulated from the final model (refitted 17 times, leaving out 1 year at a time). Observed values (solid
orange curve) and moving outbreak threshold (blue dotted curve) are included. Detecting outbreaks Year labels are included at the start of
each calendar year (e.g., in January). https://doi org/10 1371/journal pmed 1002613 g004 Fig 4. Predicted versus observed dengue incidence rates. Posterior predicted mean (dashed purple curve) and 95%
prediction interval (shaded area) for dengue incidence rates (per 100,000 population) in Barbados from June 1999 to
May 2016, simulated from the final model (refitted 17 times, leaving out 1 year at a time). Observed values (solid
orange curve) and moving outbreak threshold (blue dotted curve) are included. Year labels are included at the start of
each calendar year (e.g., in January). The outbreak detection performance of the model is summarised in Table 2, which shows
ROC and contingency table results for observed dengue cases exceeding the moving outbreak
threshold. As an indicator of the quality of the forecasting system, the area under the ROC
curve (AUC) was calculated as 0.90 for the final model. This indicates that the forecasting sys-
tem performs significantly better than randomly guessing (AUC = 0.5). For the baseline
model, AUC = 0.75 (see Table 2 and S8 Fig). The ‘optimal’ cut-off value that maximised sensi-
tivity and specificity for the moving outbreak threshold was 30% for the final model (27% for
the baseline model). Using this trigger alert threshold, we looked at a binary classification of
predicted and observed outbreaks. The final model produced more hits and fewer missed
events than the baseline model, with a hit rate (probability of detection) of 90% (miss rate =
10%) for the final model, compared with 79% (miss rate = 21%) for the baseline model. The
final model also had a lower false alarm rate of 31% compared with 57% for the baseline
model. Overall, the proportion of correctly predicted outbreaks and nonoutbreaks was 86%
(see Table 2). Climate and dengue risk in Barbados Fig 5. Predicted probability of exceeding moving outbreak thresholds. Probability of exceeding the moving outbreak
threshold (75th percentile of observed dengue cases per month, excluding the year for which the prediction is valid) from
June 1999 to May 2016, using out-of-sample predictive distributions simulated from the final model. The graduated colour
bar represents the predicted probability of observing an outbreak (ranging from 0, pale colours, to 1, deep colours). Months
in which the moving outbreak threshold was exceeded are marked with a cross. Note: the ‘dengue season’ year runs from
June to May. Fig 5. Predicted probability of exceeding moving outbreak thresholds. Probability of exceeding the moving outbreak
threshold (75th percentile of observed dengue cases per month, excluding the year for which the prediction is valid) from
June 1999 to May 2016, using out-of-sample predictive distributions simulated from the final model. The graduated colour
bar represents the predicted probability of observing an outbreak (ranging from 0, pale colours, to 1, deep colours). Months
in which the moving outbreak threshold was exceeded are marked with a cross. Note: the ‘dengue season’ year runs from
June to May. Fig 5. Predicted probability of exceeding moving outbreak thresholds. Probability of exceeding the moving outbreak
threshold (75th percentile of observed dengue cases per month, excluding the year for which the prediction is valid) from
June 1999 to May 2016, using out-of-sample predictive distributions simulated from the final model. The graduated colour
bar represents the predicted probability of observing an outbreak (ranging from 0, pale colours, to 1, deep colours). Months
in which the moving outbreak threshold was exceeded are marked with a cross. Note: the ‘dengue season’ year runs from
June to May. Fig 5. Predicted probability of exceeding moving outbreak thresholds. Probability of exceeding the moving outbreak
threshold (75th percentile of observed dengue cases per month, excluding the year for which the prediction is valid) from
June 1999 to May 2016, using out-of-sample predictive distributions simulated from the final model. The graduated colour
bar represents the predicted probability of observing an outbreak (ranging from 0, pale colours, to 1, deep colours). Months
in which the moving outbreak threshold was exceeded are marked with a cross. Note: the ‘dengue season’ year runs from
June to May. https://doi.org/10.1371/journal.pmed.1002613.g005 https://doi.org/10.1371/journal.pmed.1002613.g005 dengue outbreak. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Discussion Drought conditions were found to positively influence dengue incidence rates at longer lead
times up to 5 months, while excess rainfall increased the risk at shorter lead times between 1
and 2 months. Therefore, the modelling results suggest that a drought period followed by
intense rainfall 4 to 5 months later could provide optimum conditions for an imminent 13 / 24 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 https://doi.org/10.1371/journal.pmed.1002613.t002 Climate and dengue risk in Barbados temperature. Furthermore, including bidimensional exposure–lag–response functions of these
indicators—rather than linear effects for individual lags—more appropriately described the cli-
mate–disease associations than traditional modelling approaches. To our knowledge, DLNM
methodology has not previously been combined with a Bayesian hierarchical mixed-modelling
framework to produce probabilistic predictions of exceeding dengue outbreak thresholds. To
demonstrate the added value of our climate-driven dengue model, we formulated a baseline
model to represent current practice (i.e., monitoring dengue cases throughout the year against
historic seasonal averages). A probability trigger threshold was calculated by using the ROC
curve to select an optimal cut-off value that maximised sensitivity and specificity for the mov-
ing outbreak threshold of the upper quartile of the observed dengue distribution per month. The model successfully distinguished outbreaks from nonoutbreaks, except in the last 2 den-
gue seasons (2014–2015, 2015–2016), with an overall proportion of correct predictions (hits
and correct rejections) of 86% compared with 64% for the baseline model. The observed effect of Tmin on dengue transmission is consistent with findings from prior
studies in the Americas, which also found that Tmin was a better predictor of dengue trans-
mission than mean or maximum temperature [48–52]. Tmin appears to be a key regulating cli-
mate parameter for dengue. In Barbados, high values of Tmin were 25.5˚C, the lower end of
the optimal temperature range for endemic dengue transmission (26–29˚C) [13]. We hypothesise that the effect of excess rainfall and drought on dengue risk operated at dif-
ferent time scales due to different mechanisms associated with the availability of larval habitat
and water storage in the urban environment. Following a rainfall event, the availability of larval
habitat increases (e.g., rain-filled abandoned containers, rubbish), and within a relatively short
period of time, eggs hatch and adult mosquito densities increase (i.e., approximately 2–3
weeks after rainfall, depending on ambient temperatures). Subsequently, the risk of arbovirus
infections increases several weeks later, a lag associated with the intrinsic and extrinsic viral
incubation periods. In contrast, we observed that drought conditions, as determined by the SPI-6, were associ-
ated with dengue risk over longer periods of time (5-month lag). Barbados is among the 10
most water-scarce countries in the world [34], and water scarcity is exacerbated during periods
of drought. Drought is a slow-onset climate hazard. The use of the SPI-6, designed to monitor drought and extreme rainfall, bet-
ter explained variations in dengue risk than summary statistics of measured precipitation. dengue outbreak. The use of the SPI-6, designed to monitor drought and extreme rainfall, bet-
ter explained variations in dengue risk than summary statistics of measured precipitation. T
i
l i
d
i i
i d
i
id
h
i p
g
y
p
p
Tmin explained more variation in dengue incidence rates than mean or maximum p
g
y
p
p
Tmin explained more variation in dengue incidence rates than mean or maximum Table 2. Sensitivity and specificity results for observed dengue incidence rates exceeding the moving outbreak
threshold. Summary of ROC and contingency table analysis for observed dengue incidence rates exceeding the mov-
ing outbreak threshold (75th percentile of observed dengue cases per month, excluding the year for which the predic-
tion is valid) using out-of-sample probabilistic predictive distributions from the final and baseline models (95% CIs
included in parentheses). Performance measures
Final model
Baseline model
AUC
0.9 (0.85–0.94)
0.75 (0.71–0.79)
Probability trigger threshold
0.3
0.27
Hit rate
0.9 (0.82–0.97)
0.79 (0.69–0.9)
False alarm rate
0.31 (0.22–0.39)
0.57 (0.51–0.63)
Proportion correct
0.86 (0.81–0.91)
0.64 (0.58–0.71)
Abbreviations: AUC, area under the ROC curve; ROC, relative (receiver) operating characteristic. https://doi org/10 1371/journal pmed 1002613 t002 Table 2. Sensitivity and specificity results for observed dengue incidence rates exceeding the moving outbreak
threshold. Summary of ROC and contingency table analysis for observed dengue incidence rates exceeding the mov-
ing outbreak threshold (75th percentile of observed dengue cases per month, excluding the year for which the predic-
tion is valid) using out-of-sample probabilistic predictive distributions from the final and baseline models (95% CIs
included in parentheses). Performance measures
Final model
Baseline model
AUC
0.9 (0.85–0.94)
0.75 (0.71–0.79)
Probability trigger threshold
0.3
0.27
Hit rate
0.9 (0.82–0.97)
0.79 (0.69–0.9)
False alarm rate
0.31 (0.22–0.39)
0.57 (0.51–0.63)
Proportion correct
0.86 (0.81–0.91)
0.64 (0.58–0.71)
Abbreviations: AUC, area under the ROC curve; ROC, relative (receiver) operating characteristic. 14 / 24 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018
14 / PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 20 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Climate and dengue risk in Barbados Barbados. Ultimately, the effects of rainfall on dengue transmission depend on the local socioe-
cological conditions that determine the types of larval habitat available in the environment, as
well as water access and storage. In recent decades, the Caribbean has experienced several drought events. Notable events
during our study period include 2002–2003, 2009–2010, and 2015–2016. Drought and water
scarcity are projected to increase in the future due to climate change [54]. To address this situ-
ation, Barbados passed building regulations that mandate the construction of rainwater stor-
age receptacles under large new buildings. These receptacles have become ideal larval habitat
for A. aegypti. Although these measures were intended to increase the capacity of the country
to adapt to climate change, they may have had the unintended consequence of increasing the
overall risk of arboviral diseases during any time when rainfall is collected and stored. In
response, the Ministry of Health has issued recommendations to adequately cover all water
storage receptacles and, if necessary, apply screen mesh material to all entry sites for small and
large cisterns. Presently, the Ministry of Health is working on producing Safe Water Storage
Guidelines as a reference for the public to reduce the risk of mosquito-borne diseases. This
example highlights the need for intersectoral collaboration to address the complexities of cli-
mate and health in Barbados and in other SIDS. In Barbados, over the last 5 years, there have been 3 outbreaks of dengue fever (2013–2014,
2014–2015, and 2015–2016), 1 outbreak of chikungunya virus (2014–2015), and 1 outbreak of
Zika virus (2015–2016). Better management of arboviruses is a high priority for Caribbean
SIDS given the burden of A. aegypti–transmitted arboviruses in the region, suitable climatic
conditions for vector proliferation, and susceptible human populations, as well as the potential
for transmission via widespread intra- and extraregional travel in the Caribbean [55]. To
address this need, the CIMH—as part of its implementation of a regional multisectoral Early
Warning Information Systems Across Climate Timescales (EWISACTs) programme—has
partnered with the Caribbean Public Health Agency (CARPHA), national Ministries of Health,
National Meteorological and Hydrological Services, and an international research team to sup-
port the development of regional arbovirus forecast models using climate information. This
programme aims to codesign, codevelop, and codeliver sector-driven climate early warning
information. The depletion of surface water reservoirs
occurs over 6 or more months, and the depletion of groundwater reservoirs occurs at 9- to
12-month time scales. As households are alerted to water scarcity, they take measures to store
water in containers around the home, increasing the availability of larval habitat for A. aegypti. As a result, we hypothesise that household water storage during drought periods may increase
the risk of dengue transmission. Alternatively, we hypothesise that the increase in containers
in the environment during drought may increase the risk of dengue transmission months
later, when the rainy season begins and water storage is no longer a necessity. Prolonged
drought could result in changes in human behaviour associated with water storage, habits that
could extend beyond the period of water scarcity. In our experience, water storage containers
become a greater hazard for dengue when they are not used and maintained regularly and
instead serve as a ‘backup’ water source [14]. When a drought is followed by heavy rainfall, the
risk of dengue could increase, as observed in 2010–2011, a year of exceptionally high dengue
transmission across the Americas and an El Niño event. That year, DENV serotypes 1 and 4
were re-introduced after no circulation for several years, and an outbreak occurred with all 4
serotypes in cocirculation (2,000 suspected cases, 575 confirmed cases in Barbados) [5]. To our knowledge, there is little known about the effects of prolonged drought on dengue
transmission. Some studies have shown that rainfall shortages can increase dengue risk in
regions where people store water [14,15,53]. However, entomological and epidemiological
field studies are needed to elucidate the mechanisms linking drought to dengue risk in PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 15 / 24 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 The first forecast would be issued 3 months ahead of the target (i.e., 3-month lead
time), in July, using observed climate data (for SPI-6 and Tmin) for May and June and forecast climate data for July, August,
September, and October. The forecast would then be updated in August (i.e., 2-month lead time) using observed climate for
July and updated climate forecasts for August, September, and October. In September, 1 month before the target (i.e.,
1-month lead time), the dengue forecast would be updated again, using observed climate from May to August and updated
climate forecasts for September and October. SPI-6, 6-month Standardised Precipitation Index; Tmin, minimum
temperature. https://doi.org/10.1371/journal.pmed.1002613.g006 should reduce forecast uncertainty as one more month of climate observations becomes avail-
able and one fewer month of climate forecasts is needed. CariCOF’s SPI-6-based drought out-
looks have a prediction lead time of 3 months. For example, CariCOF’s SPI-6-based drought
outlook issued in July, which estimates the drought situation by the end of October, could be
used to predict the dengue risk in October (target month). Observed SPI-6 data for May (i.e.,
calculated from the observed rainfall totals from December of the previous year to May of the
current year) and June (i.e., 4 and 5 months before the target month, to capture the long-lag
impacts on dengue risk) would be used along with forecast SPI-6 conditions from July to
October (i.e., lags of 0–3 months with respect to the target month). This would produce a
3-month lead dengue forecast (see Fig 6). In August, the dengue forecast for October could be
updated—using the new observed SPI-6 value for July and updated SPI-6 forecasts for August
(i.e., an SPI-6 calculated from the sum of observed rainfall totals from March to July and fore-
cast rainfall totals for August) to October—to produce a 2-month-ahead dengue forecast for
October (Fig 6), and so on. The dengue model parameter estimates could be updated on an
annual basis as new dengue incidence and climate data become available. l f
d l
b
d
h
k f d
b
k b Seasonal forecast models can contribute to reducing the risk of disease outbreaks by
increasing preparation time to better allocate scarce resources. To date, there are few localised
studies in this region that quantify the associations between subseasonal to seasonal forecasts
of climate and dengue risk outcomes. Climate and dengue risk in Barbados Fig 6. The type of climate information required for a dengue forecast in a given target month. Schematic to show the
type (e.g., observed or forecast) of climate information (e.g., SPI-6 and Tmin variables, lags 0 to 5) used to produce a dengue
forecast for the target month of October. The first forecast would be issued 3 months ahead of the target (i.e., 3-month lead
time), in July, using observed climate data (for SPI-6 and Tmin) for May and June and forecast climate data for July, August,
September, and October. The forecast would then be updated in August (i.e., 2-month lead time) using observed climate for
July and updated climate forecasts for August, September, and October. In September, 1 month before the target (i.e.,
1-month lead time), the dengue forecast would be updated again, using observed climate from May to August and updated
climate forecasts for September and October. SPI-6, 6-month Standardised Precipitation Index; Tmin, minimum
temperature. https://doi org/10 1371/journal pmed 1002613 g006 Fig 6. The type of climate information required for a dengue forecast in a given target month. Schematic to show the
type (e.g., observed or forecast) of climate information (e.g., SPI-6 and Tmin variables, lags 0 to 5) used to produce a dengue
forecast for the target month of October. The first forecast would be issued 3 months ahead of the target (i.e., 3-month lead
time), in July, using observed climate data (for SPI-6 and Tmin) for May and June and forecast climate data for July, August,
September, and October. The forecast would then be updated in August (i.e., 2-month lead time) using observed climate for
July and updated climate forecasts for August, September, and October. In September, 1 month before the target (i.e.,
1-month lead time), the dengue forecast would be updated again, using observed climate from May to August and updated
climate forecasts for September and October. SPI-6, 6-month Standardised Precipitation Index; Tmin, minimum
temperature. Fig 6. The type of climate information required for a dengue forecast in a given target month. Schematic to show the
type (e.g., observed or forecast) of climate information (e.g., SPI-6 and Tmin variables, lags 0 to 5) used to produce a dengue
forecast for the target month of October. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 One avenue for communicating warnings of dengue risk is through the Carib-
bean Health-Climatic Bulletin, a new joint quarterly bulletin of the CARPHA, the Pan-Ameri-
can Health Organization, and the CIMH [56]. The bulletin offers consensus-based expert
statements of probable health outcomes extrapolated from a review of the CIMH’s suite of
monitoring and forecast products contextualised to the Caribbean health context up to 3 to 6
months in advance. Integration of quantitative probabilistic forecasts of dengue risk generated
by the modelling framework described in this paper represents a significant emerging
research-to-operations advance that has the potential to respond to the health community’s
need for empirical risk estimates for periods of operational relevance to public health. Quanti-
tative probabilistic forecasts of disease risk will enhance the quality of warning information
and provide improved guidance for decision-making. Climate anomalies in the Caribbean are associated with tropical Atlantic and Pacific sea
surface temperatures, which are usually well predictable at seasonal time scales [57]. This gives
useful predictive skill to regional and seasonal climate forecast models, especially seasonal tem-
perature and SPI-based outlooks with up to 6 months lead time. Previous studies have shown
that seasonal climate forecasts combined with dengue risk prediction models can extend pre-
dictive lead time from a few months to nearly a year [36,58], thus indicating the potential to
develop public health interventions based on climate information in the Caribbean. In practice, the CIMH could run this dengue model using their seasonal climate forecast
products. Results could be updated on a monthly basis as the target month is approached. This PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 16 / 24 Climate and dengue risk in Barbados variance in the predictions includes uncertainty that could be generated by these unknown fea-
tures of the disease system. However, while yearly effects are useful to identify missing infor-
mation and better quantify the contribution of explanatory variables to the relative risk, they
cannot contribute to out-of-sample predictions unless prior assumptions about the year ahead
can be made. The model failed to predict the peak and magnitude of the last 2 dengue transmission sea-
sons, which coincided with the first epidemics of chikungunya and Zika. The emergence of
these viruses, which are transmitted by the same mosquito vector, likely altered diagnostic,
reporting, and healthcare-seeking practices, and we suspect that this might have affected the
model performance for the last 2 dengue seasons in 2015 and 2016. This may also be true in
other countries and territories across Latin America and the Caribbean during this time
period. Dengue virus, chikungunya virus, and Zika virus have a similar clinical presentation
(e.g., mild febrile illness, rash, joint pain), and differential diagnosis is challenging without
molecular diagnostics (e.g., PCR) due to cross-reactivity of the serological assays. Only after
September 2016 was Barbados able to test suspected arbovirus infections using a triplex PCR;
prior to then, a subset of samples was sent for confirmation to CARPHA’s diagnostic labora-
tory in Trinidad and Tobago. The emergence of Zika virus may have also changed healthcare-
seeking behaviour, with more women seeking diagnostics for mild febrile illness due to con-
cerns about Zika congenital syndrome [37], resulting in an increase in the detection of dengue
and Zika infections in women. The cocirculation of dengue, chikungunya, and Zika in the
region poses a challenge for forecasting future risk, unless high-quality seroprevalence data
can be incorporated in the model [36]. Overall, the final model had a lower false alarm rate and a lower miss rate than the baseline
model. However, the model did result in false alarms 31% of the time. Repeatedly issuing false
alarms can result in a lack of trust from the general public. However, in practice, false alarms
(i.e., issuing a high probability alert of a dengue outbreak) can, in fact, result from effective
interventions in response to the alert. Furthermore, when dealing with detrimental health
events, such as disease outbreaks, false alarms are preferable to missed events. The objective of this study was to select a model to infer delayed and nonlinear impacts of
climate on dengue risk and predict the probability of exceeding an outbreak threshold relevant
to the Ministry of Health in Barbados and other islands in the Caribbean. Forecasts of the prob-
ability of exceeding outbreak thresholds allow decision-makers to quantify the level of certainty
of the model predictions. However, when using predefined outbreak and alarm trigger thresh-
olds, the translation of probabilities into discrete warnings might not always reflect the predic-
tive power of the model [46]. Outbreak and alarm trigger thresholds need to be carefully chosen
to reflect the capacity of the public health system to respond to imminent outbreaks. In the absence of a robust evidence base, the operational
use of climate information for predicting increased dengue risk remains an untapped opportu-
nity to support climate-driven dengue early warning information systems. This is an area of
further investigation, along with the transferability of this framework to model and predict the
risk of dengue and other arboviruses in other Caribbean countries. There are several limitations of the study. In Barbados, the absolute number of dengue
cases across the island is relatively small, even in outbreak years (i.e., 1,140 confirmed cases in
2013, 488 confirmed cases in 2014, and 587 confirmed cases in 2016). The time series is rela-
tively long compared with previously studied dengue records in other countries. However, it is
still limited for statistical modelling purposes because our knowledge of outbreak dynamics is
based on the behaviour of only a few outbreaks. Ideally, data on vector control activities and
other interventions or policy changes would be included in the model. In the absence of such
data, monthly and yearly random effects are included in the model to try and ensure that the PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 17 / 24 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Conclusion We are working towards implementing a climate-based early warning system for arboviral dis-
eases in all Caribbean islands and territories. Not only does this study contribute to enriching
the knowledge base, it represents a considerable opportunity to translate investment in health–
climate research into practice to improve national and regional health outcomes. The goal is to
enhance the health warnings issued in the quarterly Caribbean Health-Climatic Bulletin with
quantitative probabilistic forecasts of disease risk rather than expert statements on probable
health outcomes. Integration of an early warning information product into national and sec-
toral planning and practice has the potential to reverse the upward trend of new infections of
arboviral diseases, which currently undermines the productivity and sustainable development
of SIDS in the Caribbean. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 18 / 24 Climate and dengue risk in Barbados Supporting information S1 Fig. Site map and location of meteorological stations in Barbados. Map of Caribbean
(left) and Barbados showing population (middle) and elevation (right) and the locations of the
2 main meteorological stations (CIMH and GAIA). This figure was created in ArcGIS version
10.3.1 [59] using shapefiles from the GADM database of Global Administrative Areas version
2.8, freely available at gadm.org [60]. CIMH, Caribbean Institute for Meteorology and Hydrol-
ogy; GAIA, Grantley Adams International Airport. (TIF) S2 Fig. Smoothed rainfall seasonality at the CIMH synoptic station in Barbados. The
chance of a wet day (i.e., a calendar day with >0.85 mm; left panel) and the average rainfall
intensity on a wet day for each Julian day of the year (right panel). Source: modified from Trot-
man and colleagues [20]. CIMH, Caribbean Institute for Meteorology and Hydrology. (TIF) S3 Fig. Annual cycle of dengue incidence rates and meteorological station data. Annual
cycle of (a) dengue incidence rate (per 100,000 population), (b) precipitation (mm/month),
and (c) Tmin (˚C) averaged over CIMH and GAIA weather stations. CIMH, Caribbean Insti-
tute for Meteorology and Hydrology; GAIA, Grantley Adams International Airport; Tmin,
minimum temperature. (TIF) S4 Fig. Annual cycle of the SPI-6 and the Oceanic Niño Index. Annual cycle of (a) SPI-6 and
(b) Oceanic Niño Index, defined as the 3-month running-mean sea surface temperature depar-
tures from average in the Niño 3.4 region (120–170˚ W, 5˚ S-5˚ N), from June 1999 to May
2016. SPI-6, 6-month Standardised Precipitation Index. (TIF) S5 Fig. Annual cycle of dengue cases in Barbados during drier than normal, near-normal,
and wetter than normal seasons. Annual cycle of dengue cases given tercile categories of the
SPI-6: drier than normal (solid curve), normal (dashed curve), and wetter than normal (dotted
curve). During wetter than average years, dengue cases tended to peak earlier in the season, in
September, whereas dry years coincided with late-season peaks. SPI-6, 6-month Standardised
Precipitation Index. (TIF) S6 Fig. Overall cumulative exposure–response. Exposure–response association across all lags
(0 to 5 months) for (a) the SPI-6 relative to the baseline SPI-6 = 0 and (b) Tmin relative to the
baseline Tmin = 20˚C. SPI-6, 6-month standardised precipitation index; Tmin, minimum
temperature. (TIF) S7 Fig. Observed versus predicted dengue incidence rates. Observed versus posterior pre-
dicted mean dengue incidence rates (per 100,000 population) from the final model (purple cir-
cles) and the baseline model (red squares). Note: logarithmic scale. PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Acknowledgments The authors are grateful to Trevor Bailey, Hårvard Rue, Ben Armstrong, Charmaine Blades,
Shermaine Clauzel, Steve Daniel, Sally Edwards, Lyndon Forbes Robertson, Adrianus Vlug-
man, Marquita Gittens-St. Hilaire, and Karen Polson-Edwards for valuable expert advice dur-
ing this study. S2 Table. SPI classification used in the Caribbean from January 2011. SPI, Standardised
Precipitation Index.
(DOCX) S2 Table. SPI classification used in the Caribbean from January 2011. SPI, Standardised
Precipitation Index. (DOCX) S3 Table. Model adequacy results for single-lag univariate models. Summary statistics for a
model including monthly and yearly random effects and (a) SPI-6 and (b) Tmin at individual lags
of 0 to 5 months: posterior mean, 95% CIs, the CV mean logarithmic score, the DIC, and the like-
lihood ratio RLR
2 statistic. CI, credible interval; CV, cross-validated; DIC, deviance information
criterion; SPI-6, 6-month Standardised Precipitation Index; Tmin, minimum temperature. (DOCX) S4 Table. SPI variable at different time scales. The CV mean logarithmic score, the DIC, and
the likelihood ratio RLR
2 statistic for models including monthly and yearly random effects and
an exposure–lag–response function with time lags from 0 to 5 months for the SPI averaged
over 1 month, 3 months, 6 months, and 12 months). The best indicator is shaded in grey. CV,
cross-validated; DIC, deviance information criterion; SPI, Standardised Precipitation Index. (DOCX) S4 Table. SPI variable at different time scales. The CV mean logarithmic score, the DIC, and
the likelihood ratio RLR
2 statistic for models including monthly and yearly random effects and
an exposure–lag–response function with time lags from 0 to 5 months for the SPI averaged
over 1 month, 3 months, 6 months, and 12 months). The best indicator is shaded in grey. CV,
cross-validated; DIC, deviance information criterion; SPI, Standardised Precipitation Index. (DOCX) S5 Table. Climate variable selection. The CV mean logarithmic score, the DIC, and the likeli-
hood ratio RLR
2 statistic for models including monthly and yearly random effects and an expo-
sure–lag–response function with time lags from 0 to 5 months for individual climate variables:
precipitation, SPI (1-month, 3-month, 6-month, and 12-month), Tmin, mean temperature,
and maximum temperature. CV, cross-validated; DIC, deviance information criterion; SPI,
Standardised Precipitation Index; Tmin, minimum temperature. (DOCX) S1 Text. Prior and hyperprior distribution specification. (DOCX) Climate and dengue risk in Barbados ‘optimal’ ROC cut-off (alarm trigger threshold), defined as the point on the curve closest to the
point of perfect discrimination (0, 1). Note: false alarms are a desired outcome when the pre-
dicted probability of threshold exceedance is very low. ROC, relative (receiver) operating char-
acteristic. (TIF) ‘optimal’ ROC cut-off (alarm trigger threshold), defined as the point on the curve closest to the
point of perfect discrimination (0, 1). Note: false alarms are a desired outcome when the pre-
dicted probability of threshold exceedance is very low. ROC, relative (receiver) operating char-
acteristic. (TIF) Supporting information (TIF) S8 Fig. ROC curves to define alarm trigger threshold. ROC curve for binary event of dengue
incidence rates exceeding the moving outbreak threshold (75th percentile of observed dengue
incidence rates per month, excluding the year for which the prediction is valid) for (a) final
model (without year effect contribution) and (b) baseline model. Numbers indicate values of
probability thresholds along the curve, and the purple circle indicates the position of an S8 Fig. ROC curves to define alarm trigger threshold. ROC curve for binary event of dengue
incidence rates exceeding the moving outbreak threshold (75th percentile of observed dengue
incidence rates per month, excluding the year for which the prediction is valid) for (a) final
model (without year effect contribution) and (b) baseline model. Numbers indicate values of
probability thresholds along the curve, and the purple circle indicates the position of an PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 19 / 24 Climate and dengue risk in Barbados Formal analysis: Rachel Lowe, Antonio Gasparrini, Ce´dric J. Van Meerbeeck. Funding acquisition: Adrian R. Trotman, Anna M. Stewart-Ibarra. Investigation: Rachel Lowe, Ce´dric J. Van Meerbeeck, Catherine A. Lippi, Roche´ Mahon,
Sadie J. Ryan, Anna M. Stewart-Ibarra. Methodology: Rachel Lowe, Antonio Gasparrini, Ce´dric J. Van Meerbeeck, Roche´ Mahon. Methodology: Rachel Lowe, Antonio Gasparrini, Cedric J. Van Meerbeeck, Roche Mahon. Project administration: Anna M. Stewart-Ibarra. Project administration: Anna M. Stewart-Ibarra. Resources: Ce´dric J. Van Meerbeeck, Roche´ Mahon, Adrian R. Trotman, Avery Q. J. Hinds,
Sadie J. Ryan. Software: Antonio Gasparrini. Software: Antonio Gasparrini. Supervision: Adrian R. Trotman, Sadie J. Ryan. Supervision: Adrian R. Trotman, Sadie J. Ryan. Validation: Rachel Lowe, Ce´dric J. Van Meerbeeck, Roche´ Mahon, Adrian R. Trotman, Leslie
Rollock, Avery Q. J. Hinds, Sadie J. Ryan. Visualization: Rachel Lowe, Catherine A. Lippi. Writing – original draft: Rachel Lowe, Ce´dric J. Van Meerbeeck, Catherine A. Lippi, Roche´
Mahon, Adrian R. Trotman, Anna M. Stewart-Ibarra. Writing – review & editing: Rachel Lowe, Antonio Gasparrini, Ce´dric J. Van Meerbeeck,
Catherine A. Lippi, Roche´ Mahon, Adrian R. Trotman, Leslie Rollock, Avery Q. J. Hinds,
Sadie J. Ryan, Anna M. Stewart-Ibarra. Author Contributions Conceptualization: Rachel Lowe, Antonio Gasparrini, Ce´dric J. Van Meerbeeck, Adrian R. Trotman, Anna M. Stewart-Ibarra. Conceptualization: Rachel Lowe, Antonio Gasparrini, Ce´dric J. Van Meerbeeck, Adrian R. Trotman, Anna M. Stewart-Ibarra. Data curation: Rachel Lowe, Ce´dric J. Van Meerbeeck, Catherine A. Lippi, Leslie Rollock,
Sadie J. Ryan, Anna M. Stewart-Ibarra. Data curation: Rachel Lowe, Ce´dric J. Van Meerbeeck, Catherine A. Lippi, Leslie Rollock,
Sadie J. Ryan, Anna M. Stewart-Ibarra. 20 / 24 PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002613
July 17, 2018 Climate and dengue risk in Barbados 12. Paulo Ortiz L, Rivero A, Linares Y, Pe´rez A, Va´zquez JR. Spatial models for prediction and early warn-
ing of Aedes aegypti proliferation from data on climate change and variability in Cuba. MEDICC Rev. 2015; 17: 20–28. PMID: 26027583 13. Mordecai EA, Cohen JM, Evans MV, Gudapati P, Johnson LR, Lippi CA, et al. Detecting the impact of
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Usefulness and Impact of Big Data in Libraries: An Opportunity to Implement Embedded Librarianship
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Contents Messages
Foreword
Preface
About the Editors ( xix )
Foreword
(xi)
Preface
(xiii)
About the Editors
(xv)
1. Challenges for College Librarians’ during Pandemic at
Paschim Medinipur District of West Bengal: A Study
1
Avijit Dutta and Sanchita Bhattacharya
2. Role of Librarians in Apps Literacy
9
Chauhan Mahammadhafiz Iqbalmiyan
3. Digital Library Initiatives in India Promoting
E-Learning in Changing Technological Innovative World
16
Hafijull Mondal
4. Application of Mobile Technology in Libraries
31
Karishma K. Satarkar
5. Role of E-Resources and Services in Modern Education
38
Tripathi Sarika and Jige Sandipan Babasaheb
6. Usefulness and Impact of Big Data in Libraries:
An Opportunity to Implement Embedded Librarianship
45
Subhajit Panda
7. Digitization and Digital Preservation of Library Resources:
Challenges for the LIS Professionals
61
Sasmita Patra and Jyotshna Sahoo
8. Use of Internet in the Library of University of North Bengal:
A Case Study
70
Shubhrangshu Kumar Mistri 2. Role of Librarians in Apps Literacy
Chauhan Mahammadhafiz Iqbalmiyan 2. Role of Librarians in Apps Literacy
Chauhan Mahammadhafiz Iqbalmiyan 6. Usefulness and Impact of Big Data in Libraries:
An Opportunity to Implement Embedded Librarianship
45
Subhajit Panda 7. Digitization and Digital Preservation of Library Resources:
Challenges for the LIS Professionals
61
Sasmita Patra and Jyotshna Sahoo 8. Use of Internet in the Library of University of North Bengal:
A Case Study
70
Shubhrangshu Kumar Mistri ( xix ) 9. A Comparative Study of LIBSYS & KOHA Software for
Library Automation
Jyoti Sharma and Neha Sharma 79 10. Digital Transformation in Libraries:
A Descriptive Study
86
Neetha Devan NV 10. Digital Transformation in Libraries:
A Descriptive Study
Neetha Devan NV ( xx )
10. Digital Transformation in Libraries:
A Descriptive Study
86
Neetha Devan NV
11. Information Resource Management and Integration Practice in
P.K. Kelkar Library
92
Neha Sharma and Jyoti Sharma*
12. Mapping of Research Output on Plant Physiology
Theses Year 2009-2017: A Case Study of JNKVV, Jabalpur
95
Pradeep Kumar Dixit and Ravindra Kumar
13. Reinventing Libraries for Next-generation Users
104
Suman Kumari
14. Digital Preservation: Transforming Information
Archival and Retrieval in Digital Information Landscape
113
Garima Gujral
15. Security Systems in Libraries & Information Centers
120
Beauty Verma
16. Digital Library-Approach, Strengths, Challenges and Trends
129
Laxmi Mishra
17. E-Resources & Services
133
Pinki Sharma
18. Contents Linked Open Data: the Emerging Paradigm to Enhance the
Discoverability and Interoperability of Information in the field of LIS
142
Biswajit Saha and Rajesh Das
19. Library Consortia Approach: A Necessity at the Time of
Covid-19 Pandemic
177
Kusum Lata Malik
20. Intellectual Property Rights (IPRs) in The World of Digitization:
Opportunities & Challenges
188
Naincy Prajapati and Neelima Chaudhary 86 11. Information Resource Management and Integration Practice in
P.K. Kelkar Library
92
Neha Sharma and Jyoti Sharma* 92 12. Mapping of Research Output on Plant Physiology
Theses Year 2009-2017: A Case Study of JNKVV, Jabalpur
95
Pradeep Kumar Dixit and Ravindra Kumar 17. E-Resources & Services
Pinki Sharma 18. Linked Open Data: the Emerging Paradigm to Enhance the
Discoverability and Interoperability of Information in the field of LIS
142
Biswajit Saha and Rajesh Das 19. Library Consortia Approach: A Necessity at the Time of
Covid-19 Pandemic
177
Kusum Lata Malik ( xx )
20. Intellectual Property Rights (IPRs) in The World of Digitization:
Opportunities & Challenges
188
Naincy Prajapati and Neelima Chaudhary ( xx )
20. Intellectual Property Rights (IPRs) in The World of Digitization:
Opportunities & Challenges
188
Naincy Prajapati and Neelima Chaudhary 21. Application and Trend of Mobile Technology in
Library Science: A Study
195
Nupur Walia
22. Information Needs and Seeking Behaviour of Students:
A Case Study on Students of A Premier Private University
202
Pradyumna Kumar Panigrahi and Ravindra Kumar
23. E-Resources and Services in Modern Era
214
Sanchaita Nath and Nandita Deb 21. Application and Trend of Mobile Technology in
Library Science: A Study
195
Nupur Walia 21. Application and Trend of Mobile Technology in
Library Science: A Study
195
Nupur Walia 21. Application and Trend of Mobile Technology in
Library Science: A Study
Nupur Walia 195 22. Information Needs and Seeking Behaviour of Students:
A Case Study on Students of A Premier Private University
202
Pradyumna Kumar Panigrahi and Ravindra Kumar
23. E-Resources and Services in Modern Era
214
Sanchaita Nath and Nandita Deb 22. Information Needs and Seeking Behaviour of Students:
A Case Study on Students of A Premier Private University
202
Pradyumna Kumar Panigrahi and Ravindra Kumar 202 214 23. E-Resources and Services in Modern Era
Sanchaita Nath and Nandita Deb ( xxi )
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Antecedents of attitudes towards the use of environmentally friendly household appliance products in Zimbabwe: an extension of the theory of planned behaviour
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Antecedents of attitudes towards
the use of environmentally friendly
household appliance products in
Zimbabwe: an extension of the
theory of planned behaviour
Eugine Tafadzwa Maziriri
Department of Business Management, University of Johannesburg,
Johannesburg, South Africa
Brighton Nyagadza
Department of Marketing,
Marondera University of Agricultural Sciences and Technology,
Marondera, Zimbabwe
Tinashe Chuchu
Division of Marketing, University of the Witwatersrand,
Johannesburg, South Africa, and
Gideon Mazuruse
Teaching and Learning Institute (TLI),
Marondera University of Agricultural Sciences and Technology,
Marondera, Zimbabwe
A Antecedents of attitudes towards
the use of environmentally friendly
household appliance products in
Zimbabwe: an extension of the
theory of planned behaviour
Eugine Tafadzwa Maziriri
Department of Business Management, University of Johannesburg,
Johannesburg, South Africa
Brighton Nyagadza
Department of Marketing,
Marondera University of Agricultural Sciences and Technology,
Marondera, Zimbabwe
Tinashe Chuchu
Division of Marketing, University of the Witwatersrand,
Johannesburg, South Africa, and
Gideon Mazuruse
Teaching and Learning Institute (TLI),
Marondera University of Agricultural Sciences and Technology,
Marondera, Zimbabwe
A Platform
providers in
collaborative
consumption Platform
providers in
collaborative
consumption Received 20 March 2022
Revised 19 July 2022
5 June 2023
Accepted 22 August 2023 Declaration of interest statement: The researchers declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a potential conflict of
interest. The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/2399-1747.htm The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/2399-1747.htm The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/2399-1747.htm PSU Research Review
Emerald Publishing Limited
e-ISSN: 2398-4007
p-ISSN: 2399-1747
DOI 10.1108/PRR-03-2022-0033 Introduction
Th
d The need to encourage sustainable usage of environmentally friendly household appliance
products has been urged by the increase in the general acceptance of global climate change
(Nguyen et al., 2017). This is so because air pollution and global climate change are the main
causes of many deaths per annum (WHO, 2022). Energy consumption has proved to cause
serious environmental problems, with an expected upward trend by 2040 as alternative
cleaner-environmentally friendly energy sources are depleting (IEA, 2016). Prior research has
proved that construction is responsible for almost 50% usage of this energy around the world
(Yan et al., 2019; Shabha et al., 2023). Households and organisations, therefore, need to
embrace sustainable technologies, with proper energy management and optimised energy
use skills as a matter of urgency to counter this social responsibility issue (Zhou and
Bukenya, 2016). In line with this, several studies have depicted that consumers’ reduced
consumption of energy-efficient high-tech, environmentally friendly appliances has
contributed sustainability (Waris et al., 2021; Yan et al., 2019; De Luca et al., 2018). Despite the ability to provide important insights into the importance of environmentally
friendly household appliance products, it is essential to note that there is still a shortage of
empirical evidence of relationships between green product awareness, social influence,
perceived benefit, attitudes towards green appliances and green purchase intention. Consequently, further scholarly introspections are considered necessary. Much of what is
written on the subject is based on samples from Asian countries such as Pakistan, Malaysia,
Korea, Vietnam, China and Bangladesh (among others). For instance, Bhutto et al. (2020)
empirically investigate consumers’ intentions in Pakistan to purchase energy-efficient
appliances (EEAs). Harun et al. (2022) examined consumer purchases of energy-efficient
appliances in Malaysia. In addition, Ha and Janda (2012) predicted consumer intentions to
purchase energy-efficient products in Korea. Furthermore, Nguyen et al. (2016) determined
the influence of consumers’ values and knowledge on their attitudes and purchase behaviour
of energy efficient household appliances in Vietnam. Additionally, Hua and Wang (2019),
examined the antecedents of consumers’ intention to purchase energy-efficient appliances in
China. Moreover, Rahman and Haq (2016) investigated the factors influencing the buying
intention of energy-efficient home appliances in Bangladesh. Even in Zimbabwe, there is scant evidence in studies that have determined the
antecedents that influence attitudes towards the use of environmentally friendly household
appliance products and consumers’ green purchase intention. Abstract Purpose – This study aims to determine the antecedents that influence attitudes towards the use of
environmentally friendly household appliance products and consumers’ green purchase intention among
consumers in Harare, Zimbabwe. Design/methodology/approach – Data were collected from 329 consumers in Harare, Zimbabwe’s
commercial capital who were served from five using a structured questionnaire via an online web-based cross-
sectional survey. Hypothesised relationships were tested through structural equation modelling with the aid of
Smart PLS software. Findings – Green product awareness, social influence, perceived benefit and attitude towards gree
appliances were found to have a significant positive effect on green purchase intention. Research limitations/implications – The study’s findings may not be generalised to other contexts as
sample data was only collected in Zimbabwe. Complementary cross-sectional research studies can be done in
other parts of the world to enable cross-cultural comparisons and methodological validations. © Eugine Tafadzwa Maziriri, Brighton Nyagadza, Tinashe Chuchu and Gideon Mazuruse. Published in
PSU Research Review. 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 PSU Research Review
Emerald Publishing Limited
e-ISSN: 2398-4007
p-ISSN: 2399-1747
DOI 10.1108/PRR-03-2022-0033 Funding: This research did not receive any specific grant from funding agencies in the publi
commercial, or not-for-profit sectors. Declaration of interest statement: The researchers 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. PRR Practical implications – The green appliance and energy saving practices are vastly growing, with many
multinational appliance companies introducing green products within their product lines and adopting the
concept of sustainability through modifications in production, design and consumption of household appliance
products that encompass fewer harmful consequences on the environment in response to their concerns about
the scarcity of natural resources, environmental well-being and the potential detriment of future generations. Originality/value – Notwithstanding the limitations of the current study, the results have the potential to
contribute to an improved understanding of influence attitudes towards the use of environmentally friendly
household appliance products. Keywords Attitude, Behaviour, Environmentally friendly, Green household appliance products
Paper type Research paper Introduction
Th
d Therefore, little is known of the
same from developing parts of the world, such as African countries –Zimbabwe in particular. Hence, this lacuna deserves empirical inspection in the case of a neglected context. Local
scholars in Zimbabwe have determined the environmentally-friendly practices in hotels in
Zimbabwe (Mbasera et al., 2016). In addition, Chikosha and Potwana (2021) examined
consumer perceptions of green products, purchasing behaviour and loyalty in Zimbabwe. Furthermore, Ndofirepi (2019) explored on the gender-based dichotomies in various
psychographic attributes for environmentally friendly products in Zimbabwe. Moreover, Ndofirepi and Matema (2020) investigated on the relationship between personality and the
intention on repeat purchases for environmentally friendly products among college students
in Zimbabwe. Platform
providers in
collaborative
consumption While these international and local studies are informative, they did not examine the
association between green product awareness, social influence, perceived benefit, attitudes
towards green appliances and green purchase intention in Zimbabwe. Therefore, this article
helps tackle the gap by using a sample of consumers from a developing country context. It is
also important to note that very few (if any) researchers have used Structural
Equation Modelling (SEM) to test the relationships between green product awareness,
social influence, perceived benefit, attitudes towards green appliances and green purchase
intention. The exhaustive review of relevant literature opens a gap in research that needs to
be addressed. Regarding the conceptual model proposed in this study, it can be noted that it is
unique –as there are deficiencies in studies that have tested the variables in the proposed
model in relation to the Zimbabwean context (to the best knowledge of the researchers). Moreover, the moderating role of attitudes has also been determined by authors such as
Camacho et al. (2020), Irshad and Ahmad (2019), Abrar et al. (2019) as well as Wulandari et al. (2015). However, there is scant evidence, on studies that have explored the role of attitudes as
a mediator between green product awareness and green purchase intention; social influence
and green purchase intention; as well as perceived benefit and green purchase intention. Hence this study will be a significant contribution in addressing this gap. This article is structured as follows: first, a theoretical basis for the analysis is presented, then
a theoretical model is presented and then, the hypothesis is established. After that, the study
design and methodology are discussed, followed by the findings and discussion. Introduction
Th
d The
implications, limitations and future research directions are discussed in the article’s final sections. Platform
providers in
collaborative
consumption Contextualisation of the study y
Harare, Zimbabwe as a research setting This section details the study’s demarcation area, Harare, Zimbabwe. According to
Mwonzora (2021) Harare is the capital city of Zimbabwe with a population of 2,123,132
million. Chikosha and Potwana (2021) recommended that future research be done in more
extensive, wealthier areas like Harare to see how the results turn out. Their study examined
the impact of product quality on purchase intention for green products. Therefore, the study
by Chikosha and Potwana served as a starting point for the researchers to consider the
factors that influence attitudes toward adopting environmentally friendly household
appliance products in Harare, Zimbabwe. Electricity has a huge impact on mainly growing
economies like Zimbabwe and is considered a significant input of production (Rafindadi and
Ozturk, 2016), hence a study centred on the antecedents that influence attitudes towards the
use of environmentally friendly household appliance products in Zimbabwe is of paramount
importance. Research in this area is of even greater significance to Zimbabwe since the
country heavily depends on conventional energy production means (Samu et al., 2019). These
sources of energy production are not only depleting but they are unclean, not
environmentally friendly and unsustainable, thus the policymakers should now
incorporate renewable energy resources in the energy portfolio to ensure sustainability
(Samu et al., 2019). In Zimbabwe, noticeable green products are energy saving bulbs,
computers, refillable/reusable consumer product packages, solar energy systems, ethanol
blended fuel and washing machines, amongst others (Chikosha and Potwana, 2021). To
hasten the growth of a green economy, the Zimbabwean government has weighed in by
crafting an environmental policy aimed at integrating environmental aspects into national
development plans, embarking on specific actions that encourage the consumption of green
products (Chikosha and Potwana, 2021). Therefore, deducing from the aforementioned elucidations, the choice of Harare,
Zimbabwe as the location for the study on attitudes towards the use of environmentally
friendly household appliance products and consumers’ green purchase intention is relevant
for several reasons. Firstly, Harare is the capital city of Zimbabwe, representing a significant
urban centre and a hub of economic activity. This makes it a suitable location to understand
consumer behaviours and attitudes towards green products, as urban areas often have higher
levels of consumption and access to a wider range of products compared to rural areas. Secondly, Zimbabwe is facing various environmental challenges, including deforestation,
water scarcity and waste management issues. Contextualisation of the study As a result, the study conducted in Harare can
shed light on the specific context of a developing country facing environmental issues,
providing valuable insights into the unique challenges and opportunities that exist in such a
setting. Finally, by focussing on Harare, the study can help inform local policymakers,
businesses and organisations about the factors influencing consumer attitudes and
intentions towards environmentally friendly products, facilitating the development of
targeted interventions and strategies to promote sustainable consumption patterns in
the city. Theoretical premise Diverse models and theories exist for seeking to predict and explain human behaviour. To
comprehend the antecedents that influence attitudes towards the use of environmentally
friendly household appliance products and green purchase Intention. This study is grounded
into the theory of planned behaviour. The theory is discussed in the ensuing section. Green product awareness and green purchase intention
B i
h (201 )
l d h
b
d
i i
l Braimah (2015) revealed that green brand awareness positively and significantly influences
customer purchase intention. Similarly, a study conducted by Mahmood et al. (2014) found that
greenawarenessisoneofthemostvitalpredictorsofgreenpurchaseintentionandthat,ultimately,
green awareness has a significant and positive relationship with green purchase intention. Moreover, Suki (2016) conducted a study on consumers’ green purchase intention whereby the
findings postulate that green product awareness has a positive and significant impact on green
purchase intention. Drawing from the above discussion, it can be hypothesised that: H1. Green product awareness has a significant and positive impact on green purchase
intention. H1. Green product awareness has a significant and positive impact on green purchase
intention. Conceptual research model and hypotheses formulation Conceptual research model and hypotheses formulation
Figure 1 illustrates the conceptual model reflecting the distinct paths and connections
between the investigated constructs. The subsequent sections will then provide the
formulation of the hypotheses for the present research. Platform
providers in
collaborative
consumption The theory of planned behaviour (TPB) The theory of planned behaviour (TPB)
Ajzen (2002) proposed the Planned Behaviour Theory. The TPB improves the purchase
intention model’s predictability (Jebarajakirthy and Lobo, 2014) for green products. The TPB
model has been one of the most widely used models for studying environmental behaviours
(Fielding et al., 2008). Many researchers believe that the TPB model can explain consumers’
sustainable consumption behavioural intentions and predict their future behaviours well
(Mannetti et al., 2004). The TPB model demonstrates that three predictors guide human
intention: attitude towards behaviour, subjective norm and perceived behavioural control. However, although the TPB model has been widely used to examine the motivation of
sustainable consumption intentions, researchers have noticed that domain-specific factors
have not been included in the model (Armitage and Conner, 2001; Donald et al., 2014). An
increasing number of studies have extended the TPB model by including new constructs
(Jang et al., 2015; Maichum et al., 2016; Read et al., 2013). This study extended the TPB by
including three variables such as green product awareness, social influence, perceived
benefit, as well as traditional TPB constructs (i.e., attitude towards behaviour and
behavioural intention) to measure consumers’ green purchase intention of environmentally
friendly household appliance products. Inferring from the Theory of Reasoned Action (TRA), green purchase intention can be
rationalised by referring to Qureshi et al. (2023), who explored how to determine the factors
that led to the intention to purchase green products? Other scholars, namely, Mohd Suki
(2016) and Salam et al. (2022) have even gone further to adopt the TPB which is an extension
of the TRA through the addition of perceived behavioural control to discuss attitudes and
intention to purchase green product. Mohd Suki (2016) suggested that, done properly, green
brand positioning has the potential to increase green brand knowledge, attitude toward green
products and green brand purchase intention. Furthermore, Salam et al. (2022) justified the
use of the TPB when in it comes to green purchase behaviour because it helps explain the factors that influence attitude toward green brands and additionally, whether these attitudes
lead to purchase intention? The application of this model to this study is that, the model
provides further explanations into the connection between green product awareness, social
influence, perceived benefit, attitudes and green purchase intention. The theory of planned behaviour (TPB) Since the literature on
consumer purchase of environmentally friendly household appliance products is still in its
infancy, it is commendable to scrutinise and examine the relation of the TPB with other
critical determinants of consumer intention and purchase of environmentally friendly
household appliance products. The outcome would provide helpful insight to policymakers
and authorities to implement better mitigation plans to increase the purchase of
environmentally friendly household appliance products. Figure 1.
The conceptual model Green product awareness and attitudes Anvar and Venter (2014) findings show that environmental awareness significantly and
positively influences individuals’ attitudes towards green products. Boztepe (2012) also found Green
product
awareness
Attitude
towards
green
appliances
Green
purchase
Intention
Social
Influence
Perceived
benefit
Attitude
towards
green
appliances
Attitude
towards
H1
H6
H5
H4
H2
H3
Source(s): Authors’ own work (2022) Green
product
awareness Green
purchase
Intention Attitude
towards
green
appliances
Attitude
towards
green
appliances
Attitude
towards Social
Influence H4 Source(s): Authors’ own work (2022) PRR a positive and significant relationship between environmental awareness and consumers’
attitudes towards purchasing a green product. Moreover, green product knowledge
fundamentally encourages stronger beliefs towards the benefits attained through the use
of green products and more positively balanced opinions regarding the various impacts of
product use (Ha and Janda, 2012). Therefore, based on the above discussion along with
Ajzen’s Theory of Planned Behaviour (1991) stating that the beliefs consumers may have also
form attitudes, the following hypothesis can be drawn: H2. Green product awareness has a significant and positive impact on attitudes. Social influence and attitudes Muhammad andGhani(2016) investigates the relationship between attitude andsocial influence
on purchase behaviour. The results suggest a positive and significant relationship between
social influence and attitude. Muhammad and Ghani (2016) state that social influences
manipulate consumers to perform activities to gain approval in certain social situations. Those
personal thoughts and social influences are predictors of behaviour towards intention. Ajzen’s
Theory of Planned Behaviour looks into subjective norms as being directly linked to social
pressures and thus having a direct influence on whether or not to perform a specific behaviour
(Ajzen, 1991). Therefore, the following hypothesis can be formed: H3. Social influence has a significant and positive impact on attitudes. Perceived benefit and attitudes Ruiz-Molina and Gil-Saura (2008) found that perceived value has a significant and positive
attitude on customer attitude. Zhang and Wang (2005) also discovered a positive and a
significant relationship between perceived value and attitudes toward a given behaviour
(Zhang and Wang, 2005). According to Mostafa (2006), perceived consumer benefit is a highly
influential factor when analysing the attitude-consumer behaviour relationship. Furthermore, it was determined that concepts relating to people’s beliefs about their
impact on future outcomes and the desire to provide benefits for others provide benefits for
others may positively influence pro-environmental attitudes and behaviours (Mostafa, 2006). Thus, it is hypothesised that: H4. Perceived benefit has a significant and positive impact on attitude. The mediating role of attitudes
A
f
h
i d
l i
hi Apart from the posited relationships depicted in conceptual model (Figure 1). Alternative
hypothesis statements incorporating attitudes towards environmentally friendly household
appliance products as a mediating variable have also been included. It is imperative to
provide empirical evidence that regards attitudes towards using environmentally friendly
household appliance products as a mediating variable between green product awareness,
social influence, perceived benefit and green purchase intention. However, there are
deficiencies in empirical studies that are centred on the use of environmentally friendly
household appliance products as a mediating variable. Hence the need to close this mediation
gap. A few closely related studies such as the one conducted by Chu (2018) discovered that
consumer attitudes toward organic foods mediate the effects of health consciousness on the
intention of purchasing organic foods. Camacho et al. (2020) revealed that product attitudes
will mediate the relationship between xenocentrism and purchase intentions. Furthermore, Irshad and Ahmad (2019) discovered that consumer attitudes mediate the
relationship between hedonic value and purchase intentions, while Abrar et al. (2019) found
out that consumer attitudes mediate behavioural intentions. The attitude variable has been
proven to mediate consumer knowledge influence the purchase intention of green products
(Wulandari et al., 2015). Based on this premise, the mediating impact of attitudes still needs
further clarification as there is still limited empirical research. It is expected that attitudes
towards environmentally friendly household appliance product use can be a mechanism
through which green product awareness, social influence and perceived benefit would
influence green purchase intention. This is one of the essential empirical contributions of this
study because it offers a more nuanced explanation of the essence of attitudes towards using
environmentally friendly household appliance products as a mediator variable. Consequently, drawing from the above discussion and past empirical evidence, it can be
hypothesised that: H7. Attitudes towards the use of environmentally friendly household appliance products
positively and significantly mediates the relationship between green product
awareness and green purchase intention. H8. Attitudes towards the use of environmentally friendly household appliance products
positively and significantly mediates the relationship between social influence and
green purchase intention. Attitudes and green purchase intention Nam et al. (2017) found out that attitude has a significant and positive influence on green
purchase intention whereby it was concluded that attitude plays a key role as a mediator
between purchase intention and other relevant predictor variables (Nam et al., 2017). Furthermore, Suki (2016) postulate that consumers’ attitude toward green brands
significantly and positively influences green product purchase intention. Purchase
intention is built on consumers’ attitudes, evaluation and external factors, essentially
making it a vital factor to predict consumer behaviour (Braimah, 2015). Based on the above
findings, it is hypothesised that: H6. Attitude has a significant and positive impact on green purchase intention. Platform
providers in
collaborative
consumption Perceived benefit and green purchase intention
M h
d
l (2014)
l d
i i
d
i Perceived benefit and green purchase intention
Mahmood et al. (2014) revealed a positive and a significant relationship between green
perceived value and green purchase intention. Furthermore, Salehzadeh and Pool (2017)
found that perceived value positively and significantly influences purchase intention. Green
purchase intention is described in terms of the willingness and probability of a consumer to
choose products containing eco-friendly features over traditional products in their evoked set
(Mei et al., 2012). Chen and Chang (2012), argue that the perceived benefit and value of
products act as a signal to consumers as judgement is often built from incomplete
information, and therefore the benefit perceived will positively influence purchase intention. A consumer may find that an eco-friendly product provides high perceived benefit through
the multiple advantage of environmental protection and personal gain which would
positively influence their intention to purchase that product. Therefore, based on the above,
the following hypothesis is proposed: H5. Perceived benefit has a significant and positive impact on green purchase intention. Sample and data collection Using an online questionnaire platform, a survey was conducted online. A pilot study using
40 samples was conducted prior to the full-fledged data gathering. The pilot study’s findings
imply that the construct validity was satisfactory. The researchers divided the population
potential respondents into more relevant and significant strata based on subsets where a
random sample was drawn from each of the strata (Hair et al., 2014) such as the customers’
profiles (low, middle- and high-income earning capacities) as well as the geographical
locations (local, regional and international) to which they belong to. Stratified random
sampling technique was applied due to its accuracy and easy-to-use advantages (Cude et al.,
2016). Krejcie and Morgan (1970) formular was applied to determine the sample size, which
was necessary to construct a confidence interval (generally þ5%). The participants were
invited through the author’s networking and contacts. Survey invitations were sent to
approximately 500 potential respondents, with their anonymity and confidentiality were
guaranteed. Of the 500 questionnaires distributed, 329 questionnaires returned were useable,
resulting in a response rate of 65.8%. Results analysis Results analysis
Analysis of background variables
The subjects of this research are a total of 329 respondents. As shown in Table 1, there are 145
(44.48%) males and 184 (55.52%) females. y
f
g
The subjects of this research are a total of 329 respondents. As shown in Table 1, there are 1
(44.48%) males and 184 (55.52%) females. Measurement instrument All questionnaires were distributed through the web-based survey method to customers. Participation was voluntary and the objectives of the study were explained to the participants
in the research study before completing the questionnaire. Four constructs of the proposed
research model were adopted from existing literature and refined based on the specific topic
of this study. The respondents’ beliefs were captured using a five-point Likert-type scale
anchored on strongly agree (5) to strongly disagree (1). The items used to measure each
construct are provided in Appendix. Both descriptive and inferential statistics were used in
analysing quantitative data from the questionnaire. Ethical considerations related to
participating customers’ privacy, informed consent, freedom of response, professionalism,
integrity, accuracy and values of research have been adhered to by the researchers. Results analysis
Analysis of background variables
The subjects of this research are a total of 329 respondents. As shown in Table 1, there are 145
(44.48%) males and 184 (55.52%) females. Methodological aspects The research philosophy for this study was positivism. This study was conducted using a
quantitative research method. The design was suitable for gathering information on green
product awareness, social influence, perceived benefit, attitudes towards green appliances PRR and green purchase intention. Furthermore, the technique enables for the investigation of
causal links between the research constructs. and green purchase intention. Furthermore, the technique enables for the investigation of
causal links between the research constructs. Reliability analysis
l
f
bl
i Results from Table 3 indicates that the Cronbach’s alpha value ranges between 0.703 and
0.889, demonstrating that all the observed items are reliable and consistent. For green
product awareness, social influence, perceived benefit, attitude towards green appliances and
green purchase intention, the Cronbach’s alpha value were 0.812, 0.773, 0.703, 0.806 and 0.832
respectively. Sample adequacy and test of normality p
q
y
y
The results showed that the KMO’s measure of sampling adequacy value was 0.863, which is
by greater than the threshold value of 0.5 that indicated adequate sample size. Likewise, the
Bartlett’s test of Sphericity tested (χ2 (320) 5 24165.271, p < 0.05) and the correlation matrix Items
Background variables
Number of people
Percentage (%)
Gender
Male
145
44.48
Female
184
55.52
329
100
Source(s): Primary data (2022)
Table 1. Frequency distribution
of sample data Platform
providers in
collaborative
consumption was adequacy. The proportion of item’s variance explained by the extracted factors
(communalities) were all above 3.00, further confirming that each item shared common
variance with other items. The results indicated that the matrix was not an identity matrix
and this allowed the factor analysis to be conducted as relationships between variables
existed. Multivariate normality was examined by means of SPSS (Version 25). According to the results in Table 2, (KMO 5 0.863, p > 0.05), there is need to increase the
sample size (collect more information) or to select certain variables to include in the analysis. The correlation matrix is not an identity matrix according to the Bartlett’s test of Sphericity
(χ2 (320) 5 24165.271, p < 0.001). The results indicate that the matrix is not an identity matrix,
and this allows the factor analysis to be conducted as relationships between variables exist. Platform
providers in
collaborative
consumption Measurement model assessment SmartPLS 3.2.9 was used to evaluate the measurement model and test the main effects
Construct validity of the measurement model was assessed by investigating the construc SmartPLS 3.2.9 was used to evaluate the measurement model and test the main effects. Construct validity of the measurement model was assessed by investigating the construct
Kaiser-Meyer-Olkin measure of sampling adequacy
0.863
Bartlett’s test of sphericity
Approx. chi-square
24165.271
df
320
Sig
0.000
Source(s): Primary data (2022)
Dimension
Question
items
Skewness
Kurtosis
Factor
loadings
Cronbach’s
alpha
CR
AVE
Green product
awareness (GPA)
GPA1
1.173
0.369
0.824
0.812
0.889
0.728
GPA2
0.801
GPA3
0.785
Social influence (SI)
SI1
1.151
0.510
0.712
0.773
0.868
0.690
SI2
0.814
SI3
0.763
Perceived benefit
(PB)
PB1
1.262
0.353
0.696
0.703
0.834
0.626
PB2
0.786
PB3
0.807
Attitude towards
green appliances
(AT)
AT1
1.567
0.300
0.841
0.806
0.873
0.632
AT2
0.725
AT3
0.716
AT4
0.769
Green purchase
intention (GPI)
GPI1
1.125
0.318
0.854
0.832
0.888
0.665
GPI2
0.728
GPI3
0.742
GPI4
0.810
Source(s): Primary data (2022)
Table 2. KMO and
Bartlett’s test
Table 3. Measurement model
parameter estimation Kaiser-Meyer-Olkin measure of sampling adequacy
0.863
Bartlett’s test of sphericity
Approx. chi-square
24165.271
df
320
Sig
0.000
Source(s): Primary data (2022)
Table 2. KMO and
Bartlett’s test Dimension
Question
items
Skewness
Kurtosis
Factor
loadings
Cronbach’s
alpha
CR
AVE
Green product
awareness (GPA)
GPA1
1.173
0.369
0.824
0.812
0.889
0.728
GPA2
0.801
GPA3
0.785
Social influence (SI)
SI1
1.151
0.510
0.712
0.773
0.868
0.690
SI2
0.814
SI3
0.763
Perceived benefit
(PB)
PB1
1.262
0.353
0.696
0.703
0.834
0.626
PB2
0.786
PB3
0.807
Attitude towards
green appliances
(AT)
AT1
1.567
0.300
0.841
0.806
0.873
0.632
AT2
0.725
AT3
0.716
AT4
0.769
Green purchase
intention (GPI)
GPI1
1.125
0.318
0.854
0.832
0.888
0.665
GPI2
0.728
GPI3
0.742
GPI4
0.810
Source(s): Primary data (2022)
Table 3. Measurement model
parameter estimation PRR measures’ convergent and discriminant validity (Hair et al., 2019). The standard criteria for
convergent validity were applied, which include: (1) all standardised loadings (in PLS, outer
loadings) which statically surpassed the recommended value 0.5 for each relevant research
construct (Aldalaigan and Buttle, 2002), the average variance extracted (AVE) of each
construct is 0.5 or higher and the composite reliability (CR) value is 0.7 or higher. Convergent validity From the results displayed in Table 4, the AVE values for green product awareness (0.728),
social influence (0.690), perceived benefit (0.626), attitude towards green appliances (0.632)
and green purchase intention (0.665) (>0.50), showing that the indicators were assumed to
measure the same construct sufficiently (Collier, 2020). Correlation analysis
L
k
d A
d lli Lovakov and Agadullina (2021) gave the rule of thumb for the correlation coefficient to be as
follows: r < 0.2 (very-weak), 0.2 ≤r < 0:4 ðweakÞ; 0:4 ≤r < 0:6Þ ðmoderateÞ; r ≥0.8 (very
strong). Table 4, gives the inter-item correlation estimates: green product awareness and
attitude towards green appliances (r 5 0.718), green purchase intention and attitude towards
green appliances (r 5 0.762), perceived benefit and attitude towards green appliances
(r 5 0.860), social influence and attitude towards green appliances (r 5 0.831), green purchase
intention and green product awareness (r 5 0.979), perceived benefit and green product
awareness (r 5 0.719), social influence and green product awareness (r 5 0.694), perceived
benefit and green purchase intention (r 5 0.753), social influence and green purchase
intention (r 5 0.716) and social influence and perceived benefit (r 5 0.896). From the results,
the relationship among the variables very strong. Measurement model assessment It is
imperative to note that GPA1, GPA5, PB4, AT5, AT6 and GPI5 items were removed because
the item loadings were less than 0.500; thus, less than 50% of variance was explained and the
thresholds of equivalent or higher than 0.500 did not reach. Key: 1 – Attitude towards green appliances, 2 – Green Product awareness, 3 – Green purchase intention, 4 –
erceived benefit, 5 – Social Influence
ource(s): Primary data (2022) Discriminant validity In line with the results presented in Table 5, all the cross-loadings are exceeding 0.6 implying
that convergent validity on the measurement constructs exist (Fornell and Larcker, 1981). Moreover, discriminant validity of the constructs was also examined through Heterotrait–
Monotrait Ratio of correlations. Henseler et al. (2015) proposed a more rigorous valuation of the variables discriminant
validity by observing the heterotrait-monotrait criterion. Henseler’s HTMT criterion
recommends that all the variables are uniquely different at HTMT 0.90 cut-off point. As Construct
1
2
3
4
5
1
1
2
0.718
1
3
0.762
0.979
1
4
0.860
0.719
0.753
1
5
0.831
0.694
0.716
0.896
1
Note(s): **Correlation is significant at the 0.01 level (2-tailed)
Key: 1 – Attitude towards green appliances, 2 – Green Product awareness, 3 – Green purchase intention, 4 –
Perceived benefit, 5 – Social Influence
Source(s): Primary data (2022)
Table 4. Correlation between
constructs ( )
g
(
)
Key: 1 – Attitude towards green appliances, 2 – Green Product awareness, 3 – Green purchase intention, 4 –
Perceived benefit, 5 – Social Influence
Source(s): Primary data (2022)
Table 4. Correlation between
constructs Construct
Attitude towards
green appliances
Green product
awareness
Green purchase
intention
Perceived
benefit
Social
influence
AT1
0.795
0.789
0.721
0.712
0.574
AT2
0.782
0.694
0.669
0.593
0.685
AT3
0.811
0.645
0.654
0.694
0.701
AT4
0.793
0.703
0.734
0.564
0.594
GPA1
0.646
0.863
0.685
0.669
0.648
GPA2
0.535
0.877
0.648
0.714
0.619
GPA3
0.519
0.818
0.583
0.627
0.620
GPI1
0.593
0.756
0.739
0.568
0.591
GPI2
0.635
0.793
0.857
0.628
0.493
GPI3
0.685
0.747
0.851
0.553
0.517
GPI4
0.702
0.694
0.808
0.639
0.633
PB1
0.639
0.773
0.674
0.800
0.581
PB2
0.589
0.720
0.696
0.747
0.584
PB3
0.718
0.665
0.701
0.825
0.622
SI1
0.664
0.699
0.638
0.573
0.703
SI2
0.627
0.607
0.617
0.643
0.888
SI3
0.726
0.739
0.593
0.588
0.886
Source(s): Primary data (2022)
Table 5. Cross loadings
Platform
providers in
collaborative
consumption shown in Table 6, the HTMT values for all variables are in the range from 0.692 to 0.884 and
these indicate that are variables are uniquely different at values below HTMT 0.90 which also
confirms discriminant validity. Hair et al. (2016) suggested HTMT cut off point value of 0.85,
in this case we the research may lack discriminant validity. Structural equation model (SEM) After we have established a consistent and valid model, the next step of the analysis involves
examining the relationship between the exogenous (independent) and endogenous
(dependent) latent variables. Research hypothesis testing results The bootstrap method was used to confirm the significance of the path coefficients by
comparing β values among all the paths. The output for the analysis was presented in
Figures 1 and 2. In Table 7, the following path were statistically significant: green product
awareness →green purchase intention (β ¼ 0:885, t 5 9.372, p 5 0.025), green product
awareness →attitude towards green appliances (β ¼ 0:180, t 5 2.894, p < 0.01), social
influence →attitude towards green appliances (β ¼ 0:254, t 5 4.271, p < 0.01), perceived Construct
1
2
3
4
5
1
2
0.886
3
0.763
0.797
4
0.806
0.741
0.836
5
0.692
0.884
0.728
0.720
Source(s): Primary data (2022)
Table 6. Heterotrait–monotrait
ratio of
correlations (HTMT) Figure 2. SEM with coefficients
PRR Figure 2. SEM with coefficients
PRR PRR Path analysis
Path coefficient
t-value
p-value
Hypothesis
GPA →GPI
0.885
9.372
<0.01
H1 valid
GPA →AT
0.180
2.894
<0.01
H2 valid
SI →AT
0.254
4.271
<0.01
H3 valid
PB →AT
0.505
6.210
<0.01
H4 valid
PB →GPI
0.032
2.715
<0.01
H5 valid
AT →GPI
0.099
2.793
<0.01
H6 valid
Note(s): *Significant at p < 0.01
Source(s): Primary data (2022)
Table 7. Path analysis benefit →attitude towards green appliances (β ¼0:505, t 5 6.210, p < 0.01), perceived benefit
→green purchase intention (β ¼0:032, t 5 2.715, p < 0.01), attitude towards green appliances
→green purchase intention (β ¼ 0:099, t 5 2.793, p < 0.01). benefit →attitude towards green appliances (β ¼0:505, t 5 6.210, p < 0.01), perceived benefit
→green purchase intention (β ¼0:032, t 5 2.715, p < 0.01), attitude towards green appliances
→green purchase intention (β ¼ 0:099, t 5 2.793, p < 0.01). benefit →attitude towards green appliances (β ¼0:505, t 5 6.210, p < 0.01), perceived benefit
→green purchase intention (β ¼0:032, t 5 2.715, p < 0.01), attitude towards green appliances
→green purchase intention (β ¼ 0:099, t 5 2.793, p < 0.01). Evaluation of the structural model
B f
l di
h
h Before concluding that the measurement model was valid and reliable, measuring the
structural model outcomes was the next step. This included examining by at the Variance
Inflation Factor (VIF), coefficient of determination (R2), Effect size (f 2) and the predictive
relevance of the model (Q2) which in most cases not widely used. From our model results, the
VIFs values of the most variables were below the rules of thumb of 5 and they were ranging
from 2.124 to 5.212 which approves that there is no multicollinearity. Mediation effect analysis (Sobel’s test) y
(
)
It is defined when having a third variable (mediator) between the two variables (Cude et al.,
2016). Mediation analysis was done using Sobel’s test in this study. The Sobel’s test uses the
product of coefficients. The results are presented in Table 8. Taking for example the path GPA →AT →GPI. We find the product of 0.404 and 0.838
which are beta values for GPA →AT and AT →GPI respectively that is 0.180 3 0.099 we get
0.339. According to the results in Table 8, the relationship between green product awareness
and green purchase intention is significantly mediated by attitude towards green appliances
(β 5 0.118, p < 0.01), the relationship between social influence and green purchase intention is
significantly mediated by attitude towards green appliances (β 5 0.125, p < 0.01) and the
relationship between perceived benefit and green purchase intention is significantly Platform
providers in
collaborative
consumption mediated by attitude towards green appliances (β 5 0.150, p < 0.01). From the analysis
showing direct and indirect relationship there was no change in terms of the significance of
the constructs. The only notable change was the reduction in the beta value, and this indicates
the existence of a partial mediation. Coefficient of determination (R2) The exogenous variables in the model may have a significant effect in the dependent variable. The coefficient of determination (R2) examines the amount of variation in the dependent
variable which is caused by the exogenous variables as propounded by (Schumacher et al.,
2016). R2 values of 0.75, 0.5 and 0.25 can be considered substantial, moderate and weak
respectively (Hair et al., 2011). Very high values of R2 may result in model overfitting the data
and may result in spurious relationship provided the R2 value is greater than the Durbin
Watson. In our case, as indicated in Table 10 attitude towards green applications has an R2
value of 0.774 which is being explained by green product awareness, social influence and
perceived benefit. The predictors have a direct effect towards emotional attachment. The
green purchase intention R2 value is 0.966 contributed by attitude towards green
applications, green product awareness and perceived benefit directly. Social influence has
an indirect relationship towards green purchase intention. The developed model has a
substantial explaining power. Platform
providers in
collaborative
consumption Goodness of fit of the model The Standardised Root Mean Square Residual (SRMSR) is an index of the average of
standardised residuals between the observed and the hypothesised covariance matrices
(Chen, 2007). SRMR is a measure of the estimated model fit. When SRMSR ≤0.08, then the
study model has a good fit (Hu and Bentler, 1998), with a lower SRMSR being a better fit. According to Table 11 results, the SRMR value for the fitted model is 0.071 which is less than
the threshold value of 0.08, suggesting that the model can be accepted. Furthermore, the NFI
value for the model is 0.902 which is slightly above the recommended threshold value of 0.9. These results suggest that the fitted model is a good model, whereas the Chi-Square was
equal to 7426.162. The effect size (f 2)
2 (
)
Effect size (f 2) is a measurement that tells the impact of change in the R2 value when
a specified exogenous construct is ignored in the model (Hair et al., 2011). An effect size
f 2 ≤0.30, 0.3 < f2 ≤0.50 and f2 >0.50 is thought to represent a weak, moderate and
strong effect respectively (Bliwise, 2006). From Table 9, above, f2 values of the
relationship between perceived benefit and green purchase intention is weak. The effect
sizes of most of the relationships between the variables are moderate as depicted by
(Bliwise, 2006). Hypothesis Path
Std
beta
Std
error
t-statistics p-values Decision
Bootstrapping
confidence interval
95% CI
LL
95% CI
UL
H7
GPA →AT →GPI
0.118
0.131
2.692
<0.01
Supported
0.006
0.101
H8
SI →AT→GPI
0.125
0.108
2.740
<0.01
Supported
0.010
0.163
H9
PB →AT→GPI
0.150
0.041
3.253
<0.01
Supported
0.021
0.138
Note(s): *Significant at p < 0.01
Source(s): Primary data (2022)
Table 8. Mediating effect
analysis via Sobel test Constructs
f square
VIF (inner values)
GPA →GPI
0.394
2.246
GPA →AT
0.367
2.124
SI →AT
0.455
5.212
PB →AT
0.432
4.590
AT →GPI
0.302
4.201
PB →GPI
0.277
4.205
Note(s): GPA – Green Product awareness, GPI – Green Purchase Intention, AT – Attitude towards green
applications, SI – Social Influence, PB – Perceived benefit
Source(s): Primary data (2022)
Endogenous variables
R square
t-value
p-value
Attitude towards green applications
0.774
2.682
<0.01
Green purchase intention
0.966
2.715
<0.01
Note(s): *Significant at p < 0.01
Source(s): Primary data (2022)
Table 9. Collinearity
Table 10. The coefficient of
determination
PRR Overall assessment Goodness of Fit (GoF) defined as the geometric mean of both average variances extracted
(AVE) and the average of R2 of all endogenous variables (Akter et al., 2017). PLS results can be
assessed globally for the overall mode and locally for the measurement and structural models
(Henseler et al., 2015). The criteria of GoF to decide whether GoF values are not fit, small,
medium, or large to be considered as global valid PLS model are given by (Akter et al., 2017)
as GoF less than 0.1(not fit), GoF between 0.1 and 0.25 (small), GoF between 0.25 and 0.36 Estimated model
SRMSR
0.071
d_ULS
11.527
d_ G
15.920
Chi-Square
7426.162
NFI
0.902
Source(s): Primary data (2022)
Table 11. Goodness of fit results Estimated model
SRMSR
0.071
d_ULS
11.527
d_ G
15.920
Chi-Square
7426.162
NFI
0.902
Source(s): Primary data (2022)
Table 11. Goodness of fit results (medium) and GoF greater than 0.36 (large). The formula for calculating GoF was adopted
from (Akter et al., 2017) as follows: (medium) and GoF greater than 0.36 (large). The formula for calculating GoF was adopte
from (Akter et al., 2017) as follows: Platform
providers in
collaborative
consumption GoF ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
AVE 3 R2
p GoF ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
AVE 3 R2
p GoF ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
AVE 3 R2
p Therefore, as clearly shown in Table 12, the GoF value for this study is 0.762 which is above
0.36 as indicated (Akter et al., 2017). This proves that the developed model is large in
explaining the issues of corporate brand perception. Platform
providers in
collaborative
consumption Table 12.
Goodness of fit index
calculation Discussion
ifi
i
f Kim and Chung (2011), elucidate that environmental consciousness directs people
towards making purchase decisions that are more environmentally friendly, along with the
verdict that consumers’ environmental awareness is essentially a pre-requisite for green
consumption. Moreover, studies show that green product knowledge fundamentally
encourages stronger beliefs towards the benefits attained using green products as well as
more positively balanced opinions with regards to the various impacts of product use (Ha and
Janda, 2012). PRR Social influence →attitude towards green appliances (β ¼ 0:254, t 5 4.271, p < 0.01). A
powerful example illustrating the existence of social influence is the way in which romantic
relationship partners jointly make consumer decisions and by doing so they ultimately
influence each other’s attitudes, beliefs and judgement (Wood and Hayes, 2012). Muhammad
and Ghani (2016) state that social influences manipulate consumers to perform particular
activities to gain approval in certain social situations. Those personal thoughts and social
influences are predictors of behaviour towards intention. Therefore, highlighting that
marketing and promotional efforts should be directed at the cognitive and emotional
components of consumers’ attitudes to ensure a better understanding and support for green
products, especially in developing countries (Hamid, 2014; Nyagadza, 2021). Perceived benefit →attitude towards green appliances (β ¼ 0:505, t 5 6.210, p < 0.01). Studies concur that consumer’s look towards green products based on the environmental
benefit they may provide. In many cases consumers adopt green products for the direct
personal benefit in the form of energy saving through the use of an eco-friendly air
conditioner, perceived health benefits as a result of organic foods as well as perceived
financial gain by minimising personal electricity usage or reducing water consumption using
eco-friendly washing machines (Kong et al., 2014). According to Mostafa (2006), perceived
consumer benefit is a highly influential factor when analysing the attitude-consumer
behaviour relationship. Perceived benefit →green purchase intention (β ¼0:032, t 5 2.715, p < 0.01). A consumer
may find that an eco-friendly product provides high perceived benefit through the multiple
advantage of environmental protection and personal gain which would positively influence
their intention to purchase that product. Alshura and Zabadi (2016) indicates that perceived
benefits not only assist in maintaining long-term customer relationships but also have a
significant impact in influencing purchase intention. Discussion
ifi
i
f Justification for the study of Harare, capital city of Zimbabwe is that the country heavily
depends on conventional energy production means. Further to this, there is barely sufficient
evidence in prior research enquiries that have determined the antecedents which influence
attitudes towards the use of environmentally friendly household appliance products and
consumers’ green purchase intention. Therefore, there is paucity of the same information
from developing parts of the world, such as African countries, Zimbabwe in particular. Hence,
this lacuna deserves empirical inspection in the case of a neglected context. The sources of
energy production are not only depleting but they are unclean, not environmentally friendly
and unsustainable, thus the policymakers should now incorporate renewable energy
resources in the energy portfolio to ensure sustainability. With the above background justifying the need for carrying out research in the study area
of Harare, the result after bootstrapping, showed a positive relationship between Green
product awareness →green purchase intention (β ¼0:885, t 5 9.372, p 5 0.025). This implies
that environmental knowledge is associated with pro-environmental behaviour and that
exposure (through eco-labelling, eco-branding, environmental advertising, word of mouth
and so forth) is essentially the most important factor to environmentalism. Consumers that
have a concern for the environment often make purchasing decisions that are directly
influenced by brand image awareness and therefore it is the marketers’ responsibility to
ensure that information regarding green products, eco-labelling and green messages are
provided in order to familiarise customers with the green brand (Alshura and Zabadi, 2016). As part of a study conducted by Aman et al. (2012), it was determined that as a result of past
literature and previous studies, actual purchase behaviour has a high level of dependency on
an individual’s green purchase intention and, therefore strongly correlates with Ajzen and
Fishbein (1980, 1991) Theory of Planned Behaviour. However, there has not been a significant
amount of studies directed at green purchase behaviour, and more importantly, green
purchase behaviour within developing countries as opposed to westernised, developed
countries (Mei et al., 2012). Construct
AVE
R2
AT
0.632
0.774
GPA
0.728
PB
0.626
GPI
0.665
0.966
S1
0.690
AVE
0.668
AVE 3 R2
0.581
GoF
0.762
Source(s): Primary data (2022)
Table 12. Goodness of fit index
calculation Green product awareness →attitude towards green appliances (β ¼ 0:180, t 5 2.894,
p < 0.01). Discussion
ifi
i
f In addition, it is pointed out that
consumers are increasingly viewing the use of environmentally friendly products as more
beneficial and rewarding as opposed to conventional products and therefore encourages
consumer green purchase intention on the basis that perceived benefit is a strong influencer
of consumer trust (Alshura and Zabadi, 2016). (
,
)
Attitude towards green appliances →green purchase intention (β ¼ 0:099, t 5 2.793,
p < 0.01). Prior research findings postulate that consumers’ attitude toward green brands
significantly and positively influences green product purchase intention (Suki, 2016). These
results are in line with literature. Purchase intention is built on consumers’ attitudes, evaluation
and external factors, essentially making it a vital factor to predict consumer behaviour
(Braimah, 2015). Additionally, a previous study led by Balderjahn (1988) concluded that having
a positiveattitude towardsenvironmentally consciouslivingwillultimatelyresult in purchasing
and using these environmentally responsible products (Mostafa, 2006). As reflected in Table 8, “attitude towards green appliances” was discovered to mediate the
relationship between green product awareness and green purchase intention (β 5 0.118,
t 5 2.692, p < 0.01), social influence and green purchase intention (β 5 0.125, t 5 2.740,
p < 0.01) as well as perceived benefit and green purchase intention (β 5 0.150, t 5 3.253,
p < 0.01). These results mirror with the works of Camacho et al. (2020) who revealed that
product attitudes will mediate the relationship between xenocentrism and purchase intentions. Irshad and Ahmad (2019) also discovered that consumer attitudes mediate the
relationship between hedonic value and purchase intentions. Moreover, Abrar et al. (2019)
found out that consumer attitudes mediate behavioural intentions. intentions. Irshad and Ahmad (2019) also discovered that consumer attitudes mediate the
relationship between hedonic value and purchase intentions. Moreover, Abrar et al. (2019)
found out that consumer attitudes mediate behavioural intentions. To wrap up the discussion, in order to hasten the growth of a green economy, the
Government of Zimbabwe has weighed in by crafting a “National Environmental Policy and
Strategies” (June 2009), under the “Ministry of Environment and Natural Resources
Management”, aimed at integrating environmental aspects into national development plans,
embarking on specific actions that encourage the consumption of green products. Conclusion and implications for theory and practice
T
l d
h
d
li
h
i
i Conclusion and implications for theory and practice
To conclude the study, green appliance purchase intention can be explained as an indication
towards a potential purchase behaviour specifically at the stage where consumers evaluate
and rank considerable brands as part of the purchase intention process, bearing in mind the
effect of unexpected situational factors, attitudes of others and the subjective expectancies
attached to the purchase of green products. Moreover, consumer’s beliefs and values are
empirical in the assessment of influences towards purchase behaviour as values directly
affect people’s beliefs, ultimately influencing personal norms that induce pro-environmental
behaviours. The ideal method for environmental preservation is through the internalisation
of consumer and producer activities that potentially have an adverse effect on the
environment. These methods take the form of environmental standards, including
environmental taxes and penalties along with eco-labelling and eco-branding on products
that can be used as an effective tool for the moderation of environmental problems. Practically, environmental knowledge is associated with pro-environmental behaviour,
and exposure (through eco-labelling, eco-branding, environmental advertising, word of
mouth and so forth) is essentially the most important factor to environmentalism. The more
consumers know about environmentally friendly behavioural actions, the more they will act
positively. Green awareness is operationalised as the level of knowledge a consumer bears
regarding environmental education and consumption effects on the environment, along with
the capacity consumers must identify, recognise and pro-actively respond to environmental
concerns and green product features. Social influence as a key predictor of young consumer
purchase behaviour is highly impactful factor when assessing the purchase intention
towards environmentally friendly products. In addition, it is pointed out that consumers are
increasingly viewing the use of environmentally friendly products as more beneficial and
rewarding as opposed to conventional products and therefore encourages consumer green
purchase intention on the basis that perceived benefit is a strong influencer of consumer trust. Although consumers may have little environmental awareness, they would still express a
strong emotional attachment towards the environment’s wellbeing. F
h
l
d i
b
d
d
i
i Furthermore, several recommendations can be made to producing companies across
various sectors. Firstly, companies should prioritise research and development to innovate
and produce more environmentally friendly products that align with consumers’
sustainability values. This can involve adopting energy-efficient technologies, using
recyclable materials and reducing carbon emissions throughout the product lifecycle. Platform
providers in
collaborative
consumption Discussion
ifi
i
f Electricity
has a huge impact on mainly growing economies like Zimbabwe and is considered a
significant input of production, hence a study centred on the antecedents that influence
attitudes towards the use of environmentally friendly household appliance products in
Zimbabwe is of paramount importance. In Zimbabwe, noticeable green products are energy
saving bulbs, computers, refillable/reusable consumer product packages, solar energy
systems, ethanol blended fuel and washing machines, amongst others. Limitations of the study and future research avenues y
This study has its fair share of limitations. The usage of a cross-sectional design has
drawbacks. Future studies could adopt the longitudinal research design. This approach will
aid in investigating the behaviour of environmentally friendly household appliances over
time and not in a snapshot. In this way, time-specific solutions can be implemented over a
series of times. It is imperative to note that the results cannot be generalised to customers of
environmentally friendly household appliances in other countries. New studies should focus
on including respondents from other countries and, preferably, encompass cross-national
studies which compare attitudes of environmentally friendly household appliances between
different country samples. Nonetheless, the identified limitations offer a starting point for
future studies in the same area of interest as it provides general guidelines and suggestions
for the proposed antecedents that influence attitudes towards the use of environmentally
friendly household appliance products. Understanding the limitations mentioned above
enables future studies to address or scrutinise the above issues to expand the knowledge and
better understandability of this emerging yet exciting area of research. Akroush, M.N., Zuriekat, M.I., Al Jabali, H.I. and Asfour, N.A. (2019), “Determinants of purchasing
intentions of energy-efficient products: the roles of energy awareness and perceived benefits”,
International Journal of Energy Sector Management, Vol. 13 No. 1, pp. 128-148. Ajzen, I. and Fishbein, M. (1980), Understanding Attitudes and Predicting Social Behaviour, Prentice-
Hall, Englewood Cliffs, NJ. Ajzen, I. (2002), “Perceived behavioural control, self-efficacy, locus of control, and the theory of
planned behaviour 1”, Journal of Applied Social Psychology, Vol. 32 No. 4, pp. 665-683. Ajzen, I. (1991), “The theory of planned behaviour”, Organisational Behaviour and Human Decision
Process, Vol. 50 No. 2, pp. 179-211, doi: 10.1016/0749-5978(91)90020-T. Conclusion and implications for theory and practice
T
l d
h
d
li
h
i
i Secondly, companies should invest in robust marketing strategies to raise awareness and
educate consumers about the benefits of green products, highlighting their positive impact on PRR the environment and personal well-being. This can be done through effective communication
channels, such as social media, eco-friendly certifications and partnerships with
environmental organisations. Thirdly, companies should consider pricing strategies that
incentivise consumers to choose green products, such as offering competitive prices or
discounts for eco-friendly alternatives. Lastly, fostering collaboration and partnerships
among companies, government agencies and non-profit organisations can help establish
industry-wide standards and regulations for sustainable production practices. By adopting
these recommendations, companies can contribute to the reform of the environment on a
global scale, while also meeting the growing demand for environmentally friendly products
among consumers in Harare and beyond. g
y
The implication to theory suggests that attitude toward the behaviour relates to the
measure of the degree to which an individual has either a negative or positive outlook on his/
her performance of the behaviour. Perceived behavioural control is directly linked to
individual perceptions of whether they can perform that given behaviour or not, as well as
how easy it potentially is to perform whereas subjective norm is connected to the views and
opinions of others as to whether the individual should perform the behaviour. Thus,
perceived benefit can be explained as the expected value of a consumer relative to their
personal and environmental needs or wants whereby the more benefit a consumer receives,
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Environmental Psychology, Vol. 36, pp. 70-76. Li, Y., Siddik, A.B., Masukujjaman, M. and Wei, X. (2021), “Bridging green gaps: the buying intention
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Vol. 11 No. 21, pp. 1-21. Further reading Akkucuk, U. (2014), Handbook of Research on Developing Sustainable Value in Economics, Finance and
Marketing, IGI Global, Istanbul. Albright, J.J. and Park, H.M. (2009), “Confirmatory factor analysis using amos”, LISREL, Mplus, and
SAS/STAT CALIS. Working Paper, pp. 1-89. Brand South Africa (2014), “Poverty levels in South Africa dropping”, available at: https://www. brandsouthafrica.com/south-africa-fast-facts/poverty-040414 Platform
providers in
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consumption Li, Y., Siddik, A.B., Masukujjaman, M. and Wei, X. (2021), “Bridging green gaps: the buying intention
of energy efficient home appliances and moderation of green self-identity”, Applied Sciences,
Vol. 11 No. 21, pp. 1-21. Lin, P.-C. and Huang, Y.-H. (2012), “The influence factors on choice behaviour regarding green
products based on the theory of consumption values”, Journal of Cleaner Production, Vol. 1
No. 22, pp. 11-18. Lin, P.-C. and Huang, Y.-H. (2012), “The influence factors on choice behaviour regarding green
products based on the theory of consumption values”, Journal of Cleaner Production, Vol. 1
No. 22, pp. 11-18. Lin, P.-C. and Huang, Y.-H. (2012), “The influence factors on choice behaviour regarding green
products based on the theory of consumption values”, Journal of Cleaner Production, Vol. 1
No. 22, pp. 11-18. Platform
providers in
collaborative
consumption Social influence (Varshneya et al., 2017) SI1- My friends, often, recommend environmentally friendly household appliance products to me. SI2- My friends often go shopping for environmentally friendly household appliance products
with me. SI3- My friends often share their experiences and knowledge about environmentally friendly
household appliance products with me. Perceived benefit (Akroush et al., 2019) PB1-Environmentally friendly household appliance products give me extra value for example,
economic value, environmental value, social PB3- Environmentally friendly household appliance products can meet my requirements PB4- Environmentally friendly household appliance products give me more benefits than the costs
Attitude towards green appliances (Chen and Tung, 2014) AT6- If I had to choose between green appliances and conventional ones, I would prefer the green
version. Measurement scales
Green product awareness (Ansu-Mensah, 2021) GPA1-I have heard about green products. GPA2-I have detailed knowledge and understanding about green products. GPA2-I have detailed knowledge and understanding about green products. GPA3- I am aware of the difference between green products and conventional products. GPA3- I am aware of the difference between green products and conventional products. GPA4-I buy green products instead of common/conventional products. GPA5-I am aware that buying green products contributes to sustainable future. GPA5-I am aware that buying green products contributes to sustainable future. About the authors Eugine Tafadzwa Maziriri (Ph.D) is currently a Senior Lecturer in the Department of Business
Management at the University of Johannesburg (UJ) in Johannesburg, South Africa. He is a Y rated
scientist of the National Research Foundation (NRF). He is a entrepreneurship, small business
management and entrepreneurial marketing researcher who has produced several papers in these fields
in prestigious journals such European Journal of Innovation Management (Emerald Insight, UK),
European Journal of Management Studies (Emerald Insight, UK), Gender in Management: An
International Journal (Emerald Insight, UK), Journal of Public Affairs (Wiley, USA), Data in Brief
(Elsevier), Global Journal of Emerging Market Economies (SAGE, London, UK), Arab Gulf Journal of
Scientific Research (Emerald Insight, UK), PSU Research Review: An International Interdisciplinary
Journal (Emerald Insight, UK), Cogent Business and Management, and Cogent Psychology (Taylor &
Francis, England & Wales, UK), among others. He has also presented papers at local and international
conferences. Furthermore, he has supervised over 25 honour students, 10 master’s students and 2 PhD
students. He earned his PhD in Business Sciences from the University of Witwatersrand, Johannesburg,
South Africa. Also, he sits on the editorial board of the Southern African Journal of Entrepreneurship
and Small Business Management (SAJESBM). Moreover, he currently has over 1,500 citations to his
research works and reflect a Google scholar h-index of 20 and an i-10 index of 31. B i h
N
d
(Ph D) i
f ll i
l
d A/Ch i
f h D
f M
k i Brighton Nyagadza (Ph.D) is a full time lecturer and A/Chairperson of the Department of Marketing
(digital marketing) at Marondera University of Agricultural Sciences and Technology (MUAST),
Zimbabwe, a Research Associate of the Institute for the Future of Knowledge (IFK), University of
Johannesburg (UJ), South Africa, a full member of the Marketers Association of Zimbabwe (MAZ), an
Associate of The Chartered Institute of Marketing (ACIM), United Kingdom and a Power Member of the
Digital Marketing Institute (DMI), Dublin, Ireland. Green purchase intention (Ansu-Mensah, 2021) Green purchase intention (Ansu-Mensah, 2021) Green purchase intention (Ansu-Mensah, 2021) GPI1-I will likely purchase green products next month. GPI2- I intend to switch to a green variety of a product. GPI2- I intend to switch to a green variety of a product. GPI3- I am willing to purchase green products for personal use. PRR Gideon Mazuruse (MSc.) is a full time Mathematics and Statistics lecturer under the Teaching and
Learning Institute (TLI) at Marondera University of Agricultural Sciences and Technology (MUAST),
Zimbabwe. He holds MSc Statistics and Operations Research (NUST), BSc Hons Statistics and
Operations Research (GZU), BSc Mathematics and Computer Science (GZU), Post Graduate Diploma in
Education (ZOU). Platform
providers in
collaborative
consumption About the authors He has published several book chapters in Routledge
books, published by Taylor & Francis, New York (USA), Emerald Insight, United Kingdom (UK),
Lexington books published by Rowan & Littlefield, Maryland (USA) and in reputable international
journals such as Journal of Digital Media and Policy (Intellect), Sustainable Technology and
Entrepreneurship (Elsevier), Journal of Fashion Marketing and Management (Emerald), European
Journal of Management Studies (Emerald), Journal of Entrepreneurship in Emerging Economies
(Emerald), Journal of Environmental Media (Intellect), European Journal of Innovation Management
(Emerald), Africa Review (Brill), Tourism Critiques: Practice and Theory (Emerald), SN Social Sciences
(Springer), Journal of Asian and African Studies (SAGE), PSU Research Review (Emerald), Youth and
Society (SAGE), Quality Assurance in Education (Emerald), The Marketing Review (Westburn), among
others. Brighton sits on various corporate and academic boards including the Mashonaland East Province
Zimbabwe National Development Strategy (NDS) Committee (2021–2025) – ICT and Human Capital
Development cohort. Brighton Nyagadza is the corresponding author and can be contacted at:
brightonnyagadza@gmail.com g
y g
@g
Tinashe Chuchu (Ph.D) holds a Doctorate in Marketing from the University of the Witwatersrand. Currently, he works as a Senior Lecturer in the Marketing Division of the School of Business Sciences at
the University of the Witwatersrand, South Africa. He is a Y rated scientist of the National Research
Entrepreneurial Education Foundation (NRF). Previously, he worked as a Senior Lecturer in the
Department of Marketing Management, University of Pretoria, South Africa. He is a consumer
behaviour and tourism scholar who has published numerous studies in these fields in top journals and
has presented at international conferences. Dr Chuchu has published and reviewed for major publishing
outlets, which include Wiley, Taylor and Francis, Elsevier, Emerald Publishing, SAGE and presented at
the premier conference for marketing academics (the American Marketing Association Conference)
which was held in Chicago, USA, in 2019. He is a member of The Academy of Business and Retail
Management Conferences based in the UK. He was a recipient of the South African government’s
National Research Foundation Doctoral Scholarship in 2015. In 2019, Dr Chuchu was awarded the Best
Junior Researcher in Management Sciences at the University of Pretoria, South Africa. He sits on the
editorial board of the Retail and Marketing Review as well as the African Journal of Business and
Economic Research. About the authors Platform
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collaborative
consumption For instructions on how to order reprints of this article, please visit our website:
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Supramolecular dendrimer-containing layer-by-layer nanoassemblies for bioapplications: current status and future prospects
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Polymer chemistry
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Supramolecular dendrimer-containing layer-by-layer
nanoassemblies for bioapplications: Current status and future
prospects. 12(41), 5902-5930. Polymer Chemistry
This paper must be cited as: Sousa, C. F., Fernandez-Megia, E., Borges, J., & Mano,
J. F. Supramolecular dendrimer-containing layer-by-layer nanoassemblies
for bioapplications: Current status and future prospects. 12(41), 5902-5930.
Polymer Chemistry (2021).
https://doi.org/https://doi.org/10.1039/D1PY00988E This paper must be cited as: Sousa, C. F., Fernandez-Megia, E., Borges, J., & Mano,
J. F. Supramolecular dendrimer-containing layer-by-layer nanoassemblies
for bioapplications: Current status and future prospects. 12(41), 5902-5930. Polymer Chemistry (2021). https://doi.org/https://doi.org/10.1039/D1PY00988E Cristiana F. V. Sousa,
a Eduardo Fernandez-Megia,
b João Borges
*a and
João F. Mano
*a Dendrimers are powerful synthetic macromolecular architectures for a wide variety of bioapplications
owing to their unique and superior features, including monodispersity, well-defined and highly branched
architecture, multivalency, tunable size and shape, good water solubility, bioavailability, and precisely con-
trollable size at the nanometer-scale. However, the cationic and higher generation dendrimers have gen-
erally proven to raise cytotoxicity concerns, which leads to the disruption of cell membranes and cell
death, thus extensively limiting their use. Hence, the chemical functionalization of dendrimers’ surface
with desired functional moieties and their incorporation within supramolecular Layer-by-Layer (LbL) erally proven to raise cytotoxicity concerns, which leads to the disruption of cell membranes and cell
death, thus extensively limiting their use. Hence, the chemical functionalization of dendrimers’ surface
with desired functional moieties and their incorporation within supramolecular Layer-by-Layer (LbL) assemblies has been reported as an effective strategy to circumvent the safety issues and improve their
biological performance. Herein, we systematically review the multitude of intermolecular interactions
behind the build-up of supramolecular dendrimer-containing multifunctional LbL nanoensembles with
improved properties and enhanced functionalities for being used in a wide variety of bioapplications. We
envisage that such diversity of intermolecular interactions may increase the number of building blocks
that can be combined with dendrimers and processed into robust supramolecular multifunctional
nanoarchitectures across multiple length scales well-suited to be applied in biological and biomedical
scenarios, including in controlled drug/therapeutics/nucleic acid delivery, gene therapy, biosensing, bio-
imaging, and tissue engineering and regenerative medicine. The review also provides a glimpse on the
integration of the bottom-up LbL assembly technology with other bottom-up or top-down approaches for
shaping increasingly complex and sophisticated dendrimer-based supramolecular multifunctional
devices with high capability for being translated into practical bioapplications. aCICECO–Aveiro Institute of Materials, Department of Chemistry, University of
Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal.
E-mail: joaoborges@ua.pt, jmano@ua.pt
bCentro Singular de Investigación en Química Biolóxica e Materiais Moleculares
(CIQUS), Departamento de Química Orgánica, Universidade de Santiago de
Compostela, 15782 Santiago de Compostela, Spain Supramolecular dendrimer-containing layer-by-
layer nanoassemblies for bioapplications: current
status and future prospects Cristiana F. V. Sousa,
a Eduardo Fernandez-Megia,
b João Borges
*a and
João F. Mano
*a 1. Introduction as high biocompatibility, which turn them into highly appeal-
ing macromolecules for a wide variety of bioapplications (Fig. 1).5–13 Those include drug/gene/therapeutics delivery14–23 and
intracellular trafficking,24–27 gene therapy,28–30 gene trans-
fection,31 cancer therapy,32–34 (bio)molecular imaging,17,35–41
biosensing,9,26,42–45 and tissue engineering and regenerative
medicine16,46–51 due to their intrinsic ability to encapsulate,
protect, transport and on-demand targeted release of thera-
peutic molecules or imaging moieties at desired sites, as well as
intrinsic capacity to overcome the extra and intracellular
biological barriers.8,52–59 Dendrimers are powerful synthetic macromolecules that find
broad applicability in a wide array of fields owing to their
unique and superior structural and physicochemical pro-
perties. Those include their monodisperse nature and well-
defined hyperbranched three-dimensional polymeric globular
architecture, multivalency assigned by the high density of
terminal functional groups, high surface area, as well as their
tunable nanosize, shape, topology, chemical composition and
molecular weight.1–4 In addition, dendrimers can be designed to
denote good water solubility, non-immunogenicity, as well However, most commonly used dendrimers raise cyto-
toxicity concerns, which extensively limit their practical use in
the biological systems.60 This is particularly notorious for the
widely used amine-ended dendrimers of higher generations
(usually G ≥ 4), including poly(amidoamine) (PAMAM), poly
(ethylene imine) (PEI), poly( propylene imine) (PPI), and poly(L-
lysine),48,61–63 due to their multiple surface cationic charge and
high propensity to interact and disrupt the cell membrane Fig. 1 Histogram denoting the evolution in the number of yearly publi-
cations in the last ten years on the topics “dendrimer”, and “dendrimer
and biomedical applications” or “dendrimer and biological applications”
in the Web of Science database. ditions, with finely tuned compositions, structures, properties
and functions at the nanoscale.72 Such versatility is illustrated
by the multitude of templates,73,74 assembly methodologies,75–82
and intermolecular interactions83 that enable the precise,
controlled and elegant nanoscopic assembly of an un- precedented
array of complementary building blocks into a diverse set of
advanced functional multidimensional LbL architec- tures with
applications in a myriad of fields (Fig. 2).81,83–101 To date, the LbL technology has been by far mostly widely
employed to assemble oppositely charged polymers by resort-
ing to attractive electrostatic interactions.69,71,83,87,100 Among
them, dendrimers are very promising building blocks to be
used as ingredients within LbL assemblies owing to their
unique features and high versatility. 1. Introduction To date, the LbL technology has been by far mostly widely
employed to assemble oppositely charged polymers by resort-
ing to attractive electrostatic interactions.69,71,83,87,100 Among them, dendrimers are very promising building blocks to be
used as ingredients within LbL assemblies owing to their
unique features and high versatility. Fig. 1 Histogram denoting the evolution in the number of yearly publi-
cations in the last ten years on the topics “dendrimer”, and “dendrimer
and biomedical applications” or “dendrimer and biological applications”
in the Web of Science database. The ability of the LbL ensembles to encapsulate, protect and
preserve the biological activity of therapeutic agents until they are administered and delivered at desired locations is a
key feature of the LbL assembly technology.96,102–104 Therefore,
dendrimer-containing LbL nanoassemblies represent promi-
nent platforms for a wide array of bioapplications, overcoming
the limitations associated with the employment of free
dendrimers.105,106 Moreover, in contrast to the multilayered
systems enclosing linear polymers, the dendrimer-containing
LbL assemblies have shown that dendrimers can penetrate into
the interlayers. Although the thickness, morphology and growth
of the multilayers are strongly influenced by the assem- bling
conditions, the strong interactions between dendritic polymers
and the substrate make dendrimers able to change their
conformation to increase the adsorption point. Furthermore, the
employment of dendrimers has shown to be highly
advantageous to obtain thicker films.107 and lead to cell death. To improve the biological functionality
and, thus, performance of the cationic dendrimers under
physiological conditions, considerable efforts have been made
on the peripheral functionalization of their amine end groups
with poly(ethylene glycol) (PEG),18 or through conjugation
with natural saccharides64 or other targeting-specific ligands
or cargoes.13,19,21 Besides, the rational design and develop- ment
of
dendrimer-containing
multifunctional
multilayered
assemblies,65 by resorting to the prominent LbL assembly
technology, has been also deemed as a powerful strategy to
mitigate their cytotoxicity owing to its key enabling features. The LbL technology, firstly introduced by Iler in 196666 and
mostly known after the seminal work by Decher and co-
workers in the 1990s,67–71 has quickly emerged as a simple,
cost-effective, efficient, and highly versatile bottom-up method-
ology to functionalize surfaces and precisely assemble robust
and conformal multilayered architectures, under mild con- In this review, we systematically overview the multitude of
intermolecular interactions behind the fabrication of supra-
molecular dendrimer-based multilayered assemblies using the
LbL assembly technology to be used in a wide array of bioap- Cristiana F. V. 1. Introduction Sousa
Cristiana F. V. Sousa is a PhD
student
at
CICECO–Aveiro
Institute
of
Materials,
Department
of
Chemistry,
University of Aveiro, Portugal. She received her BSc and MSc
degrees in Biochemistry from the
University of Trás-os-Montes and
Alto Douro and Faculty of Sciences
of the University of Porto, both in
Portugal, in 2016 and 2018,
respectively. Her
doc-
toral
research, under the supervi- sion
of Prof. João F. Mano at
COMPASS Research Group, focuses on the design and development
of smart and modular dendrimer-containing multilayered assem-
blies, based on the layer-by-layer assembly technology, for targeted Cristiana F. V. Sousa Cristiana F. V. Sousa Eduardo
Fernandez-Megia
is
Full
Professor
of
Organic
Chemistry at the Universidade de
Santiago de Compostela, where he
works at the Centro Singular de
Investigación
en
Química
Biolóxica e Materiais Moleculares
(CIQUS). His lab- oratory focuses
on the interface between chemical
biology and polymer chemistry
with empha- sis on well-defined
polymeric
nanostructures
for
biomedical applications, click and
acceler- Eduardo
Fernandez-Megia
is
Full
Professor
of
Organic
Chemistry at the Universidade de
Santiago de Compostela, where he
works at the Centro Singular de
Investigación
en
Química
Biolóxica e Materiais Moleculares
(CIQUS). His lab- oratory focuses
on the interface between chemical
biology and polymer chemistry
with empha- sis on well-defined
polymeric
nanostructures
for
biomedical applications, click and
acceler- Eduardo
Fernandez-Megia
is
Full
Professor
of
Organic
Chemistry at the Universidade de
Santiago de Compostela, where he
works at the Centro Singular de
Investigación
en
Química
Biolóxica e Materiais Moleculares
(CIQUS). His lab- oratory focuses
on the interface between chemical
biology and polymer chemistry
with empha- sis on well-defined
polymeric
nanostructures
for
biomedical applications, click and
acceler- Eduardo Fernandez-Megia Eduardo Fernandez-Megia Cristiana F. V. Sousa COMPASS Research Group, focuses on the design and development
of smart and modular dendrimer-containing multilayered assem-
blies, based on the layer-by-layer assembly technology, for targeted COMPASS Research Group, focuses on the design and development
of smart and modular dendrimer-containing multilayered assem-
blies, based on the layer-by-layer assembly technology, for targeted intracellular bone therapeutics delivery. intracellular bone therapeutics delivery. ated conjugation protocols for stimuli-responsive materials, the
synthesis of dendrimers, and the multivalent functionalization of
proteins to modulate the complex mechanisms governing multi-
valent interactions. Fig. 2 Schematic illustration of the high versatility endowed by the LbL assembly technology. 1. Introduction Mano is a Full Professor
at the Chemistry Department of
University of Aveiro, Portugal,
where he is directing both the MSc
and
PhD
programs
of
Biotechnology. He is the founder
and director of the COMPASS
Research
Group,
from
the
Associated Laboratory CICECO–
Aveiro Institute of Materials. He
has been applying biomimetic and
nano/micro-technology
approaches using advanced bio-
materials in order to develop
biomedical devices with improved structural and multifunctional
properties, to be exploited clinically in advanced therapies or in
drug screening. He is the Editor-in-Chief of Materials Today Bio
(Elsevier). João Borges
João Borges is an Assistant
Researcher at CICECO–Aveiro
Institute
of
Materials,
Department
of
Chemistry,
University of Aveiro, Portugal. He
graduated and received his Ph.D. in Chemistry from the University
of Porto, Portugal, in 2008 and
2013, respectively. From 2013–
2018 he conducted post- doctoral
research at
the University of
Minho and University of Aveiro,
Portugal,
focusing
on
the
development of
advanced biomimetic nanostructured multilayer coatings for bio-
medical applications. His current research focuses on the mole-
cular design, synthesis, and development of bioinstructive soft
supramolecular multicomponent biomaterials, by resorting to
(bio)polymers and self-assembling peptides, to interface with
living systems. João F. Mano is a Full Professor
at the Chemistry Department of
University of Aveiro, Portugal,
where he is directing both the MSc
and
PhD
programs
of
Biotechnology. He is the founder
and director of the COMPASS
Research
Group,
from
the
Associated Laboratory CICECO–
Aveiro Institute of Materials. He
has been applying biomimetic and
nano/micro-technology
approaches using advanced bio-
materials in order to develop João Borges João F. Mano is a Full Professor
at the Chemistry Department of
University of Aveiro, Portugal,
where he is directing both the MSc
and
PhD
programs
of
Biotechnology. He is the founder
and director of the COMPASS
Research
Group,
from
the
Associated Laboratory CICECO–
Aveiro Institute of Materials. He
has been applying biomimetic and
nano/micro-technology
approaches using advanced bio-
materials in order to develop João Borges João F. Mano João Borges is an Assistant
Researcher at CICECO–Aveiro
Institute
of
Materials,
Department
of
Chemistry,
University of Aveiro, Portugal. He
graduated and received his Ph.D. in Chemistry from the University
of Porto, Portugal, in 2008 and
2013, respectively. From 2013–
2018 he conducted post- doctoral
research at
the University of
Minho and University of Aveiro,
Portugal,
focusing
on
the
development of João F. Mano João Borges João F. 1. Introduction The final properties, functions, performance and end-
use of the resulting multilayered structures are intrinsically dependent on the template features, building blocks, intermolecular interactions, and
assembly methodologies. Adapted from ref. 81 with permission from the American Association for the Advancement of Science, copyright 2015. Adapted from ref. 97 with permission from Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, copyright 2016. Fig. 2 Schematic illustration of the high versatility endowed by the LbL assembly technology. The final properties, functions, performance and end-
use of the resulting multilayered structures are intrinsically dependent on the template features, building blocks, intermolecular interactions, and
assembly methodologies. Adapted from ref. 81 with permission from the American Association for the Advancement of Science, copyright 2015. Adapted from ref. 97 with permission from Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, copyright 2016. Fig. 2 Schematic illustration of the high versatility endowed by the LbL assembly technology. The final properties, functions, performance and end-
use of the resulting multilayered structures are intrinsically dependent on the template features, building blocks, intermolecular interactions, and
assembly methodologies. Adapted from ref. 81 with permission from the American Association for the Advancement of Science, copyright 2015. Adapted from ref. 97 with permission from Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, copyright 2016. The last section provides a glimpse on the current status and
future perspectives on the integration of dendrimers and LbL
assembly technology with either other bottom-up and/or top-
down methodologies aiming for the rational design and devel-
opment of complex and innovative supramolecular nano-
devices, exhibiting improved properties and enhanced multi-
functionalities, for being used in a wide array of biomedical,
biological and biotechnological applications. The last section provides a glimpse on the current status and
future perspectives on the integration of dendrimers and LbL
assembly technology with either other bottom-up and/or top-
down methodologies aiming for the rational design and devel-
opment of complex and innovative supramolecular nano-
devices, exhibiting improved properties and enhanced multi-
functionalities, for being used in a wide array of biomedical,
biological and biotechnological applications. plications, including biosensing, drug/therapeutics delivery,
and gene therapy. Several examples are given on supramolecu-
lar dendrimer-containing multilayered nanoensembles fabri-
cated from electrostatic, host–guest, coordination chemistry,
and biologically specific interactions, as well as from hydrogen
bonding, and covalent bonding. Moreover, the potential impact
of the developed supramolecular LbL nanoassemblies in
biomedical and biological applications is also discussed. João F. Mano
João F. 1. Introduction Mano biomedical devices with improved structural and multifunctional
properties, to be exploited clinically in advanced therapies or in
drug screening. He is the Editor-in-Chief of Materials Today Bio
(Elsevier). biomedical devices with improved structural and multifunctional
properties, to be exploited clinically in advanced therapies or in
drug screening. He is the Editor-in-Chief of Materials Today Bio
(Elsevier). advanced biomimetic nanostructured multilayer coatings for bio-
medical applications. His current research focuses on the mole-
cular design, synthesis, and development of bioinstructive soft
supramolecular multicomponent biomaterials, by resorting to
(bio)polymers and self-assembling peptides, to interface with
living systems. 2.1 Electrostatic interactions The electrostatic interaction is the most explored mechanism
behind the fabrication of multilayered assemblies when both the
molecule
and
the
adsorbent
surface
are
electrically
charged.83,108 Recently, considerable attention has been devoted
to the combined use of dendrimers and oppositely charged
polymers (Fig. 3a) or solely oppositely charged dendri- mers
(Fig. 3b) as building blocks for the development of
electrostatically-driven supramolecular multilayered thin films
taking advantage of their charged terminal functional groups,
such as amine, carboxyl, or sulfate groups.109–116 This inter-
molecular interaction represents the most explored method to
prepare multilayered assemblies encompassing dendrimers. Indeed, LbL films encompassing dendrimers and distinct
oppositely
charged
polymers,
such
as
carboxylated
azopolymer,109,114 poly(acrylic acid) (PAA),110 sulfonated poly
(aniline) (SPANI),111 polyaniline (PANI),117 poly(styrene sulfo-
nate) (PSS),115 poly(vinyl sulfonic acid) (PVS),112 poly(diallyl-di-
methylammonium chloride) (PDDA) and carboxylated poly(gly-
cerol)113 have been developed. Among the dendrimers,
PAMAM has been the most widely used to build-up multi-
layered structures due to their straightforward synthesis, low
polydispersity and commercial availability.118 2.1.1 Factors governing the growth of LbL assemblies 2.1.1.1 Influence of solution pH. Many dendritic families
include tertiary amines at the interior and primary amines on
the surface and, as such, their conformation in aqueous solu-
tion is affected by changes in the pH, which in turn influence
the preparation of dendrimer-based LbL films assembled via
electrostatic interactions.106,107 The pKa of terminal primary
amines is 9.23 whereas the pKa of tertiary ones at the branch-
ing points is 6.3.122 In this regard, at high pH, dendrimers 2.1.1.1 Influence of solution pH. Many dendritic families
include tertiary amines at the interior and primary amines on
the surface and, as such, their conformation in aqueous solu-
tion is affected by changes in the pH, which in turn influence
the preparation of dendrimer-based LbL films assembled via
electrostatic interactions.106,107 The pKa of terminal primary
amines is 9.23 whereas the pKa of tertiary ones at the branch-
ing points is 6.3.122 In this regard, at high pH, dendrimers Fig. 3 Schematic illustration denoting the preparation of (a) dendrimer/polymer and (b) dendrimer/dendrimer LbL nanofilms on solid surfaces via
electrostatic interactions. In-between the alternate immersion in the polycation and polyanion aqueous solutions, the substrate is immersed in an
aqueous solution to remove weakly adsorbed layers and avoid the contamination of the polymer solutions by liquid adhering to the substrate. Fig. 2.
Driving forces behind the
fabrication of dendrimer-containing
LbL assemblies Besides the fabrication of dendrimer/polymer multilayered
assemblies, similar LbL systems have been also developed by
resorting to solely oppositely charged dendrimers. In this
regard, Tsukruk et al. reported the build-up of multilayered
films comprising amine- and carboxyl-terminated PAMAM
dendrimers of adjacent generations on a silicon wafer.119 The
authors assembled up to 20 layers and demonstrated that the
PAMAM dendrimers were highly compressed and flattened. Moreover, Yoshimura and co-workers developed LbL films con-
taining dendrimers surface functionalized with oppositely
charged metal nanoparticles (NPs), namely oppositely charged
gold (Au)- and silver (Ag)-dendrimer nanocomposites for being
used as thin conducting or catalytic films.120 It has been
demonstrated
that
several
physicochemical
parameters,
including the solution pH and ionic strength and the intrinsic
properties of the polyelectrolytes such as the concentration,
molecular weight and charge density play a key role in the
deposition, stability, structure, properties and functions of the
assembled multilayered structures, as described below.83,121 2.1 Electrostatic interactions 3 Schematic illustration denoting the preparation of (a) dendrimer/polymer and (b) dendrimer/dendrimer LbL nanofilms on solid surfaces via
electrostatic interactions. In-between the alternate immersion in the polycation and polyanion aqueous solutions, the substrate is immersed in an
aqueous solution to remove weakly adsorbed layers and avoid the contamination of the polymer solutions by liquid adhering to the substrate. Fig. 3 Schematic illustration denoting the preparation of (a) dendrimer/polymer and (b) dendrimer/dendrimer LbL nanofilms on solid surfaces via
electrostatic interactions. In-between the alternate immersion in the polycation and polyanion aqueous solutions, the substrate is immersed in an
aqueous solution to remove weakly adsorbed layers and avoid the contamination of the polymer solutions by liquid adhering to the substrate. denote a denser and globular conformation with lower surface
charge density because only the primary amines on the term-
inals are protonated. In contrast, at low pH, dendrimers with
an open and extended conformation and higher charge density
prevail due to the repulsions between the protonated tertiary
amines at the interior and primary amines at the surface.111 In
fact, Kim and Bruening revealed that the PAMAM/PAA
multilayered growth is dependent on the solution pH.110 The
highest thickness was obtained while assembling PAMAM and
PAA aqueous solutions at pH 8 and 4 (Fig. 4a and b), respectively,
since both PAMAM and PAA are only partially charged. Moreover, those films exhibited the highest surface roughness,
which is probably assigned to the fastest formation of the film
(Fig. 4c–f). lower surface charge and more condensed structured denoted
by PAMAM, which reduces the layer interpenetration and leads
to a highly ordered multilayered film. It was also shown that,
irrespectively of the pH of the solutions, the amount of SPANI
and PAMAM adsorbed increased while increasing the number
of bilayers. 2.1.1.2 Influence of ionic strength. The growth of multi-
layered films is also strongly influenced by the ionic strength. In this regard, Li et al. reported that, at high ionic strength
(0.4 M), charged dendrimers display a denser core confor-
mation (Fig. 5a) with a lower surface charge whereas at low
ionic strength (0.2 M), dendrimers exhibited higher charge
density and might adopt a disk-like structure (Fig. 5b) and, as
such, more dendritic molecules will be absorbed into the film
surface because of the balance of the charges.111 Moreover, the
addition of salt increased the adsorption of SPANI in SPANI/
PAMAM LbL films (Fig. 2.1 Electrostatic interactions The substrate was coated with a priming layer of PEI
solution, followed by alternating layers of PSS and PAMAM via electro-
static interactions. Adapted from ref. 123 with permission from the
American Chemical Society, copyright 2002. Fig. 4 Influence of the solution pH in the thickness of 2.5 bilayers com-
posed of (a) G4 PAMAM dendrimers and PAA and (b) G8 PAMAM dendri-
mers and PAA. Microscopy images of 2.5 bilayers of (c) G8 PAMAM ( pH
= 4) and PAA ( pH = 4) and (d) G8 PAMAM ( pH = 8) and PAA ( pH = 4). The ellipsometric thickness of the films were 80 and 4000 Å, respect-
ively. Scanning electron microscope (SEM) images of the cross section
of porous alumina (e) before and (f) after coating with 4.5 bilayers of G4
PAMAM ( pH = 8) and PAA ( pH = 4) film. Adapted from ref. 110 with per-
mission from the American Chemical Society, copyright 2003. Fig. 5 Schematic illustration of analogous dendrimers to G6 PAMAM in
the presence of (a) high salt and (b) low salt concentration. Reproduced
from ref. 124 with permission from the American Chemical Society,
copyright 1998. (c) Effect of the ionic strength on the absorbance at
475 nm in SPANI/PAMAM multilayered films as a function of the number
of layers. Reproduced from ref. 111 with permission from the American
Chemical Society, copyright 2003. (d) Evolution of the absorbance at
227 nm as a function of the number of deposited bilayers for PSS/
PAMAM multilayered films prepared in buffer containing different salt
concentrations. The substrate was coated with a priming layer of PEI
solution, followed by alternating layers of PSS and PAMAM via electro-
static interactions. Adapted from ref. 123 with permission from the
American Chemical Society, copyright 2002. Fig. 5 Schematic illustration of analogous dendrimers to G6 PAMAM in
the presence of (a) high salt and (b) low salt concentration. Reproduced
from ref. 124 with permission from the American Chemical Society,
copyright 1998. (c) Effect of the ionic strength on the absorbance at
475 nm in SPANI/PAMAM multilayered films as a function of the number
of layers. Reproduced from ref. 111 with permission from the American
Chemical Society, copyright 2003. (d) Evolution of the absorbance at
227 nm as a function of the number of deposited bilayers for PSS/
PAMAM multilayered films prepared in buffer containing different salt
concentrations. 2.1 Electrostatic interactions 5c), which can be due to the partially
neutralization of its net charge. Khopade and Caruso also
studied the influence of salt concentration in the growth of
multilayered films encompassing PAMAM and PSS, showcas-
ing that the amount of adsorbed PSS per bilayer increased while
increasing the salt concentration up to 0.5–1 M, after which it
started to decrease to values similar to the ones obtained in the
absence of salt (Fig. 5d).123 Almost at the same time, Imae and co-workers prepared
SPANI/PAMAM LbL films and investigated the influence of the
solution pH on the film growth.111 They assembled multi-
layered films composed of either SPANI and PAMAM solutions
at pH 6 (fully charged) or PAMAM at pH 9.8 ( partially charged)
and SPANI at pH 6 (fully charged) and found that the amount
of SPANI and PAMAM adsorbed during the multilayered film
growth using PAMAM solutions at pH 9.8 is larger than the one
adsorbed while resorting to solutions at pH 6 due to the Fig. 4 Influence of the solution pH in the thickness of 2.5 bilayers com-
posed of (a) G4 PAMAM dendrimers and PAA and (b) G8 PAMAM dendri-
mers and PAA. Microscopy images of 2.5 bilayers of (c) G8 PAMAM ( pH
= 4) and PAA ( pH = 4) and (d) G8 PAMAM ( pH = 8) and PAA ( pH = 4). The ellipsometric thickness of the films were 80 and 4000 Å, respect-
ively. Scanning electron microscope (SEM) images of the cross section
of porous alumina (e) before and (f) after coating with 4.5 bilayers of G4
PAMAM ( pH = 8) and PAA ( pH = 4) film. Adapted from ref. 110 with per-
mission from the American Chemical Society, copyright 2003. Fig. 5 Schematic illustration of analogous dendrimers to G6 PAMAM in
the presence of (a) high salt and (b) low salt concentration. Reproduced
from ref. 124 with permission from the American Chemical Society,
copyright 1998. (c) Effect of the ionic strength on the absorbance at
475 nm in SPANI/PAMAM multilayered films as a function of the number
of layers. Reproduced from ref. 111 with permission from the American
Chemical Society, copyright 2003. (d) Evolution of the absorbance at
227 nm as a function of the number of deposited bilayers for PSS/
PAMAM multilayered films prepared in buffer containing different salt
concentrations. 2.1 Electrostatic interactions The substrate was coated with a priming layer of PEI
solution, followed by alternating layers of PSS and PAMAM via electro-
static interactions. Adapted from ref. 123 with permission from the
American Chemical Society, copyright 2002. Fig. 4 Influence of the solution pH in the thickness of 2.5 bilayers com-
posed of (a) G4 PAMAM dendrimers and PAA and (b) G8 PAMAM dendri-
mers and PAA. Microscopy images of 2.5 bilayers of (c) G8 PAMAM ( pH
= 4) and PAA ( pH = 4) and (d) G8 PAMAM ( pH = 8) and PAA ( pH = 4). The ellipsometric thickness of the films were 80 and 4000 Å, respect-
ively. Scanning electron microscope (SEM) images of the cross section
of porous alumina (e) before and (f) after coating with 4.5 bilayers of G4
PAMAM ( pH = 8) and PAA ( pH = 4) film. Adapted from ref. 110 with per-
mission from the American Chemical Society, copyright 2003. Fig. 6 (a) QCM frequency shift against layers number of PSS/PAMAM
multilayered film. (b) Evolution of the absorbance at 227 nm as function
of the number of layers of PSS/PAMAM deposited on a quartz slide. The
inset figure shows the UV-Visible spectra after PSS (solid line) and
PAMAM dendrimer (dotted line) adsorption. Reproduced from ref. 128
with permission from the American Chemical Society, copyright 2002. Evolution of the absorbance at 227 nm in PSS/PAMAM multilayered
films, prepared with (c) G2, 3 or 4 PAMAM dendrimers and (d) PSS with a
molecular weight of 13 000, 32 000, 43 000 or 70 000 Da, as a function
of the number of layers. The first layer deposited was PEI, followed by
alternating adsorption of layers of PSS and PAMAM. Reproduced from
ref. 123 with permission from the American Chemical Society, copyright
2002. Almost fifty years ago, van den Berg and Staverman
suggested the formation of PEI-anionic surfactant sodium
dodecyl sulfate (SDS) complexes by the association of PEI, SDS
and sodium chloride (NaCl).125 Later on, Windsor et al. 2.1 Electrostatic interactions showed
that the addition of NaCl to PEI containing solutions at a low
ionic strength induced a large ordering effect on adsorbed SDS
at a lower bulk concentration at the solid–liquid interface,
attributing these conformational changes to the for- mation of a
complex between PEI and SDS.126 In this regard, Wang and co-
workers developed multilayered films containing positively
charged PEI dendrimer and oppositely charged azo- benzene
polymer (PAZO)-SDS and evaluated the effect of SDS in the
film growth behavior.127 It was found that the amount of
PAZO adsorbed decreased while increasing the concen-
tration of SDS, until the critical micelle concentration of SDS
was reached, due to the competition of SDS and PAZO for PEI
binding sites. Thus, the adsorption of SDS was followed by a
slower PAZO adsorption step, with PAZO occupying the
remaining binding sites on the PEI layer. As such, SDS interpe-
netrates through the PAZO and PEI layers, which in turn
improves the surface hydrophobicity of both. Fig. 6 (a) QCM frequency shift against layers number of PSS/PAMAM
multilayered film. (b) Evolution of the absorbance at 227 nm as function
of the number of layers of PSS/PAMAM deposited on a quartz slide. The
inset figure shows the UV-Visible spectra after PSS (solid line) and
PAMAM dendrimer (dotted line) adsorption. Reproduced from ref. 128
with permission from the American Chemical Society, copyright 2002. Evolution of the absorbance at 227 nm in PSS/PAMAM multilayered
films, prepared with (c) G2, 3 or 4 PAMAM dendrimers and (d) PSS with a
molecular weight of 13 000, 32 000, 43 000 or 70 000 Da, as a function
of the number of layers. The first layer deposited was PEI, followed by
alternating adsorption of layers of PSS and PAMAM. Reproduced from
ref. 123 with permission from the American Chemical Society, copyright
2002. 2.1.1.3 Influence of dendrimer generation, polymer molecular
weight, concentration and charge density. The effect of the den-
drimer generation, polymer molecular weight and material’s
concentration on the film growth and the architecture of the
resulting multilayered films have been also explored. For
instance, Khopade and Caruso studied the build-up of multi-
layered films composed of PAMAM dendrimers bearing term-
inal primary amines and negatively charged PSS on the quartz
crystal microbalance (QCM), which revealed a linear growth of
the multilayered film (Fig. 6a). 2.1 Electrostatic interactions However, the ultraviolet-visible
(UV-Visible) spectra of the multilayered film growth showed a
partial removal of PSS layers after the deposition of PAMAM,
revealing an adsorption–desorption trend (Fig. 6b).123,128 We
hypothesize that the different behavior observed in Fig. 6a and
b is assign to the removal of PSS from the film by the dendri-
mer solution but may also be a consequence of the PAMAM
removal from the film to the PSS solution after the deposition
of PSS. Unfortunately, the desorption of PAMAM could not be
verified in the UV-Visible spectra because PAMAM does not
absorb within the range analyzed. Moreover, it was shown that
the multilayered film growth increased linearly while increas-
ing the dendrimer generation (Fig. 6c) or PSS molecular weight
(Fig. 6d). and observed that the amount of PAZO adsorbed per bilayer
was higher when a DAB-Am of higher generation was used.114
Kim and Bruening also studied the influence of the dendrimer
generation and polymer molecular weight by developing LbL
films encompassing G4 or G8 PAMAM dendrimers and PAA of
2000 or 90 000 Da and observed that the multilayered film growth
was much smaller while resorting to the lower dendri- mer
generation and PAA with molecular weight of 2 000.110
Furthermore, the multilayered films developed by Imae and co-
workers, comprising SPANI and PAMAM, showed that the
amount of SPANI absorbed per bilayer increased while increas-
ing its concentration until a maximum of ∼1 mM and then
decreased.111 As such, when a SPANI solution of high concen-
tration (5 mM) was used, more PAMAM dendrimers absorbed
in the previous layer were removed by the SPANI chains,
leading to a decrease in the surface charge density and, there-
fore, less SPANI adsorbed. Furthermore, LbL films prepared by alternate immersion of
glass substrates into solutions of PPI dendrimer with amino
surface groups (DAB-Am) and metallic AgNPs revealed that the
multilayered film growth was higher with G5 dendrimers than
with the G1 counterparts (using the same concentration).129
Such behavior suggests that G5 dendrimers have more avail-
able amino terminal functional groups able to bind AgNPs
when compared with the G1 dendrimers. Moreover, it was
found that the number of deposited layers plays a key role in the
morphology and roughness of the multilayered films. Similarly,
Mendonça and co-workers developed multilayered films
comprising G1, 3 or 5 DAB-Am dendrimers and PAZO 2.2 Hydrogen bonding Although the electrostatic interaction between oppositely
charged building blocks is by far the most commonly employed
driving force in the build-up of multilayered films, many other
intermolecular interactions can be used, enabling the
incorporation of uncharged materials within the LbL
assemblies. Those
interactions
include
hydrogen
bonding,130,131 which is a feasible driving force to create multi- layered films, provided that the building blocks to be assembled
present moieties that can act as hydrogen bonding donors and
acceptors.83,132 Hydrogen-bonded LbL films are prepared in a
manner reminiscent of electrostatically-driven LbL films, i.e. by the alternate immersion of virtually any type of substrate in
solutions of hydrogen donor and acceptor materials.132
Although the formation of hydrogen-bonded LbL films can be
performed in aqueous solutions, which is highly desired when
working with biological materials, it can also be attempted in
organic solvents, thus enabling the incorporation of water-
insoluble materials within the assembly process.130,131 (G1)4 and An(G2)4], respectively. It was shown that even the An
(G1)4 was able to provide enough hydrogen bonding pairs for
the preparation of multilayered films. In both cases, the films
presented granular morphology with roughness of 0.254 and
0.457 nm, respectively. The difference in roughness was attrib-
uted to the molecular structure of the dendrimers. This work
demonstrated that the multilayered process can be performed
using different generations of the same dendritic family (Fig. 7e). (G1)4 and An(G2)4], respectively. It was shown that even the An
(G1)4 was able to provide enough hydrogen bonding pairs for
the preparation of multilayered films. In both cases, the films
presented granular morphology with roughness of 0.254 and
0.457 nm, respectively. The difference in roughness was attrib-
uted to the molecular structure of the dendrimers. This work
demonstrated that the multilayered process can be performed
using different generations of the same dendritic family (Fig. 7e). 2.2.2 Effect of the hydrogen bonding donor architecture on
the release rate and structure of multilayered assemblies. A
multilayered film relying on two different components was also
reported by the alternate deposition of DEN-COOH (hydrogen
bonding donor) and poly(4-vinylpyridine) (PVP) (hydrogen
bonding acceptor) (Fig. 8a).130 Surprisingly, DEN-COOH was
slowly released from PVP/DEN-COOH multi- layered films
when compared to the release rate of PAA from PVP/PAA
multilayered films, in a basic solution.135 Those films 2.2.1 Hydrogen bonding-based single component films. 2.2 Hydrogen bonding Dendrimers presenting carboxyl groups on its periphery, thus
acting as hydrogen bonding donor, as well as hydrogen bonding
acceptor have been used as film components. In this regard,
Huo et al. reported the build-up of a single component
hydrogen-bonded LbL assemblies (Fig. 7a) encompassing
solely a carboxyl-terminated polyether dendrimer (DEN-COOH) (Fig. 7b), acting both as hydrogen bonding donor and hydro-
gen bonding acceptor.133 Although the dendrimer adsorption
process was continuous and uniform during the production of
the multilayered film, the infrared reflection and transmission
spectra suggest that some rearrangements occurred in the den-
drimers after their deposition on the substrate, which may
facilitate the formation of hydrogen bonding for stabilizing
the multilayers. Two years later, Sun et al. also demonstrated
that single component multilayered assemblies can be pre- pared
by self-deposition of one or two generations of dendri- mers.134
The authors developed single component multi- layered films
using either the first (Fig. 7c) or second (Fig. 7d) generation of
poly(aryl ether) dendrimers with a diphenylan- thracene core,
and 8 or 16 carboxyl end groups, named [An Fig. 8 (a) Schematic drawing showing the growth of PVP/DEN-COOH
multilayered films on a NH2-modified quartz substrate based on hydro-
gen bonding: adsorption of (I) PVP and (II) DEN-COOH. AFM images of a
(PVP/DEN-COOH)14 multilayered film deposited onto a quartz substate after
immersion in a basic solution for (b) 10, (c) 30, (d) 60 and (e)
180 min. Adapted from ref. 130 with permission from the American
Chemical Society, copyright 2003. Fig. 7 (a) Schematic illustration of the LbL assembly process encom-
passing a single component dendrimer on a NH2-modified quartz sub-
strate via hydrogen bonding. (b) The chemical structure of DEN-COOH. Reproduced from ref. 133 with permission from the Royal Society of
Chemistry, copyright 2003. Atomic force microscopy (AFM) images of
nine bilayers of (c) An(G1)4 and (d) An(G2)4 assembled on a quartz sub-
strate. The image size is 1.5 µm × 1.5 µm. (e) UV-Visible absorption
spectra of single component films by self-deposition of either An(G1)4
or An(G2)4 with similar characteristic absorption of diphenylanthracene
in the range between 350–410 nm. Adapted from ref. 134 with per-
mission from Elsevier, copyright 2005. Fig. 7 (a) Schematic illustration of the LbL assembly process encom-
passing a single component dendrimer on a NH2-modified quartz sub-
strate via hydrogen bonding. (b) The chemical structure of DEN-COOH. Reproduced from ref. 2.2 Hydrogen bonding 133 with permission from the Royal Society of
Chemistry, copyright 2003. Atomic force microscopy (AFM) images of
nine bilayers of (c) An(G1)4 and (d) An(G2)4 assembled on a quartz sub-
strate. The image size is 1.5 µm × 1.5 µm. (e) UV-Visible absorption
spectra of single component films by self-deposition of either An(G1)4
or An(G2)4 with similar characteristic absorption of diphenylanthracene
in the range between 350–410 nm. Adapted from ref. 134 with per-
mission from Elsevier, copyright 2005. Fig. 7 (a) Schematic illustration of the LbL assembly process encom-
passing a single component dendrimer on a NH2-modified quartz sub-
strate via hydrogen bonding. (b) The chemical structure of DEN-COOH. Reproduced from ref. 133 with permission from the Royal Society of
Chemistry, copyright 2003. Atomic force microscopy (AFM) images of
nine bilayers of (c) An(G1)4 and (d) An(G2)4 assembled on a quartz sub-
strate. The image size is 1.5 µm × 1.5 µm. (e) UV-Visible absorption
spectra of single component films by self-deposition of either An(G1)4
or An(G2)4 with similar characteristic absorption of diphenylanthracene
in the range between 350–410 nm. Adapted from ref. 134 with per-
mission from Elsevier, copyright 2005. Fig. 8 (a) Schematic drawing showing the growth of PVP/DEN-COOH
multilayered films on a NH2-modified quartz substrate based on hydro-
gen bonding: adsorption of (I) PVP and (II) DEN-COOH. AFM images of a
(PVP/DEN-COOH)14 multilayered film deposited onto a quartz substate after
immersion in a basic solution for (b) 10, (c) 30, (d) 60 and (e)
180 min. Adapted from ref. 130 with permission from the American
Chemical Society, copyright 2003. exhibited different microporous morphology, which in turn
prove that the molecular structure of a building block influ-
ences the behavior of multilayered films prepared by hydrogen
bonding. However, it is important to bear in mind that those
differences can also be the result of the different number of
assembled layers. As can be seen from Fig. 8b, the immersion
of PVP/DEN-COOH multilayered films for 10 min in a basic
solution ( pH = 12.5) led to a high and homogeneous surface
coverage with granular structures, and no porous structure was
observed. However, after the immersion for longer times (Fig. 8c–e), the PVP/DEN-COOH multilayered film revealed the
formation of nanosized pores with a higher diameter and depth. More recently, Sun et al. 2.2 Hydrogen bonding developed multilayered films by the
alternate deposition of mixed solutions of two carboxyl- ended
poly(aryl ether) dendrimers [Por(G2)4 with a porphyrin core
and An(G2)4 with a diphenylanthracene core] and PVP.136 It is
worth mentioning that the composition of the two dendri- mers
in the multilayered films can be adjusted by playing with their
ratio in the mixed solution, which allows a precise control of
the composition of the multilayered film. pH 4, they are positively charged, which aids PMAA in binding
to PAMAM-COOH. In contrast, at pH 2, PMAA is completely
protonated, so without electrostatic affinity to the dendrimer. On
the other hand, at pH 7, a large fraction of carboxylate groups
in PMAA and PAMAM dendrimers are negatively charged and,
thus, no complex formation has been observed. Therefore,
PMAA/PAMAM-COOH films are stable only when hydrogen
bonding and electrostatic interactions are simul- taneously
available. Thus, the pH-sensitive nature of LbL films based on
hydrogen bonding is highly useful for the develop- ment of pH-
triggered delivery systems. In another interesting contribution, Park et al. reported the
preparation of multilayered films enclosing block copolymer
micelles derived from amphiphilic poly(2-ethyl-2-oxazoline)-
block-poly(ε-caprolactone) (PetOz-PCL) and PAMAM-COOH at
pH 3 via hydrogen bonding.139 The PEtOZ outer shell of the
micelle afforded the pH-responsive hydrogen bonding sites to
the PAMAM-COOH. It was observed that, after the immersion
in an aqueous solution at pH 3, the multilayered film showed
densely packed micelles. On the other hand, the dipping of
such films in an aqueous solution at pH ≥5.6 revealed that the
multilayered film was removed in a reversible manner since
the hydrogen bonding between the PEtOZ shell of the PEtOZ-
PCL micelle and the PAMAM end groups was vanished. Moreover, PEtOz-PCL micelle was loaded with a dye (disperse
red 1), showcasing that most of the micelles were released from
the multilayered film after 1 min of immersion in an aqueous
solution at pH 5.6. Such pH-sensitive behavior turns the
multilayered films into very appealing carrier vehicles to
control the encapsulation and release of the drugs in different
pH environments. 2.2.3 Influence of solution pH. 2.2 Hydrogen bonding Weak poly(carboxylic acid)
s, such as poly(methacrylic acid) (PMAA) and PAA, are often
employed as hydrogen bonding donors to construct hydrogen
bonding-based LbL films by combining them with hydrogen
bonding acceptor polymers, such as PAMAM-COOH.137 LbL
films encompassing PMAA and PAMAM-COOH were success-
fully prepared by combining electrostatic interactions and
hydrogen bonding in acidic media ( pH 4).138 Although the main
interaction responsible for the successful deposition of these films
at pH 4 (Fig. 9a) is hydrogen bonding, the films decomposed in
strongly acidic ( pH 2) (Fig. 9b) and neutral pH (Fig. 9a). The
decomposition of LbL films at pH 2 is unex- pected because even
at this pH the hydrogen bonding should be formed, which
confirms that besides the hydrogen bonding, electrostatic
interactions play a key role in the devel- opment of
PMAA/PAMAM-COOH multilayered films. The ter- tiary amines
in PAMAM have a pKa of 6.3, and consequently at 2.3 Covalent bonding The use of covalent bonds to assemble LbL films provides a
mean to enhance the stability and strength of the multilayered
structure. Covalent bonding provides flexibility in the selection
of molecules, where moderately charged or even uncharged
molecules can be employed within the multilayered structure,
allowing the development of multifunctional assemblies.83,140
Although covalent-based multilayers are remarkably stable and
have been investigated in a high number of applications, this
approach requires chemical affinity between the molecules to be
adsorbed, which limits their choice.141 Moreover, if one
envisions the preparation of stimuli-responsive and dynamic
multilayers, the covalent-based systems are not generally
appropriate due to their irreversibility. Fig. 9 (a) QCM data for the creation of a (PMAA/PAMAM-COOH)5 mul-
tilayered film at pH 4 and its decomposition at pH 7. The quartz resona-
tor was exposed to (a) PEI, (b) PMAA, (c) PAMAM-COOH solutions and
(d) 50 mM phosphate buffer, pH 7. (b) QCM response for the decompo-
sition of a (PMAA/PAMAM-COOH)5 multilayered film in strong acidic
conditions. Adapted from ref. 138 with permission from Elsevier, copy- right
2008. The preparation of covalent-based polymeric assemblies was
firstly proposed by Crooks and co-workers, who reported the LbL
assembly of multilayered thin films through the alter- nate
deposition of poly(maleic anhydride)-c-poly(methyl vinyl ether)
(Gantrez) and amine-, hydroxyl-terminated PAMAM or amine-
terminated PPI dendrimers, resulting in a densely func- tionalized
and
organized
film.142,143
The
current resulting from
multilayered films encompassing hydroxyl-terminated PAMAM-
coated electrode was larger when compared with the response of
composite films comprising amine-terminated Fig. 9 (a) QCM data for the creation of a (PMAA/PAMAM-COOH)5 mul-
tilayered film at pH 4 and its decomposition at pH 7. The quartz resona-
tor was exposed to (a) PEI, (b) PMAA, (c) PAMAM-COOH solutions and
(d) 50 mM phosphate buffer, pH 7. (b) QCM response for the decompo-
sition of a (PMAA/PAMAM-COOH)5 multilayered film in strong acidic
conditions. Adapted from ref. 138 with permission from Elsevier, copy- right
2008. Fig. 11 (a) Development of LbL films composed of PAMAM-COOH and NDR,
and conversion of the interlayer linkage bonds from ionic to covalent upon
UV irradiation. (b) UV-Visible absorption spectra of a (PAMAM-
COOH/NDR)12 multilayered film irradiated during (top to bottom) 1, 2, 3,
4, 5, 6, 10, 20 and 30 min. 2.3 Covalent bonding The inset figure shows the photodecomposition
of the film, where A0 and At represent the absor- bance of the film before
and after UV irradiation at different times, respectively. Adapted from ref. 146 with permission from the Royal Society of Chemistry, copyright 2000. (c) UV-Visible absorption spectra of an Ag-PAMAM-COOH/DR multilayered
film (a) before and (b) after UV irradiation. The inset shows the UV-Visible
absorption spectra of the multilayered film without Ag nanoclusters. AFM
images of an Ag- PAMAM-COOH/DR multilayered film (d) before and (e)
after UV irradiation. Adapted from ref. 147 with permission from Wiley-
VCH Verlag GmbH & Co. KGaA, Weinheim, copyright 2003. PAMAM and PPI dendrimers. Moreover, it was shown that
films composed of amine-terminated dendrimers revealed a pH-
dependent behavior as the result of the internal film mor-
phology owing to the different linking chemistry and their
amphoteric character once they simultaneous present amine and
carboxylic groups, which were derived from the unreacted
primary and tertiary amine groups of dendrimers, and amic acid
groups resulting from Gantrez linking, respectively. Besides,
the effect of the thermal treatment was also assessed,
showcasing that the heating causes changes in the functional
groups’ composition, and decreases the film thickness. In the
case of films comprising Gantrez and amine-terminated
PAMAM, the heating led to a decrease in the amide band
intensity and the emergence of two imide peaks at 1710 and
1722 cm. Similarly, films incorporating Gantrez and amine-ter-
minated PPI showed the disappearance of the amide peaks and
the appearance of two new bands typical of imides. In which
concerns to the thermal treatment of the films devel- oped with
Gantrez and hydroxyl-terminated PAMAM, the authors
denoted the formation of two imide bands, which increase in
intensity with increasing the heating time. The authors
hypothesized that the imide groups were formed between the
amic acid groups derived from the reaction of Gantrez with
NH2-modified surface and the amine end groups in the
dendrimers. This methodology is a novel approach for the
preparation of thin films, where the coupling of a reactive
surface to a polyfunctional polymer leads to a covalently
attached reactive brush, that in turn allows the preparation of
a multilayered structure through the incorporation of a reactive
dendrimer substrate. PAMAM and PPI dendrimers. 2.3 Covalent bonding Moreover, it was shown that
films composed of amine-terminated dendrimers revealed a pH-
dependent behavior as the result of the internal film mor-
phology owing to the different linking chemistry and their
amphoteric character once they simultaneous present amine and
carboxylic groups, which were derived from the unreacted
primary and tertiary amine groups of dendrimers, and amic acid
groups resulting from Gantrez linking, respectively. Besides,
the effect of the thermal treatment was also assessed,
showcasing that the heating causes changes in the functional
groups’ composition, and decreases the film thickness. In the
case of films comprising Gantrez and amine-terminated
PAMAM, the heating led to a decrease in the amide band
intensity and the emergence of two imide peaks at 1710 and
1722 cm. Similarly, films incorporating Gantrez and amine-ter-
minated PPI showed the disappearance of the amide peaks and
the appearance of two new bands typical of imides. In which
concerns to the thermal treatment of the films devel- oped with
Gantrez and hydroxyl-terminated PAMAM, the authors
denoted the formation of two imide bands, which increase in
intensity with increasing the heating time. The authors
hypothesized that the imide groups were formed between the
amic acid groups derived from the reaction of Gantrez with
NH2-modified surface and the amine end groups in the
dendrimers. This methodology is a novel approach for the
preparation of thin films, where the coupling of a reactive
surface to a polyfunctional polymer leads to a covalently
attached reactive brush, that in turn allows the preparation of
a multilayered structure through the incorporation of a reactive
dendrimer substrate. Fig. 11 (a) Development of LbL films composed of PAMAM-COOH and NDR,
and conversion of the interlayer linkage bonds from ionic to covalent upon
UV irradiation. (b) UV-Visible absorption spectra of a (PAMAM-
COOH/NDR)12 multilayered film irradiated during (top to bottom) 1, 2, 3,
4, 5, 6, 10, 20 and 30 min. The inset figure shows the photodecomposition
of the film, where A0 and At represent the absor- bance of the film before
and after UV irradiation at different times, respectively. Adapted from ref. 146 with permission from the Royal Society of Chemistry, copyright 2000. (c) UV-Visible absorption spectra of an Ag-PAMAM-COOH/DR multilayered
film (a) before and (b) after UV irradiation. The inset shows the UV-Visible
absorption spectra of the multilayered film without Ag nanoclusters. 2.3 Covalent bonding AFM
images of an Ag- PAMAM-COOH/DR multilayered film (d) before and (e)
after UV irradiation. Adapted from ref. 147 with permission from Wiley-
VCH Verlag GmbH & Co. KGaA, Weinheim, copyright 2003. PAMAM or ferrocenyl-tethered dendrimers and an alde-
hyde-enzyme were also used for the fabrication of LbL films
driven by covalent bonding.144 In this regard, the hydroxyl
groups in the surface of glucose oxidase (GOx) were oxidized,
by periodate, into aldehyde groups and then reacted with the
amine groups on an Au electrode surface through a Schiff base
reaction. The remaining aldehyde groups of GOx reacted with
amino groups on PAMAM dendrimers or ferrocenyl-tethered
dendrimers (Fig. 10), resulting in a well-ordered and stable
multilayered film on the Au electrode surface. (Fig. 11a) for different times (Fig. 11b) in order to change the
linkage between the multilayers from ionic to covalent, which
improves considerably the robustness and stability of the
films.145,146 It was shown by AFM measurements that the
(PAMAM-COOH/NDR)4 multilayered film was rather flat, with a LbL films composed of PAMAM-COOH and nitro-contain-
ing diazoresin (NDR) were submitted to UV irradiation mean roughness of only 1.4 nm, confirming the successful for-
mation of a uniform multilayered film encompassing PAMAM-
COOH and NDR. In contrast, prior to UV irradiation, the
molecules were compressed in the films due to the strong ionic
interactions between PAMAM-COOH and NDR, and the soft
nature of dendrimer architecture. Similarly, multilayered films
encompassing Ag-dendrimer nanoclusters and DR were
developed (Fig. 11c), denoting better electric conductivity
when compared to the LbL films without Ag nanoclusters.147
Besides, AFM measurements disclosed that, prior to the UV
irradiation, the LbL films were destroyed after 1 h of etching
(Fig. 11d) whereas the irradiated film showed good resistance
to the same process (Fig. 11e). Fig. 10 Illustrative scheme of the preparation of multilayered films based
on the adsorption of an aldehyde-enzyme mediated Schiff base reaction
onto a flat solid substrate. Reproduced from ref. 144 with per- mission
from the American Chemical Society, copyright 2015. Fig. 10 Illustrative scheme of the preparation of multilayered films based
on the adsorption of an aldehyde-enzyme mediated Schiff base reaction
onto a flat solid substrate. Reproduced from ref. 144 with per- mission
from the American Chemical Society, copyright 2015. irradiation, the LbL films were destroyed after 1 h of etching
(Fig. 11d) whereas the irradiated film showed good resistance
to the same process (Fig. 11e). 2.4 Coordination chemistry interactions 2.4 Coordination chemistry interactions Furthermore, the covalent bonding driven LbL assembly
affords cell surface engineering. Gattás-Asfura et al. reported
the efficient preparation of conformal and covalently stabilized
nanocoatings on pancreatic islets by functionalizing them with
oppositely charged hyperbranched alginate (ALG) bio-
polymer and PAMAM dendrimer enclosing complementary
Staudinger ligation groups (another click reaction), respectively
azide groups and methyl-2-(diphenylphosphino)terephthalate
(Fig. 12).154,155 The functionalization of the PAMAM dendrimer
with triethoxysilane reduced the dendrimer charge density and
triggered its self-assembly capability during the layer for-
mation, further enabling the stable covalent-based LbL
nanoencapsulated islets to maintain high cell viability and
functionality, lack foreign body responses, and enable stable
normoglycemia after implantation in diabetic mice. Such con-
formal and stable covalent-based LbL nanocoatings have the
ability to bioorthogonally tether supplemental agents to the
surface, including proteins, via Staudinger ligation chemistry
offering the possibility to modulate the implant microenvi-
ronment and be used in the immunoprotection of islets for
the treatment of diabetes. Coordination chemistry are strong intermolecular interactions
established between a wide selection of organic ligands and tran-
sition metal ions, enabling the design and preparation of novel,
well-ordered, and highly oriented LbL assemblies. This approach
allows controlling the surface structure at the molecular level via
the synthesis of deliberately designed ligands.83,156 Despite the great interest in combining dendritic growth on
surfaces with coordination chemistry, there are only a few
studies in the literature that report the formation of multi-
layered thin films by exploiting the coordination of metal ions
to dendrimers. Watanabe and Regen developed LbL films con-
sisting of PAMAM dendrimers and Pt2+ via LbL assembly
driven by coordination chemistry (Fig. 13).65 They hypoth-
esized that by resorting to appropriately-size dendrimers,
attractive gels could be developed and applied to fabricate cat-
alysts or as a basis for optical filters and devices. Blasini and
co-workers prepared multilayered films encompassing terpyri-
dyl -pendant PAMAM dendrimers and Co2+ onto a silicon oxide
(SiO2) surface and demonstrated that the coordination between
the dendrimers and Co2+ ions is strong, conducting to a
compression or deformation of the film with decreased height
of the dendrimer.157 Furthermore, metal ions have been also
successfully employed to fabricate multilayered films
encompassing peptide dendrimers on Au surfaces func-
tionalized with 11-mercaptoundecanoic acid. 2.3 Covalent bonding Z-stack projection confocal images of
layer formation on pancreatic islets, building from (c) three and (d) four
alternating layers of functionalized PAMAM and ALG. Whole mount
immunohistochemistry imaging of insulin (green), f-actin (red), and nuclei
(blue) (c) uncoated control and (d) four-layer coated islets. Scale Fig. 12 (a) Schematic representation of the LbL assembly process com-
prising alternating layers of functionalized PAMAM and ALG assembled
on islets via covalent bonding. (b) Confocal laser scanning microcopy
(CLSM) images of live (green) and dead (red) staining on (a) uncoated
(control) and (b) coated islets. Z-stack projection confocal images of
layer formation on pancreatic islets, building from (c) three and (d) four
alternating layers of functionalized PAMAM and ALG. Whole mount
immunohistochemistry imaging of insulin (green), f-actin (red), and nuclei
(blue) (c) uncoated control and (d) four-layer coated islets. Scale
bar = 50 μm. (e) Dynamic glucose stimulated insulin release for
uncoated control (red) and four-layer coated (blue) islets. Adapted from
ref. 155 with permission from the American Chemical Society, copyright
2020. These results emphasize the potential of the covalent
bonding-based LbL assembly via CuAAC for preparing uniform polymer/NP hybrid thin films with controlled thickness. Moreover, this is an attractive strategy to develop multilayered
assemblies with noncharged polymers and NPs. 2.3 Covalent bonding irradiation, the LbL films were destroyed after 1 h of etching
(Fig. 11d) whereas the irradiated film showed good resistance
to the same process (Fig. 11e). Fig. 12 (a) Schematic representation of the LbL assembly process com-
prising alternating layers of functionalized PAMAM and ALG assembled
on islets via covalent bonding. (b) Confocal laser scanning microcopy
(CLSM) images of live (green) and dead (red) staining on (a) uncoated
(control) and (b) coated islets. Z-stack projection confocal images of
layer formation on pancreatic islets, building from (c) three and (d) four
alternating layers of functionalized PAMAM and ALG. Whole mount
immunohistochemistry imaging of insulin (green), f-actin (red), and nuclei
(blue) (c) uncoated control and (d) four-layer coated islets. Scale
bar = 50 μm. (e) Dynamic glucose stimulated insulin release for
uncoated control (red) and four-layer coated (blue) islets. Adapted from
f 1
i h
i
i
f
h
A
i
Ch
i
l S
i
i h In 2001, Sharpless and co-workers introduced the concept of
click chemistry in an effort to focus on the easy production of
properties rather than challenging structures.148 The idea was to
confine the whole range of chemical transformations to a set of
reactions with a high thermodynamic driving force, allowing
the efficient and easy transformation of “spring-loaded” starting
materials into new structures with useful properties.149 The
impact of click chemistry has been significant in polymer syn-
thesis, an area where reaction efficiency and product purity are
significantly challenged.150 Among the click reactions, the
copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) has
emerged as the archetypal example owing to its high orthogon-
ality, and reactivity and regioselectivity, leading exclusively to
1,4-disubstituted 1,2,3-triazoles.151,152 Li et al. reported the
preparation of hybrid multilayered films encompassing an
azide-functionalized poly(4-vinyl benzaldehyde) (P(VBA-AA)) and
alkyne-functionalized polyphenylene dendrimer-capped AuNPs
(G2SH-AuNPs).153 It was found that the deposition process is
linearly dependent on the number of bilayers, which was con-
firmed by the absorption peak at 260 nm, corresponding to the
complex formation between Au and the carboxyl group. The
multilayered film can be potentially applied as vapor-sorption
and electrical chemiresistors for the three-dimensional (3D)
structure of polyphenylene dendrimers. Fig. 12 (a) Schematic representation of the LbL assembly process com-
prising alternating layers of functionalized PAMAM and ALG assembled
on islets via covalent bonding. (b) Confocal laser scanning microcopy
(CLSM) images of live (green) and dead (red) staining on (a) uncoated
(control) and (b) coated islets. 2.5 Host–guest interactions LbL films can also be created using highly selective and
specific host–guest interactions by exploring the strong inter-
actions between host (e.g., cyclodextrins, calixarenes, crown
ethers, porphyrins) and guest (e.g., ferrocene, adamantane,
azobenzene) molecules.83,158 Huskens and co-workers reported
the build-up of organic/inorganic hybrid multilayered films
with controlled thickness based on selective host–guest inter-
actions between host cyclodextrins-modified AuNPs (CD-
AuNPs) and guest adamantyl-functionalized PPI dendri- mers
(Fig. 14a).159 CD-AuNPs and PAMAM or PPI dendrimers
decorated with ferrocenes can also be used to create thin films
that take advantage of the superior features of dendrimers and
the function of metals (Fig. 14b).160 In both cases, the multi-
layers were deposited onto CD self-assembled monolayers
(SAMs) on Au or SiO2 surfaces and, the multivalent host–guest
interactions between the dendrimer end groups and the CD-
AuNPs, led to the formation of well-defined and stable surface
patterns by “supramolecular microcontact printing” (Fig. 14c
and d). control when combined with top-down surface patterning
methods, such as soft lithography, for x, y control, allows the
creation of 3D supramolecular multilayered assemblies. As
such, these films are very appealing in the fabrication of elec-
tronic and optical devices, in which the structural design at the
nanometer scale level leads to superior performance and new
functionalities. 2.4 Coordination chemistry interactions 14 (a) Schematic illustration of the LbL assembly process compris-
ing alternating layers of adamantyl-functionalized PPI dendrimer and
CD-AuNPs onto CD SAMs via host–guest interactions. Adapted from ref. 159 with permission from the American Chemical Society, copyright
2005. (b) G2 PPI dendrimer with ferrocene end groups complexed with
CD. Adapted from ref. 160 with permission from the Royal Society of
Chemistry, copyright 2008. AFM images of (c) one bilayer and (d) four
bilayers composed of adamantyl-functionalized PPI dendrimer and CD-
AuNPs deposited onto a CD SAM. The image size in (c) is 20 nm ×
1.2 µm and in (d) is 10 nm × 1.2 µm. Adapted from ref. 159 with per-
mission from the American Chemical Society, copyright 2005. 2.4 Coordination chemistry interactions In this regard,
Appoh and Kraatz observed that metal ions prefer to coordi- In conclusion, covalent-based LbL assembly is a powerful
and efficient approach to construct highly stable, well-ordered
and robust ultrathin multilayered films and nanocomposites
with structural precision and flexible design, thus demonstrat-
ing its potential to tailor the film properties by the careful
selection of its components. Fig. 14 (a) Schematic illustration of the LbL assembly process compris
ing alternating layers of adamantyl-functionalized PPI dendrimer and
CD-AuNPs onto CD SAMs via host–guest interactions. Adapted from ref
159 with permission from the American Chemical Society, copyrigh
2005. (b) G2 PPI dendrimer with ferrocene end groups complexed with
CD. Adapted from ref. 160 with permission from the Royal Society o
Chemistry, copyright 2008. AFM images of (c) one bilayer and (d) fou
bilayers composed of adamantyl-functionalized PPI dendrimer and CD
AuNPs deposited onto a CD SAM. The image size in (c) is 20 nm × Fig. 13 Schematic representation of the LbL assembly process for the
alternate adsorption of dendrimers and metal ions onto a modified Au
substrate via coordination chemistry. Reproduced from ref. 156 with
permission from Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, copy-
right 2008. Fig. 13 Schematic representation of the LbL assembly process for the
alternate adsorption of dendrimers and metal ions onto a modified Au
substrate via coordination chemistry. Reproduced from ref. 156 with
permission from Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, copy-
right 2008. nate to the carboxylic acid residues when compared to the
peptide backbone.156 The good compatibility of those films
could arouse much interest for the design of NPs for
nanomedicine. Fig. 14 (a) Schematic illustration of the LbL assembly process compris-
ing alternating layers of adamantyl-functionalized PPI dendrimer and
CD-AuNPs onto CD SAMs via host–guest interactions. Adapted from ref. 159 with permission from the American Chemical Society, copyright
2005. (b) G2 PPI dendrimer with ferrocene end groups complexed with
CD. Adapted from ref. 160 with permission from the Royal Society of
Chemistry, copyright 2008. AFM images of (c) one bilayer and (d) four
bilayers composed of adamantyl-functionalized PPI dendrimer and CD-
AuNPs deposited onto a CD SAM. The image size in (c) is 20 nm ×
1.2 µm and in (d) is 10 nm × 1.2 µm. Adapted from ref. 159 with per-
mission from the American Chemical Society, copyright 2005. Fig. 2.6 Biologically specific interactions Although the prepa-
ration of LbL assemblies incorporating DNA have been predo-
minantly accomplished via electrostatic interactions, owing to
its negative charge, DNA can also be incorporated in LbL
systems by resorting to biologically specific interactions,
including DNA hybridization that allow the assembly of species
with the same charge. In this regard, Feng et al. reported the
DNA hybridization driven encapsulation of a dye molecule
(Cy5) into multilayered microcapsules with shells composed of
alternating layers of phosphorous dendrimers and PSS.167,168
The hybridized double stranded DNA can be further
dehybridized by applying an external stimulus, such as SDS or
sodium phosphate which would allow to control the release of
Cy5. Therefore, this approach would be useful for the
encapsulation of low molecular weight molecules that are
difficult to encapsulate by other methods due to their easy
diffusion through the capsule’s shells. Moreover, the encapsu-
lation of molecules into well-defined dendrimer-based cap-
sules is possible without adjusting parameters, such as shell
permeability by solvents, chemical oxidation, ionic strength or
pH, which considerably improve the probability of destroying
the shell or denature encapsulated biomolecules. demonstrated that one monolayer of avidin (tetrameric protein)
is adsorbed per each PAMAM layer (Fig. 15a).166 In contrast,
multilayered films developed with linear and branched
polymeric chains, such as PAA and PEI, respectively, showed
that for each layer of PAA (Fig. 15b) or PEI (Fig. 15c),
multilayers of avidin are deposited. These results can be
explained by the globular molecular geometry exhibited by
PAMAM when compared with the branched structure denoted
by PEI. Moreover, it was found that the films did not form when
they were assembled with biotin-free polymers, thus cor-
roborating that the binding between avidin and biotin is key
for the formation of the multilayered films. The authors also studied the effect of the concentration of
the materials in the preparation of FITC-avidin/biotin-poly
(amine)s films. It was seen that, in the case of the FITC-avidin/
biotin-PEI films, the PEI can accommodate FITC-avidin mole-
cules either inside or on the surface of the biotin-PEI layer,
providing a disheveled multilayered surface, where the loading
of FITC-avidin depends on its concentration in the solution. The deposition with a solution of 100 µg mL−1 led to the for-
mation of 5–6 layers of FITC-avidin on the slide upon each
deposition whereas a solution of 10 µg mL−1 enabled nearly
monomolecular deposition of FITC-avidin. 2.6 Biologically specific interactions Furthermore, Huskens and co-workers developed patterned
LbL assemblies on the CD SAMs by nanotransfer printing
(nTP) and nanoimprint lithography (NIL) once the LbL assem-
bly was prevented on chemically patterned SAMs by microcon-
tact printing or NIL owing to the low specificity of the adsorp-
tion of the dendrimer.161 The differences in thickness of the LbL
films produced by nTP or NIL approaches were attributed to
rinsing or remove procedures, as well as to the differences in
wetting. Overall, the preparation of organic/inorganic hybrid
multilayered films based on multivalent host–guest interactions
between host-modified AuNPs and dendritic guest molecules
was successfully reported. Furthermore, the z Biologically specific interactions can also be used to create
multilayered films. These intermolecular interactions encom-
pass a broad range of interactions, such as electrostatic and
hydrophobic interactions, and hydrogen bonding, enabling
high specificity and functionality to the target molecules.83
Biological specific interactions include avidin–biotin,162 anti-
body–antigen,163 lectin–carbohydrate interactions,164 and DNA
hybridization.165 2.6.1 Avidin–biotin interactions to build-up multilayered
systems. LbL films prepared by the alternate deposition of flu-
orescein isothiocyanate (FITC)-avidin and biotin-labeled
PAMAM dendrimers have been reported by Anzai et al. who Fig. 15 Possible structure of multilayered films composed of (a) FITC-
avidin/biotin-PAMAM, (b) FITC-avidin/biotin-PAA and (c) FITC-avidin/
biotin-PEI. Reproduced from ref. 166 with permission from the American
Chemical Society, copyright 1999. static repulsion between biotin-PEI and FITC-avidin led to a
lower adsorption rate than in the case of biotin-PAMAM films. As such, a judicious choice of the polymeric materials allows
the regulation of the loading of the biomaterials in multi- layered
films driven by biological specific interactions. Moreover, the
functionality of those films can be improved using modified
avidin residues, namely avidin-enzyme and avidin-antibody
conjugates or poly(amine)s containing biotin and other
functional groups in their side chains which allow the
development of functional films with a controlled density of
functional groups. Fig. 15 Possible structure of multilayered films composed of (a) FITC-
avidin/biotin-PAMAM, (b) FITC-avidin/biotin-PAA and (c) FITC-avidin/
biotin-PEI. Reproduced from ref. 166 with permission from the American
Chemical Society, copyright 1999. 2.6.2 DNA hybridization to assemble and growth multi-
layered assemblies. DNA has aroused much interest due to its
high biocompatibility and biodegradability and, as such, many
systems enclosing DNA have been developed for addressing
applications in the biomedical and biological fields, including
diagnostics, therapeutics and biosensing. 2.6 Biologically specific interactions Multilayered films
comprising biotin-PAA showed a similar behavior but with a
lower loading of FITC-avidin when compared to the FITC-
avidin/biotin-PEI films. Interestingly, the authors observed that
the loading of FITC-avidin in the FITC-avidin/biotin- PAMAM
films was slightly dependent on the concentration of FITC-avidin
in solution, which led to a nearly monomolecular layer of FITC-
avidin absorbed upon each deposition step, con- firming that the
driving force is the avidin/biotin complexa- tion. The different
behavior was attributed to the molecular architecture of the
polymers. Besides, the adsorption of FITC- avidin is faster in
biotin-PAMAM than in biotin-PEI films. In fact, just 10 min are
needed for the complete adsorption of FITC-avidin onto a biotin-
PAMAM layer whereas 60 min are needed for the adsorption onto
biotin-PEI. The faster adsorp- tion of avidin onto biotin-PAMAM
is due to the globular archi- tecture of PAMAM, providing
increasing accessibility sites for the adsorption of avidin and
formation of avidin–biotin com- plexes. Moreover, PEI has a
higher positive surface charge density than PAMAM and once the
FITC-avidin is also posi- tively charged under the experimental
conditions, the electro- The construction of supramolecular multilayered assem-
blies by resorting to biologically specific interactions enables
the incorporation of a wide array of materials, resulting in films
with enhanced stability and orientation. These films denote
great potential for being applied in the fabrication of biosensors,
as well as in diagnostics and molecular biology. 3.1 Biosensing Dendrimers have been used in the preparation of bio- sensors
by three main strategies: (1) producing surface mono- layers as
scaffolds for the immobilization of proteins, (2) creat- ing LbL
films
comprising
dendrimers
and
proteins
and,
(3)
encapsulating or covalent binding metal NPs and electron
transfer mediators. In fact, dendrimers have been used to
modify the surface of electrodes with monolayer films, on
which proteins are further immobilized. For instance, bio-
sensors encompassing monolayers of PAMAM dendrimers on
the surface of Au or Ag substrates further modified with pro-
teins or single-stranded DNA have been reported.172,173 Metal NPs-encapsulating dendrimers have been also suc-
cessfully and intensively employed to create sensitive enzyme-
containing LbL films for biosensing. For example, PPI-Au
nanoclusters were successfully assembled with Mb into PPI-
Au/Mb LbL films on the surface of graphite electrodes and
showed better electrochemical properties and catalytic activity
than similar PPI/Mb and PAMAM/Mb LbL films due to the
good conductivity assigned to the AuNPs, which play a key role
in the electron transfer of Mb in the LbL films.177 Almost at
the same time, Li and co-workers developed a glucose bio-
sensor through the LbL alternating deposition of platinum NPs
(PtNPs)-encapsulating PAMAM dendrimer and oppositely
charged GOx on Pt electrodes and showed that the response of
the biosensor was significantly enhanced in the presence of the
PtNPs, indicating that these procedures can be useful for
creating efficient microelectrodes.178 Moreover, an electro-
analytical glucose biosensor was also developed through a
polypyrrole (PPy) film doped with PAMAM dendrimers-encap-
sulating PtNPs (Pt-PAMAM-PPy) and GOx, aiming for triggering
the electrocatalytic oxidation of glucose.179 The resulting film
showed good stability with improved electrocatalytic activity,
thus suggesting that glucose can easily permeate porous com-
posite films. Furthermore, multilayered films composed of dendrimers
and enzymes have also been proposed for biosensing. In this
regard, multilayered films encompassing PAMAM and GOx
were developed via electrostatic interactions and it was
observed that, after the assembly of two bilayers, the further
deposition of GOx layers did not improve the response of the
glucose biosensors (Fig. 16a).174 Similarly, LbL films compris-
ing PAMAM layers covalently coupled to GOx via a Schiff base
linkage on the surface of Au electrodes showed that the
response of glucose biosensors increased while increasing the
number of GOx/PAMAM bilayers up to 5 (Fig. 3.1 Biosensing Monitoring biological and biochemical processes is essential
when aiming for biological and biomedical applications. In
fact, the development of highly sensitive, specific, and cost-
effective biosensors contributes to the creation of an accurate
diagnosis and personalized medicine. Nowadays, sensors
allow the detection of not only the more traditional class of
biomarkers (e.g., nucleic acids, proteins) but also metabolites
and pathogens, thus increasing the range of available tech-
niques for an individual’s diagnosis.169 Biosensors are usually
developed using electrodes or transducers coupled with bio-
recognition elements, such as enzymes, antibodies, DNA or
aptamers. Moreover, the immobilization of proteins on the
surface of transducers without losing their biological activity is
a key issue while designing biosensors. As such, LbL films
enclosing proteins have been widely studied to improve the
recognition and transducer processes in biosensors.170,171 Fig. 16 (a) Catalytic current response as a function of the cycle number in
multilayered films comprising PAMAM and GOx on Au surfaces via
electrostatic interactions. Adapted from ref. 174 with permission from
Elsevier, copyright 2006. (b) Evolution of the ellipsometric thickness of
the GOx/PAMAM multilayers assembled on Au surfaces via covalent
bonding as a function of the number of layers. Open circles show the
thickness of GOx-terminated multilayers. Reproduced from ref. 175 with
permission from the American Chemical Society, copyright 2000. films showed better stability than PAMAM/Cat films, without
significant decrease of the peak current. PAMAM/Cat multi-
layered films disclosed a decreased of ca. 20% compared to
the initial peak current value. The decrease in the reduction
overpotential together with the good stability and sensitivity of
the LbL films suggest their usefulness as electrochemical bio-
sensors or bioreactors without the need of mediators, in which
the electrochemical and electrocatalytic activity can be regu-
lated by adjusting the thickness of the films. films showed better stability than PAMAM/Cat films, without
significant decrease of the peak current. PAMAM/Cat multi-
layered films disclosed a decreased of ca. 20% compared to
the initial peak current value. The decrease in the reduction
overpotential together with the good stability and sensitivity of
the LbL films suggest their usefulness as electrochemical bio-
sensors or bioreactors without the need of mediators, in which
the electrochemical and electrocatalytic activity can be regu-
lated by adjusting the thickness of the films. 3.
Bioapplications of dendrimer-
containing LbL assemblies The supramolecular dendrimer-containing LbL assemblies offer
an enhanced control over the multilayer composition, structure,
properties and functions in comparison to the con- ventional
linear polymer-based LbL systems due to the well- defined
hyperbranched and globular architecture, high surface area, and
tunable nanosize, molecular weight, chemical com- position
and surface functionality of dendrimers. As such, Fig. 16 (a) Catalytic current response as a function of the cycle number in
multilayered films comprising PAMAM and GOx on Au surfaces via
electrostatic interactions. Adapted from ref. 174 with permission from
Elsevier, copyright 2006. (b) Evolution of the ellipsometric thickness of
the GOx/PAMAM multilayers assembled on Au surfaces via covalent
bonding as a function of the number of layers. Open circles show the
thickness of GOx-terminated multilayers. Reproduced from ref. 175 with
permission from the American Chemical Society, copyright 2000. dendritic-based LbL architectures have aroused much interest
in a wide variety of biological and biomedical applications,
including in biosensing, bioimaging, drug/therapeutics deliv-
ery, and gene therapy. 3.2 Drug/therapeutics delivery The design of smart modular platforms that could promote the
efficient encapsulation, protection, transport, and orche- strated
intracellularly controlled and sustained release of bio- active
agents would represent a hallmark in advanced thera- peutic
systems. In particular, the unique features of dendri- mers,185
and the high versatility of LbL assemblies104 have contributed
to the development of novel surface-mediated drug delivery
systems. Dendrimer-containing multilayered films provide
enhanced binding sites to drugs via the internal cavities of
dendrimers and their terminal functional groups, as well as
those of the multilayered film. Moreover, their inte- gration
with stimulus-sensitive materials has gathered con- siderable
attention in the development of controlled drug delivery
systems. To achieve this goal, LbL films sensitive to pH,186,187
salts,188 temperature,189,190 sugars,191,192 and electri- cal
signals193–195 have been widely exploited. For instance, Sato and
Anzai created sugar-sensitive multilayered films enclosing Furthermore, an important tool in biomedicine is bio-
imaging. The first in vivo applications of dendrimers were as
macromolecular magnetic resonance imaging (MRI) contrast
agents.5,181 In this regard, the incorporation of dendrimers
in multilayered films has aroused much attention in biome-
dicine, in particular, in the field of diagnostics. For instance,
iron oxide (Fe3O4) NPs functionalized with LbL films have
been prepared for targeting and imaging of cancer cells. For
this purpose, Fe3O4 nanoparticles were coated with PSS and
folic acid (FA)- and FITC-tagged PAMAM dendrimer layers
via electrostatic interactions, fol- lowed by an acetylation
reaction to neutralize the still present amine groups on the
surface of the dendrimers.182 Fig. 18 CLSM images of KB-KFAR cells treated with (a) phosphate buffered
saline, (b) Fe3O4/PSS/G5-NHAC-FITC NPs and (c) Fe3O4/PSS/ G5-NHAC-
FITC-FA NPs. The nucleus of cells was stained in blue with Hoescht 33342. The green color was originated from the FITC-conju- gated dendrimers. Reproduced from ref. 182 with permission from Wiley-VCH Verlag GmbH &
Co. KGaA, Weinheim, copyright 2007. Fig. 17 (a) Oversimplified representation of the structure, operation
principle and chemical reaction of the penicillin biosensor based on an
EIS structure functionalized with multilayered films encompassing
PAMAM and SWNT. (b) Capacitance-voltage curves and (c) constant-
capacitance response at different pH values for an EIS-NT sensor. Adapted from ref. 180 with permission from Elsevier, copyright 2009. Fig. 17 (a) Oversimplified representation of the structure, operation
principle and chemical reaction of the penicillin biosensor based on an
EIS structure functionalized with multilayered films encompassing
PAMAM and SWNT. 3.1 Biosensing 16b), confirming
that the catalytic activity was maintained in the LbL film.175
These results indicated that the design of glucose biosensors
through covalent bonding driven GOx/PAMAM LbL films is
stronger than similar films assembled by electrostatic
interactions. Heme proteins, including hemoglobin (Hb), myoglobin
(Mb), and catalase (Cat) have been also used to create electro-
statically-driven multilayered films with PAMAM dendrimers
on an array of solid surfaces.176 In this regard, PAMAM/protein
multilayered films were developed and exploited as enhanced
systems to catalyze substrates with biological and/or environ-
mental significance. PAMAM/Hb and PAMAM/Mb multilayered Besides, LbL films composed of dendrimers have been also
applied as gate materials for field effect transistor biosensors. Siqueira et al. reported the preparation of multilayered films
encompassing PAMAM and carboxylate single-walled carbon
nanotubes (SWNT), further modified by the immobilization of penicillinase on the top of the film surface for producing peni-
cillin biosensors (Fig. 17a).170,180 The LbL films showed to be
highly porous due to the interpenetration of SWNTs into the
dendrimer layers. Moreover, a good sensitivity to pH (Fig. 17b
and c) and biosensing ability towards penicillin was shown by
an electrolyte-insulator-semiconductor (EIS) structure modi-
fied with carbon nanotubes (EIS-NT)-penicillinase biosensor. The authors showed that Fe3O4/PSS/G5-NHAc-FITC-FA NPs can
target FA receptors that are overexpressed in the surface of
cancer cells (Fig. 18). As such, this study describes a striking methodology for
creating various nanosized imaging agents for biosensing and
therapeutic applications, since these type of materials have
ability to be specifically located in tumors via enhanced per-
meation and retention effect that in turn allow an enhanced
imaging sensitivity and specificity.183,184 As such, there is no doubt about the potential of dendri- mer-
derived LbL assemblies for biosensing. Despite the different
mechanisms involved in the development of bio- sensor-based
LbL films, those enclosing metal NPs-encapsu- lating
dendrimers showed to be promising owing to their better
electrocatalytic properties and stability, thus suggesting their
potential use as efficient electrodes. Therefore, a careful
selection of the methodology and build- ing blocks to be used
enables the design of multilayered films able to be applied
in various analytical applications in the biotechnology field. 3.2 Drug/therapeutics delivery (c) PBA-
PAMAM release profile of (a) (PAH/GOx)3(PBA-PAMAM/PVA)10, (b)
(PAH/GOx)2(PBA-PAMAM/PVA)10, (c) (PAH/GOx)1(PBA-PAMAM/PVA)10 and (d) (PBA-PAMAM/PVA)10 multilayered films in the presence of an
1 mM glucose aqueous solution. Adapted from ref. 199 with permission from
Springer Nature, copyright 2017. reported that such films also decomposed in the presence of
glucose owing to the oxidative scission of the carbon-boron
bond in the PBA residues of PBA-PAMAM by enzymatic pro-
duction of hydrogen peroxide (H2O2). Anzai and co-workers
developed later PBA-PAMAM/PVA multilayered films showcas-
ing their stability at pH 9 and decomposition after the addition
of H2O2 (Fig. 20a).199 In fact, the exposure of the films to 0.5
and 1 mM H2O2 during 30 min triggered the release of 75% and
90% of the PBA-PAMAM from the (PBA-PAMAM/PVA)10 films
(Fig. 20b). Thus, PBA-PAMAM/PVA films containing GOx
enzymatically produced H2O2 after being exposed to 1 mM
glucose aqueous solution at pH 7.4 (Fig. 20c). Therefore, those
multilayered assemblies show great promise for being used in
drug/therapeutics delivery in response to reactive oxygen
species and oxidative stress. As aforementioned, hydrogen-bonded driven LbL films are
usually pH-sensitive and decompose in strongly acidic and
neutral pH. In this regard, PAMAM-COOH/PMAA films demon-
strated to be sensitive to pH changes and decomposed upon
exposition to buffer solutions at pH 2 and 7.138 As such, these
films have been used to load model dyes, such as Rose Bengal
and sulfonated tetraphenylporphyrin, where the release is pre-
cisely controlled by pH changes. Besides, the construction of
electrostatic-driven multilayered films composed of a nega-
tively charged carboxylated Fréchet-type poly(aryl ether) den-
dronized polymer (denpol) loaded with water-insoluble pyrene
dye and oppositely charged PDDA has been reported (Fig. 21a). It was shown that the release of pyrene from the multilayered
films can be tuned by adjusting the ionic strength of the
immersing solutions (Fig. 21b).200 Moreover, it was shown that
the pH of the polyelectrolyte solution influences the surface
morphology and roughness of the dendrimer layer in the
resulting films (Fig. 21c and d). Interestingly, the authors found
that the amount of pyrene released from the multi- layered films
can be reloaded by dipping the pyrene-released film into a
saturated pyrene aqueous solution, disclosing the potential of
the multilayered films for on-demand encapsula- tion and
targeted release. Fig. 3.2 Drug/therapeutics delivery (b) Capacitance-voltage curves and (c) constant-
capacitance response at different pH values for an EIS-NT sensor. Adapted from ref. 180 with permission from Elsevier, copyright 2009. Fig. 18 CLSM images of KB-KFAR cells treated with (a) phosphate buffered
saline, (b) Fe3O4/PSS/G5-NHAC-FITC NPs and (c) Fe3O4/PSS/ G5-NHAC-
FITC-FA NPs. The nucleus of cells was stained in blue with Hoescht 33342. The green color was originated from the FITC-conju- gated dendrimers. Reproduced from ref. 182 with permission from Wiley-VCH Verlag GmbH &
Co. KGaA, Weinheim, copyright 2007. Fig. 20 (a) Schematic illustration of the H2O2-induced release of PBA-
PAMAM from PBA-PAMAM/PVA multilayered films. (b) UV-Visible
absorption spectra of a (PBA-PAMAM/PVA)10 multilayered film before
(blue) and after (red) exposure to 1 mM H2O2 in pH 9 for 30 min. (c) PBA-
PAMAM release profile of (a) (PAH/GOx)3(PBA-PAMAM/PVA)10, (b)
(PAH/GOx)2(PBA-PAMAM/PVA)10, (c) (PAH/GOx)1(PBA-PAMAM/PVA)10 poly(vinyl alcohol) (PVA) and phenylboronic acid-bearing
PAMAM dendrimer (PBA-PAMAM) via boronate ester chemistry
between the boronic acid moiety in PBA-PAMAM and the 1,3-
diol units in PVA.196 These LbL films were used as a sacrificial
layer for preparing free-standing multilayered membranes
composed of PSS and poly(allylamine hydrochloride) (PAH)
(Fig. 19). The authors demonstrated that external stimulus,
including pH changes, salts and sugar induced the release of the
free-standing PSS/PAH multilayered membranes from PVA/
PBA-PAMAM/PSS/PAH films showcasing the highest release
rate in the presence of solutions of higher sorbitol concen-
trations (5–15 mM), pH 9 and lower NaCl concentrations (150
mM). As such, this is certainly a useful approach for developing
free-standing multilayered membranes enclosing pH-sensitive
materials, including proteins. Similar multilayered films, responsive to pH and sugar, have
been reported. Those include PVA/4-PBA-PAMAM multi-
layered assemblies, which decompose in response to glucose
or fructose at pH 7.4 and 9,197 and alginic acid/PBA-PAMAM
multilayered films, which disassemble when exposed to 5–
30 mM fructose at pH 7.5.121 Moreover, multilayered films
encompassing GOx and PBA-PAMAM were found to be stable
in pH 8.5, while decomposing in neutral and acidic media due
to the cleavage of the boronate ester bond.198 Suwa et al. Fig. 20 (a) Schematic illustration of the H2O2-induced release of PBA-
PAMAM from PBA-PAMAM/PVA multilayered films. (b) UV-Visible
absorption spectra of a (PBA-PAMAM/PVA)10 multilayered film before
(blue) and after (red) exposure to 1 mM H2O2 in pH 9 for 30 min. 3.2 Drug/therapeutics delivery (d)
Release profiles of AO in water from (a) non-crosslinked and (b) cross-
linked (PAH/PAMAM)7.5 multilayered films. The inset figure in (b) shows
the growth of absorbance at 268 nm as a function of the release time. Adapted from ref. 201 with permission from the Royal Society of
Chemistry, copyright 2015. Fig. 22 (a) Schematic representation of the preparation of PAH/PAMAM
multilayered films and their stabilization with DAS, followed by the
adsorption of the hydrophobic molecules. (b) UV-Visible absorption
spectra of (PAH/PAMAM)7.5 multilayered films before (black) and after
(red) immersion in NaOH aqueous solution at pH 12 on a modified quartz
substrate. (c) AFM images of a (PAH/PAMAM)7.5 multilayered film Shi and co-workers have developed a strategy for combining
covalent interlayer linkages and drug reservoirs with a model
drug.201 For this purpose, LbL films composed of PAMAM-
COOH dendrimers, serving as the drug reservoir, and PAH
(Fig. 22a) were prepared. As expected, the immer- sion of the
film in a sodium hydroxide (NaOH) aqueous solu- tion at pH
12 for 30 min led to the almost complete disas- Shi and co-workers have developed a strategy for combining
covalent interlayer linkages and drug reservoirs with a model
drug.201 For this purpose, LbL films composed of PAMAM-
COOH dendrimers, serving as the drug reservoir, and PAH
(Fig. 22a) were prepared. As expected, the immer- sion of the
film in a sodium hydroxide (NaOH) aqueous solu- tion at pH
12 for 30 min led to the almost complete disas- (a) before and (b) after DAS infiltration and (c) after UV irradiation. (d)
Release profiles of AO in water from (a) non-crosslinked and (b) cross-
linked (PAH/PAMAM)7.5 multilayered films. The inset figure in (b) shows
the growth of absorbance at 268 nm as a function of the release time. Adapted from ref. 201 with permission from the Royal Society of
Chemistry, copyright 2015. sembly of the PAMAM/PAH films (Fig. 22b) via deprotonation
of the ammonium groups at PAH and fading of the attractive
electrostatic attraction with the carboxyl groups in PAMAM. To improve the stability of the multilayers, a small bifunc-
tional photoactive molecule, namely 4,40-diazostilbene-2,20-
disulfonic acid disodium salt (DAS) was post-infiltrated in acidic
conditions between the multilayers. This increased the
protonation of PAH, thus contributing for improving the
adsorption of DAS. The authors also observed that the infil- tration
process preserves the surface morphology of the layers, once
that either before (Fig. 3.2 Drug/therapeutics delivery 22c-a) or after DAS infil- tration (Fig. 22c-
b), as well as after UV irradiation (Fig. 22c- c), the substrate
presented similar surface morphology and roughness. The
obtained crosslinked PAH/PAMAM multi- layers showed good
stability and were organized in a rigid and compact structure,
being able to retard the load and release of the model drug from
the multilayers. The release profiles of small hydrophobic drug
molecules, modeled by acridine orange (AO),
through
crosslinked and non-cross- linked multilayered films differed
significantly, revealing similar trends to their absorption profiles. As such, the non- crosslinked loaded multilayered films released
around 98% of the AO during the first 5 min (Fig. 22d-a) whereas
the crosslinked ones retarded the release of AO (Fig. 22d-b),
with sembly of the PAMAM/PAH films (Fig. 22b) via deprotonation
of the ammonium groups at PAH and fading of the attractive
electrostatic attraction with the carboxyl groups in PAMAM. To improve the stability of the multilayers, a small bifunc-
tional photoactive molecule, namely 4,40-diazostilbene-2,20-
disulfonic acid disodium salt (DAS) was post-infiltrated in acidic
conditions between the multilayers. This increased the
protonation of PAH, thus contributing for improving the
adsorption of DAS. The authors also observed that the infil- tration
process preserves the surface morphology of the layers, once
that either before (Fig. 22c-a) or after DAS infil- tration (Fig. 22c-
b), as well as after UV irradiation (Fig. 22c- c), the substrate
presented similar surface morphology and roughness. The
obtained crosslinked PAH/PAMAM multi- layers showed good
stability and were organized in a rigid and compact structure,
being able to retard the load and release of the model drug from
the multilayers. The release profiles of small hydrophobic drug
molecules, modeled by acridine orange (AO),
through
crosslinked and non-cross- linked multilayered films differed
significantly, revealing similar trends to their absorption profiles. As such, the non- crosslinked loaded multilayered films released
around 98% of the AO during the first 5 min (Fig. 22d-a) whereas
the crosslinked ones retarded the release of AO (Fig. 22d-b),
with approximately 95% of the AO being released during the first 30
min. The crosslinking of PAH/PAMAM LbL films retained AO
molecules for a much longer time than non-crosslinked ones
due to the rigid globular shape of dendrimers, which imposes a
powerful physical barrier for diffusion of AO both in-and-out of
the film. 3.2 Drug/therapeutics delivery 19 (a) Schematic drawing showing the development of (PVA/ PBA-
PAMAM)5/(PSS/PAH)15 multilayered films (top) and sorbitol-induced
release of the PSS/PAH film from the substrate (bottom). (b) Photograph
of a released (PSS/PAH)15 film. Adapted from ref. 196 with permission
from Elsevier, copyright 2017. Fig. 19 (a) Schematic drawing showing the development of (PVA/ PBA-
PAMAM)5/(PSS/PAH)15 multilayered films (top) and sorbitol-induced
release of the PSS/PAH film from the substrate (bottom). (b) Photograph
of a released (PSS/PAH)15 film. Adapted from ref. 196 with permission
from Elsevier, copyright 2017. The development of assembling methodologies for simul-
taneously enabling the covalent crosslinking of the multilayers
and embedding drug reservoirs for retarded drug release has
raised much interest in the biomedical field. In this regard, Fig. 21 (a) Schematic illustration of the build-up of electrostatic-driven
multilayered films comprising pyrene-loaded dendronized polymer and
PDDA multilayers. (b) Effect of the ionic strength on the pyrene release
kinetics from (PDDA/denpol)10 multilayered films. AFM images of the
outmost denpol layer in (PDDA/denpol)10 LbL films with PDDA and
denpol at (c) pH 6 and (d) pH 8. The scale bar is 500 nm × 500 nm. b–d:
Adapted from ref. 200 with permission from Elsevier, copyright 2008. Fig. 22 (a) Schematic representation of the preparation of PAH/PAMAM
multilayered films and their stabilization with DAS, followed by the
adsorption of the hydrophobic molecules. (b) UV-Visible absorption
spectra of (PAH/PAMAM)7.5 multilayered films before (black) and after
Shi and co-workers have developed a strategy for combining
covalent interlayer linkages and drug reservoirs with a model
drug.201 For this purpose, LbL films composed of PAMAM-
COOH dendrimers, serving as the drug reservoir, and PAH
(red) immersion in NaOH aqueous solution at pH 12 on a modified quartz
substrate. (c) AFM images of a (PAH/PAMAM)7.5 multilayered film
(a) before and (b) after DAS infiltration and (c) after UV irradiation. (d)
Release profiles of AO in water from (a) non-crosslinked and (b) cross-
linked (PAH/PAMAM)7 5 multilayered films. The inset figure in (b) shows Fig. 22 (a) Schematic representation of the preparation of PAH/PAMAM
multilayered films and their stabilization with DAS, followed by the
adsorption of the hydrophobic molecules. (b) UV-Visible absorption
spectra of (PAH/PAMAM)7.5 multilayered films before (black) and after
(red) immersion in NaOH aqueous solution at pH 12 on a modified quartz
substrate. (c) AFM images of a (PAH/PAMAM)7.5 multilayered film
(a) before and (b) after DAS infiltration and (c) after UV irradiation. 3.2 Drug/therapeutics delivery (d)
Release profiles of AO in water from (a) non-crosslinked and (b) cross-
linked (PAH/PAMAM)7.5 multilayered films. The inset figure in (b) shows
the growth of absorbance at 268 nm as a function of the release time. Adapted from ref. 201 with permission from the Royal Society of
Chemistry, copyright 2015. Fig. 21 (a) Schematic illustration of the build-up of electrostatic-driven
multilayered films comprising pyrene-loaded dendronized polymer and
PDDA multilayers. (b) Effect of the ionic strength on the pyrene release
kinetics from (PDDA/denpol)10 multilayered films. AFM images of the
outmost denpol layer in (PDDA/denpol)10 LbL films with PDDA and
denpol at (c) pH 6 and (d) pH 8. The scale bar is 500 nm × 500 nm. b–d:
Adapted from ref. 200 with permission from Elsevier, copyright 2008. Fig. 21 (a) Schematic illustration of the build-up of electrostatic-driven
multilayered films comprising pyrene-loaded dendronized polymer and
PDDA multilayers. (b) Effect of the ionic strength on the pyrene release
kinetics from (PDDA/denpol)10 multilayered films. AFM images of the
outmost denpol layer in (PDDA/denpol)10 LbL films with PDDA and
denpol at (c) pH 6 and (d) pH 8. The scale bar is 500 nm × 500 nm. b–d:
Adapted from ref. 200 with permission from Elsevier, copyright 2008. Fig. 22 (a) Schematic representation of the preparation of PAH/PAMAM
multilayered films and their stabilization with DAS, followed by the
adsorption of the hydrophobic molecules. (b) UV-Visible absorption
spectra of (PAH/PAMAM)7.5 multilayered films before (black) and after
(red) immersion in NaOH aqueous solution at pH 12 on a modified quartz
substrate. (c) AFM images of a (PAH/PAMAM)7.5 multilayered film
(a) before and (b) after DAS infiltration and (c) after UV irradiation. (d)
Release profiles of AO in water from (a) non-crosslinked and (b) cross-
linked (PAH/PAMAM)7.5 multilayered films. The inset figure in (b) shows
the growth of absorbance at 268 nm as a function of the release time. Adapted from ref. 201 with permission from the Royal Society of
Chemistry, copyright 2015. Fig. 22 (a) Schematic representation of the preparation of PAH/PAMAM
multilayered films and their stabilization with DAS, followed by the
adsorption of the hydrophobic molecules. (b) UV-Visible absorption
spectra of (PAH/PAMAM)7.5 multilayered films before (black) and after
(red) immersion in NaOH aqueous solution at pH 12 on a modified quartz
substrate. (c) AFM images of a (PAH/PAMAM)7.5 multilayered film
(a) before and (b) after DAS infiltration and (c) after UV irradiation. 3.2 Drug/therapeutics delivery In another example, Oliveira and co-workers reported the
fabrication of porous multilayered films incorporating dendri-
mers and liposomes showcasing their ability to interact with
proteins, including bovine serum albumin.207 Therefore, such
films may be useful for releasing ions and small particles from
liposomes that will diffuse through the dendrimer’s layers. The design of hollow multilayered microcapsules through
the sequential deposition of polymeric materials on the surface
of sacrificial colloidal particles, followed by dissolution of the
core template, has been attracting growing attention as
reservoirs for the encapsulation and on-demand controlled
release of drugs/therapeutics.132,208–210 For instance, PAMAM-
loaded LbL microcapsules, through the sequential deposition of
PAH and PVS layers onto the surface of calcium carbonate
(CaCO3) microparticles in which dendrimer is loaded followed
by core template dissolution, have been developed (Fig. 24a and
b) to study the loading and release of 1-anilinonaphtha- lene-8-
sulfonic acid (ANS) as a model drug.211 The binding of ANS to
the PAMAM dendrimers highly increased the ANS loading in
the microcapsules compared to a control lacking PAMAM as
seen by a large difference in the fluorescence emis- sion of the
dye. The rate of ANS uptake (Fig. 24c) was deter- mined by the
rate of transport of ANS across the capsule, while the
dissociation of ANS (Fig. 24d) from PAMAM was the rate-
determining step for ANS release out of the microcapsules. The
reproducibility over the particle size and shape, as well as the
versatility in the choice of LbL ingredients for the core– shell
capsules constitute an innovative approach to produce modular
multilayered microcapsules able to bind and release small
molecules. Fig. 23 (a) Schematic illustration of the preparation of crosslinked PAH/
PAMAM LbL films on a piezoelectric-dielectric composite substrate. (b) SEM
image of the cross section of (PAH/PAMAM)7.5@rGO-PEI/PVDF-HFP LbL
films. The inset figure is a digital photo of the flexible film. (c) Preparation of
the multilayers on the disposable gastric lavage tube. (d) Schematic
representation of the piezoelectric generation mechanism during the
application. (e)
Metoclopramide
release
profiles
from
(PAH/
PAMAM)10.5@rGO-PEI/PVDF-HFP LbL films on the disposable gastric
lavage tube. Adapted from ref. 206 with permission from Wiley-VCH Verlag
GmbH & Co. KGaA, Weinheim, copyright 2018. lowed by core template dissolution, and evaluated the loading
and release profile of the anticancer drug doxorubicin (DOX). 3.2 Drug/therapeutics delivery Moreover, the crosslinked multi- layered films showed
the ability to withstand rising with ethanol, the most often used
sterilization procedure, thus holding great promise for being
used in a variety of bio- medical scenarios. Thus, the covalent interlayer linkages and the photochemi-
cal crosslinking of the multilayered films encompassing drug
reservoirs contributed to the creation of multilayered films with
retarded loading-release profiles without compromising the
stability, surface morphology and roughness of films. Other
authors reported a similar approach for combining post-
infiltration, photochemical crosslinking and selective etching
to develop porous multilayered films encompassing weak
polyelectrolytes.202–205 Therefore, these systems show great
promise in polymer science, since they enable an well-defined Fig. 23 (a) Schematic illustration of the preparation of crosslinked PAH/
PAMAM LbL films on a piezoelectric-dielectric composite substrate. (b) SEM
image of the cross section of (PAH/PAMAM)7.5@rGO-PEI/PVDF-HFP LbL
films. The inset figure is a digital photo of the flexible film. (c) Preparation of
the multilayers on the disposable gastric lavage tube. (d) Schematic
representation of the piezoelectric generation mechanism during the
application. (e)
Metoclopramide
release
profiles
from
(PAH/
PAMAM)10.5@rGO-PEI/PVDF-HFP LbL films on the disposable gastric
lavage tube. Adapted from ref. 206 with permission from Wiley-VCH Verlag
GmbH & Co. KGaA, Weinheim, copyright 2018. control over the film structure, as well as the encapsulation
and controlled release of drugs. Using a similar strategy, Zhang et al. prepared a multi-
layered film comprising DAS crosslinked PAH/PAMAM multi-
layers, built on a piezoelectric-dielectric flexible film with
reduced graphene oxide fillers (rGO-PEI/PVDF-HFP) (Fig. 23a)
for improving the release of antinausea drugs.206 These authors
observed that the photochemical crosslinking with DAS
provided a stable drug matrix while preserving the piezo-
electric-dielectric film properties (Fig. 23b). The multilayered
film was also assembled on the surface of a disposable gastric
lavage tube (Fig. 23c), showcasing that mimetic forces in the
same range of human swallowing were transformed by the
piezoelectric network into an electrostatic field, leading to an
antiemetic drug release increase by 200% within 60 min (Fig. 23d and e). Therefore, the multilayered film can activate
electrically triggered releases that were formerly only per-
formed using complicated electrochemical setups and allows
the attenuation of nausea, facilitating the intubation process
itself. Thus, it is expected that this kind of multilayered plat-
forms could simplify drug applications in various emergent
situations and, at the same time, maximize drug release from
a matrix, reducing the wasting drugs. 3.2 Drug/therapeutics delivery (PDT) and as light-harvesting sensitizers (PSs) owing to their
optical properties and long triplet excited state lifetime.221–224
Briefly, this therapeutic approach involves the selective localiz- ation of a non-toxic photoactivatable dye (the PS) in the neo-
plastic tissue and its irradiation with harmless visible light to
generate highly reactive oxygen species that lead to the destruc-
tion of the tumor cells.225 Recently, several approaches have
been employed towards the incorporation of PS into new drug
delivery systems, which improve the selectivity and efficacy of
PDT, as well as reduce the undesirable side effects, such as skin
hyperphotosensitivity.226–228 For instance, Koh and co- workers
prepared core–shell LbL nanocapsules encompassing anionic
dendrimer porphyrin and cationic PAH multilayered shells on a
sacrificial polystyrene template.229 The resulting capsules
revealed high potential for encapsulating DOX for cancer
therapy applications. In addition, it was found that the hollow
capsules can be used as PS for PDT, whereas the loaded-
capsules, after the irradiation, were much more cyto- toxic than
either chemotherapy or PDT alone, suggesting its application as
nano-devices for cancer therapy. As such, the molecular design
of dendritic structures can result in the development of
innovative and sophisticated multilayered plat- forms with
emergent properties and functions across multiple scale lengths
for being used in biomedicine. PEG-modified LbL films reduced the phagocytosis of the
microcarriers and adsorption of human serum albumin, as well
as their adhesion to the macrophage cell line TPH-1,
responsible for granuloma formation and infection at an implant
site, when compared to the unmodified LbL films, which prove
the biocompatibility of the surface-modified mul- tilayered
films. In
another
study,
multilayered
microcapsules
encompassing N,N-disubstituted hydrazine phosphorus-con-
taining dendrimers coupled with oppositely charged linear
polyelectrolytes or DNA shells were developed. When com-
pared to the microcapsules developed with solely linear poly-
electrolytes, the dendrimer-based microcapsules were found to
be much softer, although these microcapsules could be stiffened
by the treatment with organic solvents.214–216 In addition, the
effect of the type of surface charges of the multi- layered films
encompassing either dendrimers and oppositely charged
polymers or oppositely charged dendrimers was evalu- ated on
the adhesion and maturation of fetal cortical rat neurons. It was
demonstrated that neurons attached preferen- tially and matured
faster in the films having positively charged hydrazine
phosphorous dendrimers as the outermost layer (Fig. 25a)
when compared with similar films ended in nega- tively
charged ones (Fig. 25b).115 3.2 Drug/therapeutics delivery It was shown that more than 90% of the loaded DOX was
released from the microcapsules after 4–5 h in a 0.154 M NaCl
solution.212 The same authors also studied the adsorption of
biological materials, namely cells and proteins on top of the
PAMAM/PSS multilayered films adsorbed on the surface of flat
substrates and microparticles and modified with PEG-bearing
lipids via glutaraldehyde cross-linking, to evaluate the biocom-
patibility of the multilayered films.213 It was found that the Khopade and Caruso developed hollow multilayered micro-
capsules by coating melamine formaldehyde colloidal particles
with alternating PSS and G4 PAMAM multilayered shells fol- Fig. 24 (a) Preparation of LbL microcapsules encapsulating dendrimers. The LbL assembly process is developed on CaCO3 particles, followed by
core dissolution to achieve PAMAM-loaded microcapsules. (b)
Fluorescence microscope image of PAMAM-loaded PAH/PVS micro-
capsules, where PAMAM was labeled with tetramethylrhodamine iso-
thiocyanate. (c) ANS uptake into (a) PAMAM-loaded microcapsules and
(b) PAMAM-free microcapsules, at pH 4. (d) ANS release profile from
PAMAM-loaded microcapsules. b–d: Adapted from ref. 211 with per-
mission from Elsevier, copyright 2009. Fig. 25 Immunofluorescence staining of neurons cultured in film sur-
faces terminated with (a) positively and (b) negatively charged hydrazine
phosphorus dendrimers, after 5 days. Adapted from ref. 115 with per-
mission from Elsevier, copyright 2008. Fig. 25 Immunofluorescence staining of neurons cultured in film sur-
faces terminated with (a) positively and (b) negatively charged hydrazine
phosphorus dendrimers, after 5 days. Adapted from ref. 115 with per-
mission from Elsevier, copyright 2008. Fig. 24 (a) Preparation of LbL microcapsules encapsulating dendrimers. The LbL assembly process is developed on CaCO3 particles, followed by
core dissolution to achieve PAMAM-loaded microcapsules. (b)
Fluorescence microscope image of PAMAM-loaded PAH/PVS micro-
capsules, where PAMAM was labeled with tetramethylrhodamine iso-
thiocyanate. (c) ANS uptake into (a) PAMAM-loaded microcapsules and
(b) PAMAM-free microcapsules, at pH 4. (d) ANS release profile from
PAMAM-loaded microcapsules. b–d: Adapted from ref. 211 with per-
mission from Elsevier, copyright 2009. 3.3 Gene therapy 26b) and
higher transfection levels in comparison with the control
encompassing PEI-based DNA multilayered films (Fig. 26c). It
was found that the GSH concentration influenced the transfec-
tion efficiency of the G3/DNA films in 293T cells. The highest
concentrations of GSH tested (5 and 10 mM) accelerated the
degradation of the films in small fragments, which promoted
the detachment of the adhered cells, leading to low transfec-
tion levels. Conversely, the highest transfection activity was
obtained in the presence of 2.5 mM GSH (Fig. 26c) with ca. 36% GFP-positive 293T cells after 5 days of co-culture. Therefore, the cleavage of the disulfide bonds in the polycation
by the cell attachment on G3/DNA LbL films induced the
release of the content into the cytoplasm in a sustained Electrospun polymer fibers have been also deeply studied
as platforms for the controlled delivery of therapeutics.232–234
Tomás and co-workers have combined electrospinning and LbL
assembly for developing a gene delivery platform for tissue
engineering.235 The biologically active surface was created by
surface functionalization of biodegradable electro- spun
poly(lactic-co-glycolic acid) (PLGA) fibers with chitosan and
ALG multilayers (Fig. 27). It was shown that the developed
system protects pDNA from endonucleases degradation, serving
as a platform for the controlled delivery of pDNA/den- drimer
complexes. As such, the attachment, growth, and differ-
entiation of human mesenchymal stem cells towards the osteo-
genic lineage were supported by the functionalized fiber when manner. Interestingly, it was found that the pDNA within the
G3/DNA multilayered films could be also internalized by 293T
cells without any reducing agent because the plasma mem-
brane surface and intracellular environment could trigger the
disassembly of the LbL films and release the incorporated
pDNA locally. Fig. 27 (a) Preparation of PLGA fiber mats and immobilization of pDNA/
dendrimer complexes in those platforms. Adapted from ref. 234 with
permission from the American Chemical Society, copyright 2020. (b)
SEM images of electrospun PLGA fiber mats (a and b) before and (c and
d) after coating with alternating layers of chitosan and ALG. (b) and (d)
are magnified images of (a) and (c), respectively. Small pores intercon-
nected are observed in the fiber structure resulting from the electro-
spinning process. Reproduced from ref. 235 with permission from the
Royal Society of Chemistry, copyright 2016. Recently, the focus has been on the synthesis of bio-
degradable dendrimers,231 and their incorporation in multi-
layered assemblies. 3.3 Gene therapy Gene therapy is considered as a powerful approach to prevent
and/or treat numerous diseases via the introduction of exogen-
ous DNA into cells. This concept has been broadened and, cur-
rently, promising strategies incorporating dendrimers and
nucleic acids in multilayered films have been reported. For
instance, electrostatically-driven multilayered films composed
of G3 amine-terminated poly(disulfide amine) dendronized
polymer and oppositely charged plasmid DNA ( pDNA) have been
proposed for in vitro localized gene delivery.230 Although the
disulfide bonds on the polycation are stable during the
preparation and storage of the multilayered films, their degra-
dation can be triggered by reductive agents, such as gluta-
thione (GSH). Indeed, the multilayered assemblies were dis-
assembled in the presence of GSH, triggering the release of Polymeric micelles, obtained through the spontaneous
arrangement of amphiphilic block copolymers in aqueous
solutions, have been appointed as powerful for the encapsula-
tion of poorly water-soluble cancer drugs. As such, the develop-
ment of polyion complex (PIC) micelles have raised much
attention.217 However, the instability of PIC micelles towards
ionic strength under physiological conditions has led research-
ers to prepare stable PIC micelles by increasing its rigidity
through the introduction of dendrimers in their structure.15,218–
220 PIC micelles containing dendritic photosen- sitizers have
been intensively studied in photodynamic therapy developed multilayered films to control the release of incorpor-
ated nucleic acids.230 the complexed DNA, which in turn affected the corresponding
gene expression. The G3/DNA multilayered films exhibited
good biocompatibility (Fig. 26a), low cytotoxicity (Fig. 26b) and
higher transfection levels in comparison with the control
encompassing PEI-based DNA multilayered films (Fig. 26c). It
was found that the GSH concentration influenced the transfec-
tion efficiency of the G3/DNA films in 293T cells. The highest
concentrations of GSH tested (5 and 10 mM) accelerated the
degradation of the films in small fragments, which promoted
the detachment of the adhered cells, leading to low transfec-
tion levels. Conversely, the highest transfection activity was
obtained in the presence of 2.5 mM GSH (Fig. 26c) with ca. 36% GFP-positive 293T cells after 5 days of co-culture. Therefore, the cleavage of the disulfide bonds in the polycation
by the cell attachment on G3/DNA LbL films induced the
release of the content into the cytoplasm in a sustained the complexed DNA, which in turn affected the corresponding
gene expression. The G3/DNA multilayered films exhibited
good biocompatibility (Fig. 26a), low cytotoxicity (Fig. 3.3 Gene therapy In particular, the preparation of multi-
layered films incorporating biodegradable dendrimers and
nucleic acids has been on the spotlight. In this regard, Peng
et al. proposed a completely new approach, highly attractive for
gene therapy and tissue engineering owing the ability of the Fig. 26 (a) Cell attachment and proliferation on the surfaces of (G3/ DNA)9.5
and (PEI/DNA)9.5 multilayered films for 1 day (a and d), 3 days (b and e) and
5 days (c and f). (b) CLSM images of live (green) and dead (red) 293T cells
after 24 h culture on the surfaces of (a) (G3/DNA)9.5 and
(b) (PEI/DNA)9.5 multilayered films. The scale bar is 5 µm. (c) Transfection
efficiencies of (G3/DNA)9.5 multilayered films in 293T cells as a function
of the GSH concentration. (PEI/DNA)9.5 multilayered films were incubated
without GSH (shown as control 1) and in 5 mM GSH (shown as control 2). Adapted from ref. 230 with permission from Elsevier, copyright 2019. Fig. 26 (a) Cell attachment and proliferation on the surfaces of (G3/ DNA)9.5
and (PEI/DNA)9.5 multilayered films for 1 day (a and d), 3 days (b and e) and
5 days (c and f). (b) CLSM images of live (green) and dead (red) 293T cells
after 24 h culture on the surfaces of (a) (G3/DNA)9.5 and
(b) (PEI/DNA)9.5 multilayered films. The scale bar is 5 µm. (c) Transfection
efficiencies of (G3/DNA)9.5 multilayered films in 293T cells as a function
of the GSH concentration. (PEI/DNA)9.5 multilayered films were incubated
without GSH (shown as control 1) and in 5 mM GSH (shown as control 2). Adapted from ref. 230 with permission from Elsevier, copyright 2019. Fig. 26 (a) Cell attachment and proliferation on the surfaces of (G3/ DNA)9.5
and (PEI/DNA)9.5 multilayered films for 1 day (a and d), 3 days (b and e) and
5 days (c and f). (b) CLSM images of live (green) and dead (red) 293T cells
after 24 h culture on the surfaces of (a) (G3/DNA)9.5 and
(b) (PEI/DNA)9.5 multilayered films. The scale bar is 5 µm. (c) Transfection
efficiencies of (G3/DNA)9.5 multilayered films in 293T cells as a function
of the GSH concentration. (PEI/DNA)9.5 multilayered films were incubated
without GSH (shown as control 1) and in 5 mM GSH (shown as control 2). Adapted from ref. 230 with permission from Elsevier, copyright 2019. Fig. 3.3 Gene therapy 26 (a) Cell attachment and proliferation on the surfaces of (G3/ DNA)9.5
and (PEI/DNA)9.5 multilayered films for 1 day (a and d), 3 days (b and e) and
5 days (c and f). (b) CLSM images of live (green) and dead (red) 293T cells
after 24 h culture on the surfaces of (a) (G3/DNA)9.5 and Fig. 27 (a) Preparation of PLGA fiber mats and immobilization of pDNA/
dendrimer complexes in those platforms. Adapted from ref. 234 with
permission from the American Chemical Society, copyright 2020. (b)
SEM images of electrospun PLGA fiber mats (a and b) before and (c and
d) after coating with alternating layers of chitosan and ALG. (b) and (d)
are magnified images of (a) and (c), respectively. Small pores intercon-
nected are observed in the fiber structure resulting from the electro-
spinning process. Reproduced from ref. 235 with permission from the
Royal Society of Chemistry, copyright 2016. (b) (PEI/DNA)9.5 multilayered films. The scale bar is 5 µm. (c) Transfection
efficiencies of (G3/DNA)9.5 multilayered films in 293T cells as a function
of the GSH concentration. (PEI/DNA)9.5 multilayered films were incubated
without GSH (shown as control 1) and in 5 mM GSH (shown as control 2). Adapted from ref. 230 with permission from Elsevier, copyright 2019. versatility imparted by the LbL assembly technology in terms
of the unprecedented choice of both inanimate and animate
substrates to be coated and building blocks to be assembled via
a multitude of intermolecular interactions and assembly
methodologies. In fact, the LbL technology enables shaping a
wide array of innovative and highly controlled multifunctional
bioarchitectures, ranging from simple to more complex and
elaborated structures spanning from 0D to 3D, not attainable
with LbL-free dendrimers. The integration of dendrimers as
ingredients in LbL assemblies enables processing them not only
into core–shell particles and hollow multilayered capsules or
tubes, but also into multilayered thin films, free-standing
membranes, among others depending on the template to be
coated and envisioned bioapplication. Besides, the adaptable
and tunable chemistry denoted by the dendrimers turn them into
promising nanoreservoirs for enabling high payload carry- ing
and controlled release at a target specific site. 4.
Concluding remarks and future
perspectives Dendrimers are very attractive building blocks for a wide array
of biomedical, biological and biotechnological applications,
such as biosensing, diagnosis, bioimaging, drug/therapeutics
delivery, gene therapy, tissue engineering and regenerative
medicine owing to their unique and superior structural and
physicochemical properties. Those include, their well-defined
and highly branched structure, monodisperse and homo-
geneous 3D globular architecture, multivalency, tunable mole-
cular weight, chemical composition and surface functionality,
and precisely controllable nanosize. In this regard, they have
been appointed as promising ingredients for the bottom-up
assembly of innovative LbL multifunctional systems denoting
enhanced structural properties and multifunctionalities not
attainable with linear polymers. Those include their intrinsic
ability to penetrate the interlayers, produce thicker polymeric
multilayered films, and foster stronger interactions between
the assembled building blocks. Furthermore, the dendrimer-
containing LbL assemblies provide enhanced binding sites to
drugs/therapeutics/bioactive agents either via the internal cores
of the dendrimers or their high density of peripheral functional
groups, as well as those of the multilayered systems,
representing enhanced nanocarriers for the encapsulation,
protection, transport and on-demand site-specific and con-
trolled delivery of cargoes. However, despite the benefits and significance of the devel-
oped dendrimer-based multifunctional LbL systems for bioap-
plications, there are still major bottlenecks that inhibit their
translation into practical clinical applications. Among them,
most of the higher generation dendrimers assembled into LbL
systems are cationic, thus raising cytotoxicity concerns and
biological cell membrane disruption. This drawback ought to be
addressed by the LbL community by taking advantage of the
already successfully functionalized amine-ended dendri- mers
with PEG, natural saccharides, or other ligands. Furthermore, a
major hurdle pertains to the nonbiodegradabil- ity of the mostly
used and LbL assembled dendrimers under biologically relevant
conditions, which elicit cytotoxicity effects by transposing the cell
membrane and accumulating intra- cellularly. In this regard,
biodegradable dendrimers have been gathering much interest
and
hold
great
promise
as
smart
nanocarriers
for
bioapplications, including for on-demand cell-regulated
intracellularly controlled and sustained thera- peutics delivery
and tissue regeneration. For this purpose, the In this review, we provide a comprehensive overview of the
different intermolecular interactions to create dendrimer-con-
taining nanostructured LbL assemblies, including electrostatic
interactions, hydrogen bonding, covalent bonding, coordi-
nation chemistry, host–guest and biological specific inter-
actions, for bioapplications (Table 1). 3.3 Gene therapy In addition, the
multivalency assigned by the high density of surface func-
tional groups allows tethering targeting-specific cargoes or
ligands in a precise controllable manner for improving their
biocompatibility, site-specific controlled delivery, circulation
time in the bloodstream and readily interact with the cell
membrane via the cell specific receptors. Although these fea-
tures are commonly exploited in LbL-free dendrimers, the inte-
gration of dendrimers into LbL systems allows an enhanced
protection of themselves and their cargo from undesirable
interactions with the biological environment and improve the
aqueous solubility and biodistribution of the cargo with reduced
side effects. Moreover, the LbL architecture itself could be
easily modulated and imparted with stimuli-respon- sive
materials, thus rendering the LbL systems smart systems for on-
demand site-specific sustained delivery of dendrimer and
encapsulated cargoes. As such, there is no doubt that the
development of supramolecular dendrimer-containing multi-
layered systems is highly beneficial when compared to the LbL-
free dendrimers and LbL ensembles prepared with linear
polymers. a pDNA codifying for human bone morphogenetic protein-2
(hBMP-2) was used. A similar approach has been proposed by
Chen et al., who reported the preparation of electrostatic-based
multilayered films consisting of cationic PAMAM dendrimers/
EGFP-hBMP-2 pDNA complex and anionic naked plasmid on
titanium substrates, denoting a great potential to functionalize
titanium implants with hBMP-2 gene.236 Moreover, the multi-
layered films can be used for the delivery of functional genes
in a localized and timely manner at the target site. The gene-
functionalized titanium implant improved the osteogenic
differentiation of osteoblasts, promoting early bone formation
around implant, thus suggesting its possible application in gene-
stimulating biomaterials. • Multilayered thin
films
• Hollow multilayered
capsules • Single component
multilayered thin
films
• Multilayered thin
films relying on
different components • Multilayered thin
films 4.
Concluding remarks and future
perspectives This has been fueled by the simpli-
city, cost-effectiveness, mild processing conditions, and high Table 1 Intermolecular interactions driving the LbL assembly of dendrimer-containing LbL nanoassemblies and their corresponding main features
and bioapplications
Intermolecular
Interactions
Main features
• The most explored mechanism to
Dendrimer-based LbL
nanoassemblies
Bioapplications
Representative references
• Core–shell particles
•Biosensing
83, 108–116, 119–123,
develop dendrimer-containing
multilayered assemblies
• The LbL assembly process can be
performed with virtually any charged
and water-soluble molecules
• The structure, composition and
properties of the multilayered
assemblies can be precisely tailored
by adjusting several parameters (e.g.,
pH, ionic strength, polymer
molecular weight)
• Multilayered thin
films
• Robust free-standing
multilayered
membranes
• Hollow multilayered
capsules
•Diagnostics
•Drug/therapeutics
delivery
•Cancer therapy
•Gene therapy
•Tissue engineering
128, 174, 182, 196, 199,
211, 212, 229, 230, 235
and 236 Table 1 Intermolecular interactions driving the LbL assembly of dendrimer-containing LbL nanoassemblies and their corresponding main features
and bioapplications
Intermolecular
Interactions
Main features
• The most explored mechanism to
Dendrimer-based LbL
nanoassemblies
Bioapplications
Representative references
• Core–shell particles
•Biosensing
83, 108–116, 119–123,
develop dendrimer-containing
multilayered assemblies
• The LbL assembly process can be
performed with virtually any charged
and water-soluble molecules
• The structure, composition and
properties of the multilayered
assemblies can be precisely tailored
by adjusting several parameters (e.g.,
pH, ionic strength, polymer
molecular weight)
• Multilayered thin
films
• Robust free-standing
multilayered
membranes
• Hollow multilayered
capsules
•Diagnostics
•Drug/therapeutics
delivery
•Cancer therapy
•Gene therapy
•Tissue engineering
128, 174, 182, 196, 199,
211, 212, 229, 230, 235
and 236
•Enables the incorporation of
charged and uncharged molecules
within the multilayers
•Fabrication of more stable and
robust films than the ones developed
by electrostatic interactions
•The LbL films growth by assembling
polymers bearing moieties that can
act as hydrogen bonding donors and
hydrogen bonding acceptors and can
be accomplished in aqueous
solutions or in organic solvents
•The LbL films created using
hydrogen bonding are highly
sensitive to pH
• Single component
multilayered thin
films
• Multilayered thin
films relying on
different components
• Drug/therapeutics
delivery
• Biosensing
130–134 and 137–139
• Flexibility in the selection of the
• Multilayered thin
• Immunoprotection
140, 141, 144–146,
molecules to be assembled, allowing
the development of multifunctional
assemblies that cannot be prepared
by electrostatic-driven LbL assembly
methods
• Fabrication of more stable and
robust multilayered films than the
ones developed by electrostatic
interactions and hydrogen bonding
• The exposure of multilayered films
to UV irradiation induces changes in
the properties of the films, increasing
films
• Hollow multilayered
capsules
• Biosensing
• Drug/therapeutics
delivery
153–155, 175 and 201 • Drug/therapeutics
delivery
• Biosensing
130–134 and 137–139 • Single component
multilayered thin
films
• Multilayered thin
films relying on
different components •Enables the incorporation of
charged and uncharged molecules
within the multilayers
•Fabrication of more stable and
robust films than the ones developed
by electrostatic interactions
•The LbL films growth by assembling
polymers bearing moieties that can
act as hydrogen bonding donors and
hydrogen bonding acceptors and can
be accomplished in aqueous
solutions or in organic solvents
•The LbL films created using
hydrogen bonding are highly
sensitive to pH • Multilayered thin
• Immunoprotection
140, 141, 144–146,
films
• Hollow multilayered
capsules
• Biosensing
• Drug/therapeutics
delivery
153–155, 175 and 201 • Multilayered thin
• Immunoprotection
140, 141, 144–146,
films
• Hollow multilayered
capsules
• Biosensing
• Drug/therapeutics
delivery
153–155, 175 and 201 • Multilayered thin
films
• Hollow multilayered
capsules 4.
Concluding remarks and future
perspectives Although most of the
studies reported in the literature focused on the employment
of free dendrimers, herein we have shown that there has been
an increasing interest in the molecular design and develop-
ment of supramolecular dendrimer-based multilayered assem-
blies for addressing multiple biological and biomedical appli-
cations, including biosensing, bioimaging, drug/therapeutics
delivery, and gene therapy. • Flexibility in the selection of the molecules to be assembled, allowing
the development of multifunctional
assemblies that cannot be prepared
by electrostatic-driven LbL assembly
methods • Fabrication of more stable and
robust multilayered films than the
ones developed by electrostatic
interactions and hydrogen bonding
• The exposure of multilayered films
to UV irradiation induces changes in
the properties of the films, increasing
their robustness and stability
• The design of stimuli-responsive
multilayers is not appropriate due to
the irreversibility of the covalent-
based systems • Multilayered thin
films
• Biosensing
65, 83, 156 and 157 65, 83, 156 and 157 65, 83, 156 and 157 • Design of well-ordered, stable and
highly oriented multilayered films
encompassing organic ligands and
transition metal ions
• Enables the control over surface
structure at the molecular level • Design of well-ordered, stable and
highly oriented multilayered films
encompassing organic ligands and
transition metal ions • Enables the control over surface
structure at the molecular level Table 1 (Contd.) Main features • Preparation of multilayered films
based on highly selective and strong
interactions between host and guest
molecules • Multilayered thin
films
• Hollow multilayered
capsules
• Biosensors
162–168
• Diagnostics
• Molecular biology 162–168 • Encompass specific biological
interactions (e.g., avidin–biotin, DNA
hybridization) • Polymers with the same charge can
be assembly within the multilayers
provided that they entail
complementary biospecific
interactions Note: The schematic illustrations represent dendrimer-polymer and dendrimer-dendrimer (in the case of the electrostatic interactions and
hydrogen bonding) interactions. cal conditions and after being internalized by cells and reach- Note: The schematic illustrations represent dendrimer-polymer and dendrimer-dendrimer (in the case of the electrostatic interactions and
hydrogen bonding) interactions. cal conditions and after being internalized by cells and reach- use of biorthogonal coupling reactions, namely the strain-pro-
moted azide–alkyne cycloaddition or thiol–ene, among others,
represents a powerful strategy for the synthesis and covalent
decoration of dendrimers in the absence of metal catalysts,
additives and organic solvents.149 The possibility of developing
biocompatible and biodegradable smart dendrimer-containing
LbL nanoassemblies is particularly attractive for the nanoen-
capsulation of living cells, aiming to protect them and their
cargo from extreme conditions in the biological environment
and enable their on-demand delivery at the target site. Furthermore, those smart biocompatible and biodegradable
nanoassemblies also open new pathways in coating core–shell
particles and produce hollow multilayered nano/micro-cap-
sules able to bind and release payloads on-demand and be
used for cellular uptake. Acknowledgements 12 S. Mignani, J. Rodrigues, H. Tomas, M. Zablocka, X. Shi,
A. M. Caminade and J. P. Majoral, Chem. Soc. Rev., 2018,
47, 514–532, DOI: 10.1039/c7cs00550d. This work was supported by the Marine Biotechnology ERA-NET
project “BLUETEETH” (ERA-MBT/0002/2015) funded by the
Portuguese Foundation for Science and Technology (FCT) under
EU FP7 (Grant Agreement number: 604814), as well as by the
Programa Operacional Regional do Centro–Centro 2020, in the
component FEDER, and by national funds (OE) through
FCT/MCTES, in the scope of the project “SUPRASORT”
(PTDC/QUI-OUT/30658/2017, CENTRO-01-0145-FEDER-030658). This work was developed within the scope of the project CICECO–
Aveiro Institute of Materials, UIDB/50011/2020 & UIDP/
50011/2020, financed by national funds through the FCT/
MCTES. Financial aid was also obtained from the Spanish
Ministry of Science and Innovation (RTI2018-102212−B-I00), the
Xunta de Galicia (ED431C 2018/30, and Centro singular de
investigación de Galicia accreditation 2019-2022, ED431G2019/
03), Axencia Galega de Innovación (IN845D 2020/09), and the
European Union (European Regional Development Fund-ERDF). Cristiana F. V. Sousa and João Borges gratefully acknowledge
the financial support by FCT through the individual PhD grant
(2020.04408.BD) and individual Assistant Researcher contract
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innovation, and challenges that ought to be addressed while
the field is expanding to construct advanced and sophisticated
devices for diverse applications in the biological and biomedical
fields. Therefore, we foresee that this review will provide a fresh
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materials to be assembled into multilayered nanoassemblies
dictate their final end-use. For instance, the multilayered
nanoshells of hollow nano/micro-capsules could be enlisted
with “intelligent” ingredients, i.e. materials that are (multi)
stimuli-responsive (e.g., pH-, temperature-, ionic strength-,
light-sensitive, among others), whose response can be modu-
lated by playing with stimuli. An illustrative example pertains
to the development of hollow nanocapsules entailing pH-sensi-
tive multilayered shells that would be stable under physiologi- ing the endosomes, denoting a slightly acidic pH, would be
degraded and release the encapsulated bioactive agents. Besides, we envisage that the possibility of using biocompati-
ble and biodegradable dendrimers as own nanocarrier
vehicles and coating them with stimuli-responsive LbL
nanoshells con- stitutes an innovative approach that would
afford them with superior performance and improved control
at the nanoscale tackling a diverse set of bioapplications. Of great interest is also the capacity to accelerate the pro-
duction and scale-up of dendrimers, with novel strategies and
reactions challenging the conventionally tedious stepwise
syn- thesis of dendrimers.237 The noncovalent synthesis of
dendri- mers by the self-assembly of small amphiphilic
dendrimer building units is a revolutionary approach for
bioapplications, being able to mimic the covalently
developed dendrimers in their structure and functions.238
Furthermore, we envision that the integration of the bottom-
up LbL assembly technology with other prominent nano- and
micro-fabrication
method-
ologies,
including
photolithography, photopatterning and 3D bioprinting, holds
great promise for tailoring the construction of functional,
stable and well-defined nanostructures. Moreover, it would
also enable the design of complex, sophisti- cated, and
multiscale devices exhibiting emergent properties and
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and multi stimuli responsive behavior as well as self and multi-stimuli responsive behavior, as well as self- healing ability, thus opening new avenues to increase the
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CHANGES OF MORPHOLOGY AND THERMOOPTIC CHARACTERISTICS OF COMPOSITE WITH CRYSTALLINE SILICON DIOXIDE AT VACUUM-THERMAL EXPOSURE
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ИЗМЕНЕНИЕ МОРФОЛОГИИ И ТЕРМООПТИЧЕСКИХ ХАРАКТЕРИСТИК
КОМПОЗИТА С КРИСТАЛЛИЧЕСКИМ ДИОКСИДОМ КРЕМНИЯ
ПРИ ВАКУУМНО-ТЕПЛОВОМ ВОЗДЕЙСТВИИ Аннотация. В данной работе изучено воздействие вакуммного ультрафиолетового (ВУФ) излу-
чения на полимерные композиты на основе полиалканимида с кристаллическим диоксидом кремния. Рассмотрено изменение морфологии и термооптических характеристик полимерного композита при
вакуумно-тепловом воздействии. Облучение образцов полимерных композитов вакуумным ультрафи-
олетовым излучением проводилось в специализированной установке для испытаний образцов из поли-
меркомпозитов в условиях, приближенных к околоземному космическому пространству. Изучена мик-
роскопия поверхности полимерного композита методами растровой (сканирующей) электронной
микроскопией и атомной-силовой (зондовой) микроскопией до и после облучения ВУФ. Установлено,
что микроскопия поверхности необработанного композита, характеризуются непрерывным распре-
делением кристаллического SiO2 по всему объему полиалканимидной матрицы, однако также наблю-
дается неоднородность распределения кристаллического SiO2 неорганического компонента в объеме
композита. После 24-часовой обработки ВУФ микроскопия поверхности практически не изменилась. Отчетливо видны гр333аницы кристаллического SiO2, которые также покрыты полиалканимидом. Изначальная поверхность композита достаточно гладкая и ровная, а шероховатость находится в
нанодиапазоне. Установлено, что ВУФ приводит к эффективной функционализации поверхностного
слоя полимерного материала (65 % мас. наполнителя) и сглаживанию поверхности: средняя шерохо-
ватость уменьшается с 20 до 11 нм. Показано, что изменение параметров шероховатости поверх-
ности композита после обработки ВУФ указывает на травление поверхностного слоя, не затрагива-
ющее нижние слои композита. После облучения композита с 65 % содержанием наполнителя при
температуре 125 °С в течение 24 часов интегральный коэффициент поглощения увеличивается на 9,6
% с 0,09 до 0,096 по сравнению с исходным, что меньше критерия стойкости к ВУФ-излучению. р
р
р
у
Ключевые слова: вакуумный ультрафиолет, полимерный комопозит, кристаллический SiO2, зон-
довая микроскопия, шероховатость. Введение. Известно, что ультрафиолет (УФ)
и вакуумный ультрафиолетовый (ВУФ) значи-
тельно способствуют деградации материалов
космических аппаратов на околоземной орбите. УФ/ВУФ-излучение вездесущее – широко рас-
пространено практически на всех орбиталях вы-
соты. В то же время в присутствии атомарного
кислорода (например, на низкой околоземной ор-
бите) эффекты деградации могут быть более вы-
раженным на определенных высотах [1–2]. Температура космического аппарата про-
порциональна отношению его поглощающей
солнечной энергии (αsolar) к его излучательной
способности (ε). Tspacecraft=αsolar/ε (1) (1) Tspacecraft=αsolar/ε Как правило, излучательная способность
терморегулирующих покрытий не изменяется. Поэтому, если поглощающая способность мате-
риалов
терморегулирования
возрастает
со временем, будет сопутствующее повышение
температуры космического корабля. Действи-
тельно, данные как по летным экспериментам,
так и по лабораторным испытаниям, указывают
на серьезные последствия деградации полимер-
ных материалов после воздействия ВУФ [5-7]. Например, органические материалы, в частности
пленки Kapton® и Teflon® снятые со спутника
SolarMaximumMission имели серьезные повре-
ждения [8]. 2018, №11 2018, №11 Вестник БГТУ им. В.Г. Шухова ИЗМЕНЕНИЕ МОРФОЛОГИИ И ТЕРМООПТИЧЕСКИХ ХАРАКТЕРИСТИК
КОМПОЗИТА С КРИСТАЛЛИЧЕСКИМ ДИОКСИДОМ КРЕМНИЯ
ПРИ ВАКУУМНО-ТЕПЛОВОМ ВОЗДЕЙСТВИИ Тепловые покрытия, которые находи-
лись на околоземной орбите в течение четырех Материалы, используемые в конструкции
космических аппаратов в качестве терморегули-
рующих покрытий, показывают наиболее значи-
тельное ухудшение функциональных свойств
под воздействием ВУФ. Эта деградация проявля-
ется
как
поверхностная
эрозия
в
полимерах, так и в увеличении абсорбционной
способности
(потемнение)
в
полимерах
и термических красок [3–4]. Именно такое изме-
нение
абсорбции
ставит
под
угрозу
терморегулирующие свойства материалов. 83 Вестник БГТУ им. В.Г. Шухова 2018, №11 дробно способ получения и свойства кристалли-
ческого диоксида кремния описаны в [17]. дробно способ получения и свойства кристалли-
ческого диоксида кремния описаны в [17]. лет и двух месяцев, показали деградацию, харак-
теризующуюся интенсивной эрозией, возникаю-
щей на поверхностях материала подвергавшегося
воздействию как ВУФ, так и атомарного кисло-
рода. Обнаружена наиболее серьезная деграда-
ция для образцов, прикрепленных к передней
кромке спутника, который получил преимуще-
ственно ВУФ-облучение. Кроме того, краски для
терморегулирования, применяемые к задней
кромке показали сильное затемнение. Измене-
ния, вызванные ВУФ в этих материалах, могут
значительно меняют их поглощающую способ-
ность и контрольные характеристики. Понятно,
что долгосрочное космическое воздействие на
эти материалы значительно ухудшит их целост-
ность теплового контроля. Композитные диски получали следующим
образом. Полиалканимид растворяли в полярном
апротонном растворителе (толуоле). Растворение
полиалканимида необходимо для смешения мат-
рицы и модифицированного наполнителя в жид-
кой среде с использованием ультразвуковой ка-
витации. Использование ультразвуковой обра-
ботки (частота 22 Гц) позволяет достичь высокой
однородности распределения наполнителя в мат-
рице и не дает возможности образованию агломе-
ратов частиц наполнителя [18–21]. После выпа-
ривания растворителя получали порошкообраз-
ную смесь матрицы и наполнителя. Полученную смесь полиалканимида и кри-
сталлического диоксида кремния загружали в
стальную пресс-форму, нагревали до темпера-
туры размягчения полиалканимида и поддержи-
вали ее в течение часа. После нагрева происхо-
дило прессование при высоком давлении 1 ГПа. Полученный композит представлял собой диски
круглой формы диаметром 3 см и толщиной
1–5 мм. ВУФ-излучение длин волн от 115 до 200 нм,
создаваемое солнцем в космической среде, мо-
жет вызвать деградацию терморегулирующих
покрытий, производя изменения оптических,
механических и химических свойств [9–12]. Эти
эффекты особенно важны для полимера, по-
скольку для большинства полимеров ВУФ-излу-
чение
поглощается
тонким
поверхностным
слоем [13–14]. Вакуумный ультрафиолет содей-
ствует десорбции молекул СО, СО2 , Н2О и Н2 с
поверхности полимера: энергии ВУФ-излучения
достаточно для разрыва связей С–С, С–О и функ-
циональных групп [15–16]. Большинство иссле-
дований посвящено изучению изменений опти-
ческих характеристик под воздействием ВУФ на
чистые полимеры. ИЗМЕНЕНИЕ МОРФОЛОГИИ И ТЕРМООПТИЧЕСКИХ ХАРАКТЕРИСТИК
КОМПОЗИТА С КРИСТАЛЛИЧЕСКИМ ДИОКСИДОМ КРЕМНИЯ
ПРИ ВАКУУМНО-ТЕПЛОВОМ ВОЗДЕЙСТВИИ Однако чистые полимеры
редко применяются для терморегулирующих по-
крытий класса «Солнечные отражатели», так как
они обладают низкими исходными оптическими
характеристиками. Облучение образцов полимерных компози-
тов вакуумным ультрафиолетовым излучением
проводилось в специализированной установке
для испытаний образцов из полимеркомпозитов
в условиях, приближенных к околоземному кос-
мическому пространству (рис. 2). Установка из-
готовлена фирмой ООО «Вакуумные системы и
электроника» (Новосибирск, Россия) по заказу
Центра
радиационного
мониторинга
BSTU
named after V.G. Shukhov (Belgorod, Russia). Установка имеет следующие технологиче-
ские параметры: вакуум (давление не более 10-3
Па), температура -190 °С до +160 ºС, ионизирую-
щее излучение (ВУФ с длиной волны λ = 90…115
нм, интенсивность 0.5 Вт/м2). Установка позво-
ляет одновременно облучать 12 образцов диамет-
ром не более 5 см. Облучение в камере прово-
дили при повышенной 125°С температуре в тече-
ние 24 часов, поглощенная доза в каждом экспе-
рименте составляла 9,54 МГр. В данной работе изучено воздействие ВУФ
на полимерные композиты на основе полиалка-
нимида с кристаллическим диоксидом кремния. Рассмотрено изменение морфологии и термооп-
тических характеристик полимерного композита
при вакуумно-тепловом воздействии. Методология. В качестве полимерной мат-
рицы для синтеза композитов использовали тер-
мопластичный полиалканимид, получаемый по-
ликонденсацией
диангидридапиромеллитовой
кислоты с 1,12 – додекаметилендиамином. Сканирующая (растровая) электронная мик-
роскопия была использована для изучения мор-
фологии поверхности до и после облучения
ВУФ. Микроскопию проводили с использова-
нием электронного микроскопа TescanMira 3
LMU. В качестве наполнителя использовали кри-
сталлический диоксид кремния. Синтез кристал-
лического диоксида кремния производили гидро-
термальным способом в щелочной среде путем
автоклавирования в реакторе высокого давления
(GSA-0.3). Рабочий объем автоклава способен
вместить в себя до 300 мл вещества жидкого со-
стояния, создавать рабочее давление до 70 МПа,
рабочий интервал температур до 350оС.Более по- Зондовую микроскопию поверхности ком-
позитов до и после облучения ВУФ изучали с ис-
пользованием сканирующего зондового микро-
скопа Ntegra-aura (изготовитель ЗАО «НТ-МДТ»,
Россия, 2007). Интегральный коэффициент пoглощени-
ясoлнечногoизлучeния рассчитывали исходя из 84 Вестник БГТУ им. В.Г. Шухова 2018, №11 Для более тонкого анализа поверхностных
изменении композита после обработки ВУФ на
рис. 2 представлено АСМ-изображение поверх-
ности исходного полимерного композита с 65 %
содержанием кристаллического SiO2, а также по-
верхности после обработки ВУФ в течение 24 ча-
сов при температуре 125 °С. значений спектров отражения композитов при
определенной длине волны. Спектры отражения
композитов (rλ) регистрировали с помощью спек-
трофотометра UV-3600. Спектры rλ регистриро-
вали в диапазоне 0.24–2.02 µm по точкам с шагом
0.5 nm. значений спектров отражения композитов при
определенной длине волны. Спектры отражения
композитов (rλ) регистрировали с помощью спек-
трофотометра UV-3600. ИЗМЕНЕНИЕ МОРФОЛОГИИ И ТЕРМООПТИЧЕСКИХ ХАРАКТЕРИСТИК
КОМПОЗИТА С КРИСТАЛЛИЧЕСКИМ ДИОКСИДОМ КРЕМНИЯ
ПРИ ВАКУУМНО-ТЕПЛОВОМ ВОЗДЕЙСТВИИ 1
( , )
a
A
S
z x y dx dy
A
(4) Одним из основных параметров ТРП явля-
ется коэффициент поглощения солнечного излу-
чения αs. Интегральный коэффициент поглоще-
ния αs, вычисляли исходя из величин коэффици-
ентов отражения по формуле: (4) 4) Среднеквадратичная шероховатость (Sq)
представляет собой среднеквадратичное значе-
ние значений ординат в области определения. Она эквивалентна стандартным отклонениям вы-
сот. 4) Среднеквадратичная шероховатость (Sq)
представляет собой среднеквадратичное значе-
ние значений ординат в области определения. Она эквивалентна стандартным отклонениям вы-
сот. 2
1
2
1
1
1
1
1
n
i
i
s
s
J
d
R
n
J
d
, (6) 2
1
( , )
q
A
S
z
x y dx dy
A
(5) , (6) где Rs – интегральный коэффициент отражения
солнечного излучения rλ – значение коэффици-
ента отражения композита при длине волны λ;
n – число равноэнергетических участков солнеч-
ного спектра (n=24). (5) 5) Поверхностная ассиметрия (Ssk) представ-
ляет собой степень смещения формы шерохова-
тости (неровности). 5) Поверхностная ассиметрия (Ssk) представ-
ляет собой степень смещения формы шерохова-
тости (неровности). 5) Поверхностная ассиметрия (Ssk) представ-
ляет собой степень смещения формы шерохова-
тости (неровности). Арбитражный критерий стойкости к воздей-
ствию ВУФ для ТРП в космическом простран-
стве состоит в увеличении интегрального коэф-
фициента поглощенияне более, чем на 25 % по
ГОСТ. После облучения композита с 65 % содер-
жанием наполнителя при температуре 125 °С в
течение 24 часов коэффициент αsувеличился на
9,6 % с 0,09 до 0,096 по сравнению с исходным,
что меньше критерия стойкости к ВУФ-излуче-
нию. Ssk<0: распределение по высоте смещено над
средней плоскостью. Ssk<0: распределение по высоте смещено над
средней плоскостью. Ssk = 0: распределение высоты (пики и ямы)
симметрично относительно средней плоскости. Ssk> 0: распределение по высоте смещено
ниже средней плоскости. 6) Коэффициент резкости пика, (Ska)является
мерой резкости профиля шероховатости. Ska<3:Распределение по высоте смещено над
средней плоскостью. Выводы. В ходе проведения исследований
был выявлен характер изменения поверхностных
совйств полимерных композитов на основе по-
лиалканимида и кристаллического диоксида
кремния до и после облучения ВУФ. Установ-
лено, что микроскопия поверхности необрабо-
танного композита, характеризуются непрерыв-
ным распределением кристаллического SiO2 по
всему объему полиалканимидной матрицы, од-
нако также наблюдается неоднородность распре-
деления кристаллического SiO2 неорганического Ska = 3: Нормальное распределение. Ska> 3: Распределение по высоте с шипами. ИЗМЕНЕНИЕ МОРФОЛОГИИ И ТЕРМООПТИЧЕСКИХ ХАРАКТЕРИСТИК
КОМПОЗИТА С КРИСТАЛЛИЧЕСКИМ ДИОКСИДОМ КРЕМНИЯ
ПРИ ВАКУУМНО-ТЕПЛОВОМ ВОЗДЕЙСТВИИ Шухова максимумов плюс средняя высота пяти наимень-
ших локальных минимумов: максимумов плюс средняя высота пяти наимень-
ших локальных минимумов: [23], в то время как радиолиз приводит к иониза-
ции и возбуждению электронов в синглетные и
триплетные состояния. Поэтому обработка поли-
мерного композита ВУФ привела к разрыву раз-
личных химических связей в полимерных моле-
кулах, включая C-C, C-H, C-O. Расщепление
связи и рекомбинация радикалов, образованных
фотолизом ВУФ, могут приводить к двойным
связям и поперечным связям. Поэтому ВУФ мо-
жет модифицировать полимерные поверхности,
не повреждая объемные свойства, поскольку ха-
рактерная глубина проникновения ВУФ в поли-
меры не превышает нескольких сотен нм из-за
высокой ионизации поглощения (104–105 см-1)
[22]. [23], в то время как радиолиз приводит к иониза-
ции и возбуждению электронов в синглетные и
триплетные состояния. Поэтому обработка поли-
мерного композита ВУФ привела к разрыву раз-
личных химических связей в полимерных моле-
кулах, включая C-C, C-H, C-O. Расщепление
связи и рекомбинация радикалов, образованных
фотолизом ВУФ, могут приводить к двойным
связям и поперечным связям. Поэтому ВУФ мо-
жет модифицировать полимерные поверхности,
не повреждая объемные свойства, поскольку ха-
рактерная глубина проникновения ВУФ в поли-
меры не превышает нескольких сотен нм из-за
высокой ионизации поглощения (104–105 см-1)
[22]. 5
5
1
1
5
pi
vi
i
i
z
z
z
S
(3) 5
5
1
1
5
pi
vi
i
i
z
z
z
S
5
5
1
1
5
pi
vi
i
i
z
z
z
S
(3) 5 (3) где zpi и zvi – высота i-го максимального локаль-
ного максимума и i-го младшего локального ми-
нимума соответственно. Если не существует пяти
допустимых максимумов или пяти действитель-
ных минимумов, параметр не определен. 3) Средняя шероховатость обозначается как
Ra (средняя арифметическая высота линии). Этот
параметр выражает абсолютную величину раз-
ницу в высоте каждой точки по сравнению с
арифметическим средним для поверхности. Этот
параметр обычно используется для оценки шеро-
ховатости поверхности. 3) Средняя шероховатость обозначается как
Ra (средняя арифметическая высота линии). Этот
параметр выражает абсолютную величину раз-
ницу в высоте каждой точки по сравнению с
арифметическим средним для поверхности. Этот
параметр обычно используется для оценки шеро-
ховатости поверхности. Такое комплексное воздействие приводит к
эффективной функционализации поверхност-
ного слоя полимерного материала и сглажива-
нию поверхности, что отчетливо видно по дан-
ным таблицы 1: средняя шероховатость умень-
шается с 20 до 11 нм. ИЗМЕНЕНИЕ МОРФОЛОГИИ И ТЕРМООПТИЧЕСКИХ ХАРАКТЕРИСТИК
КОМПОЗИТА С КРИСТАЛЛИЧЕСКИМ ДИОКСИДОМ КРЕМНИЯ
ПРИ ВАКУУМНО-ТЕПЛОВОМ ВОЗДЕЙСТВИИ Спектры rλ регистриро-
вали в диапазоне 0.24–2.02 µm по точкам с шагом
0.5 nm. значений спектров отражения композитов при
определенной длине волны. Спектры отражения
композитов (rλ) регистрировали с помощью спек-
трофотометра UV-3600. Спектры rλ регистриро-
вали в диапазоне 0.24–2.02 µm по точкам с шагом
0.5 nm. Основная часть. По проведенным ранее
данным оптимальными физико-механическими
характеристиками
обладали
композиты
с
65 мас. % содержанием наполнителя. На рис. 1
представлены микрофотографии полимерного
композита до облучения (рис. 1, а) и после облу-
чения ВУФ (рис. 1, б). Микроскопия поверхности
необработанного композита, характеризуются
непрерывным распределением кристаллического
SiO2 по всему объему полиалканимидной мат-
рицы, однако также наблюдается неоднород-
ность распределения кристаллического SiO2 не-
органического компонента в объеме композита
(рис. 1, а). Анализ микроструктуры поверхности
необработанного композита показал тонкую
структуру зерен. Частицы SiO2 покрыты термо-
пластичным покрытием из полиалканимидного
полимера (рис. 1, а). Хотя изображение СЭМ на
рис. 1, а показывает неоднородное распределение
наполнителя из-за агломерации частиц, но ника-
ких микротрещин или расколов обнаружено не
было. После 24-часовой обработки ВУФ микро-
скопия поверхности практически не изменилась. Отчетливо видны границы кристаллического
SiO2, которые также покрыты полиалканимидом. а
б
Рис. 2. АСМ-изображение поверхности
исходного полимерного композита до (а) и после
облучения ВУФ (б) а а б а
б
Рис. 1. Микроструктура поверхности
полимерного композита до (а) и
после облучения ВУФ (б) а а б Рис. 2. АСМ-изображение поверхности
исходного полимерного композита до (а) и после
облучения ВУФ (б) Анализ параметров шероховатости поверх-
ности проводился на площади 10×10 мкм. По па-
раметрам, представленным в табл. 1, можно су-
дить о степени шероховатости поверхности. Ана-
лиз статистических параметров поверхности по-
казывает, что изначальная поверхность компо-
зита достаточно гладкая и ровная, а шерохова-
тость находится в нанодиапазоне. а б Ниже представлены параметры шероховато-
сти поверхности и формулы по которым они вы-
числялись: 1) Высота пикового пика обозначается Sy. Параметр определяется как разность высот
между самым высоким и самым низким изобра-
жением. (2) Sy=zmax-zmin (2) Sy=zmax-zmin Sy=zmax-zmin б y
( )
2) Sz-10 точек по высоте, определяется как
средняя высота пяти максимальных локальных y
2) Sz-10 точек по высоте, определяется как
средняя высота пяти максимальных локальных Рис. 1. Микроструктура поверхности
полимерного композита до (а) и
после облучения ВУФ (б) Рис. 1. Микроструктура поверхности
полимерного композита до (а) и
после облучения ВУФ (б) 85 2018, №11 2018, №11 Вестник БГТУ им. В.Г. ИЗМЕНЕНИЕ МОРФОЛОГИИ И ТЕРМООПТИЧЕСКИХ ХАРАКТЕРИСТИК
КОМПОЗИТА С КРИСТАЛЛИЧЕСКИМ ДИОКСИДОМ КРЕМНИЯ
ПРИ ВАКУУМНО-ТЕПЛОВОМ ВОЗДЕЙСТВИИ Изменение параметров шероховатости по-
верхности композита после обработки ВУФ ука-
зывает на травление поверхностного слоя, не за-
трагивающее нижние слои композита. Энергия
фотонов от 9,6 до 15 эВ достаточно высока,
чтобы возбуждать электроны до высоких уров-
ней вблизи порога ионизации [22]. Вакуумное
ультрафиолетовое облучение ведет главным об-
разом к селективному возбуждению электронов в
синглетных состояниях в молекулах полимера 86 2018, №11 Вестник БГТУ им. В.Г. Шухова не было. После 24-часовой обработки ВУФ мик-
роскопия поверхности практически не измени-
лась. Отчетливо видны границы кристалличе-
ского SiO2, которые также покрыты полиалкани-
мидом. не было. После 24-часовой обработки ВУФ мик-
роскопия поверхности практически не измени-
лась. Отчетливо видны границы кристалличе-
ского SiO2, которые также покрыты полиалкани-
мидом. компонента в объеме композита. Хотя изображе-
ние СЭМ на показывает неоднородное распреде-
ление наполнителя из-за агломерации частиц, но
никаких микротрещин или расколов обнаружено компонента в объеме композита. Хотя изображе-
ние СЭМ на показывает неоднородное распреде-
ление наполнителя из-за агломерации частиц, но
никаких микротрещин или расколов обнаружено Таблица 1
Значения основных статистических параметров шероховатости поверхности исходного
полимерного композита до и после облучения ВУФ
№
п/п
Наименование параметра шероховатости
Значение
До обработки ВУФ
После обработки ВУФ
1. Объем выборки
65536
65536
2. Максимум, нм
439
189
3. Минимум, нм
0
0
4. Пиковый пик, Sy, нм
439
189
5. 10 точек по высоте, Sz, нм
222
96
6. Среднее, нм
294
128
7. Средняя шероховатость, Sa, нм
20
11
8. Второй момент
295
129
9. Среднеквадратичная шероховатость, Sq, нм
28
15
10. Поверхностная ассиметрия, Ssk, нм
-1,18
-0,34
11. Коэффициент резкости пика, Ska
7,7
2,5
12. Энтропия
10,1
9,3 Значения основных статистических параметров шероховатости поверхности и
полимерного композита до и после облучения ВУФ A.T., Baklanov M.R. Synergistic effect of VUV pho-
tons and F atoms on damage and etching of porous
organosilicate films // Plasma Processes and Poly-
mers. 2018. Vol. 15. Is. 4,
1700213
DOI:10.1002/ppap.201700213. В работе показано, что изначальная поверх-
ность композита достаточно гладкая и ровная, а
шероховатость находится в нанодиапазоне. Из-
менение параметров шероховатости поверхности
композита после обработки ВУФ указывает на
травление поверхностного слоя, не затрагиваю-
щее нижние слои композита. Установлено, что
ВУФ приводит к эффективной функционализа-
ции поверхностного слоя полимерного матери-
ала (65 % мас. наполнителя) и сглаживанию по-
верхности: средняя шероховатость уменьшается
с 20 до 11 нм. 4. Шувалов В.А., Токмак Н.А., Резниченко
Н.П. Физическое моделирование воздействия
атомарного кислорода и вакуумного ультрафио-
лета на полимеры в ионосфере Земли // Приборы
и техника эксперимента. ИЗМЕНЕНИЕ МОРФОЛОГИИ И ТЕРМООПТИЧЕСКИХ ХАРАКТЕРИСТИК
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ные исследования. 2010. № 1. C. 5–16. 21.Tanahashi M. Development of Fabrication
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sistance to ultraviolet radiation of POSS–TiO2/epoxy
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polymer materials in the space environment // Pro-
gress in Aerospace Sciences. 2016. No 83. pp. 37-56. DOI: 10.1016/j.paerosci.2016.02.002. 22. Skurat V.E., Dorofeev Yu.I. The transfor-
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rect bonding of polymers by vacuum ultraviolet light
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419. pp. 319–327. DOI:
10.1016/j.ap-
susc.2017.04.032 15. Hashimoto Yu., Yamamoto T. Solid state di-
rect bonding of polymers by vacuum ultraviolet light
below 160 nm // Applied Surface Science. 2017. No
419. pp. 319–327. DOI:
10.1016/j.ap-
susc.2017.04.032 23. Fozza A.C., Klemberg-Sapieha J.E.,
Wertheimer M.R. Vacuum Ultraviolet Irradiation of
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© Павленко В.И., Черкашина Н.И., Манаев В.А., Сидельников Р.В., 2018 Информация об авторах Павленко Вячеслав Иванович, доктор технических наук, профессор, директор химико-технологического ин-
ститута. Е-mail: belpavlenko@mail.ru. Белгородский государственный технологический университет им. В.Г. Шу-
хова. Россия, 308012, Белгород, ул. Костюкова, д. 46. Черкашина Наталья Игоревна, кандидат технических наук, доцент кафедры теоретической и прикладной хи-
мии. Е-mail: cherkashina.ni@bstu.ru. Белгородский государственный технологический университет им. В.Г. Шу-
хова. Россия, 308012, Белгород, ул. Костюкова, д. 46. Манаев Владимир Алексеевич, научный сотрудник кафедры теоретической и прикладной химии. Е-mail: xxi-vek.2015@yandex.ru. Белгородский государственный технологический университет им. В.Г. Шухова. Россия, 308012, Белгород, ул. Костюкова, д. 46. Сидельников Роман Владимирович, студент (бакалавр) кафедры теоретической и прикладной химии. Белго-
родский государственный технологический университет им. В.Г. Шухова. Россия, 308012, Белгород, ул. Костю-
кова, д. 46 Поступила в августе 2018 г. © Павленко В.И., Черкашина Н.И., Манаев В.А., Сидельников Р.В., 2018 88 2018, №11 Вестник БГТУ им. В.Г. Шухова 1Pavlenko V.I., 1,*Cherkashina N.I., 1Manaev V.A., 1Sidelnikov R.V. 1Belgorod State Technological University named after V.G. Shukhov
Russia, 308012, Belgorod, st. Kostyukova, 46
*E-mail: cherkashina.ni@bstu.ru REFERENCES "Functional nanomaterials for space technology",
2010, pp. 105–109. 1. Koontz S., Leger L., Albyn K., Cross J. Vacuum
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348. DOI:10.2514/3.26146 1. Koontz S., Leger L., Albyn K., Cross J. Vacuum
ultraviolet
radiation/atomic
oxygen
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Spacecraft and Rockets, 1990, vol. 27, no. 3. pp. 346-
348. DOI:10.2514/3.26146 6. Peng G., Hao W., Yang D., He S. Degradation of polyimide film under vacuum
ultraviolet irradiation. Journal of Applied Polymer
Science,
2004,
no. 94,
pp. 1370–1374. DOI: 10.1002/app.20920. 6. Peng G., Hao W., Yang D., He S. Degradation of polyimide film under vacuum
ultraviolet irradiation. Journal of Applied Polymer
Science,
2004,
no. 94,
pp. 1370–1374. DOI: 10.1002/app.20920. 2. Shuvalov V.A., Pismenny N.I., Kochubei
G.S., Tokmak N.A. Loss of mass of polyimide films
of space vehicles under the influence of atomic
oxygen and vacuum ultraviolet radiation. Cosmic
Investigations, 2014, vol. 52, no. 2, pp. 106–112. 7. Celina M.C., Dargaville T.R., Jones G.D.,
Elliott J.M. Verification and Operation of Adaptive
Materials in Space. Prepared by Sandia National
Laboratories Albuquerque, New Mexico 87185 and
Livermore, California 94550, 2006, 28 p. 3. Lopaev D.V., Zyryanov S.M., Zotovich A.I.,
Rakhimova T.V., Mankelevich Y.A., Rakhimov
A.T., Baklanov M.R. Synergistic effect of VUV
photons and F atoms on damage and etching of
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2018,
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1700213
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Vacuum-Ultraviolet Radiation Effects on Spacecraft
Thermal Control Materials. In: DeWitt R.N., Duston
D., Hyder A.K. (eds) The Behavior of Systems in the
Space Environment. NATO ASI Series (Series E:
Applied Sciences), vol 245. Springer, Dordrecht,
1993, pp. 259-266. DOI: 10.1007/978-94-011-2048-
7 11. 4. Shuvalov V.A., Tokmak N.A., Reznichenko
N.P. Physical modeling of the effect of atomic
oxygen and vacuum ultraviolet on polymers in the
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S0032816216020269. _
9. Mikhailov M.M., Dvoretsky M.I. A
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predicting
the
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M.S., Bednyakov S.A., Gaidar A.I., Zaitsev S.V.,
Chernik V.N. Investigation of the structure of
nanocomposites
based
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polymer
matrices. Proceedings of the 1 st all-Russian school-seminar of
students, graduate students and young scientists 10. THE CHANGES IN THE MORPHOLOGY AND THERMO-OPTICAL
CHARACTERISTICS OF THE COMPOSITE WITH CRYSTALLINE SILICA UNDER
VACUUM-THERMAL EFFECT Abstract. The effect of vacuum ultraviolet (VUV) radiation on polymer composites based on polyalkan-
imide with crystalline silicon dioxide is studied in this work. The changes in the morphology and thermos-optic
characteristics of the polymer composite under vacuum-thermal influence are considered. The irradiation of
polymer composite samples by vacuum ultraviolet radiation is carried out in a specialized facility for testing
polymer composite samples in conditions close to the near-Earth space. The microscopy of the polymer com-
posite surface by scanning electron microscope and atomic probe force microscopy is studied before and after
the VUV irradiation. It is established that microscopy of the raw composite’s surfaces are characterized by a
continuous distribution of SiO2 in the entire volume of polyalkylimide matrix, also non-uniform distribution of
SiO2 inorganic component in the volume of the composite is observed. After a 24-hour VUV treatment, the
surface microscopy remained practically unchanged. The boundaries of SiO2 are clearly visible and covered
with polyalkanimide. The initial surface of the composite is quite smooth, even; the roughness is in the na-
noscale. It is found that the VUV leads to an effective functionalization of the surface of the polymer material
(65 % by weight filling) and smoothing of the surface: the average roughness decreases from 20 to 11 nm. A
change in the surface roughness parameters after VUV treatment indicates etching of the surface that does not
affect the lower layers of the composite. After irradiation of the composite with 65 % filler content at 125 °C
for 24 hours, the integral absorption coefficient increases by 9.6 % from 0.09 to 0.096 compared to the origi-
nal, which is less than the criterion of resistance to VUV radiation. f
Keywords: vacuum ultraviolet, polymeric comoposite, crystalline SiO2, probe microscopy f
: vacuum ultraviolet, polymeric comoposite, crystalline SiO2, probe microscopy, roughness. "Functional nanomaterials for space technology",
2010, pp. 105–109. REFERENCES Cherkashina N.I., Pavlenko A.V.Changes in
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M.M. Photostability
of 89 Вестник БГТУ им. В.Г. Шухова 2018, №11 2018, №11 thermoregulatory coatings
of space
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Odintsov S.L., Krotkov V.A. Smoothing of the
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neutron studies, 2010, no. 1, pp. 5–16. 19. Goncharov V.D., Novik A.A. Application of
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ultradispersed materials. Izvestiya SPbGETU LETI,
2013, no. 5, pp. 98–103. 13. Peng D., Qin W., Wu X. A study on
resistance to ultraviolet radiation of POSS–
TiO2/epoxy nanocomposites. Acta Astronautica,
2015, no. 111, pp. 84–88. DOI: 10.1016/j.actaastro. 2015.02.004. 20. Manning A., Fricker A., Thompson R. The
anomalous effect of high intensity ultrasound on
paper fibre‐filler combinations. Pigment and Resin
Technology, 2009, vol. 38, is. 4, pp. 218–229,
DOI: 10.1108/03699420910973305. 14. Chen J., Ding N., Li Zh.,Wang W. Organic
polymer materials in the space environment. Progress in Aerospace Sciences, 2016, no. 83, pp. 37-56. DOI: 10.1016/j.paerosci.2016.02.002. 21.Tanahashi M. Development of Fabrication
Methods of Filler / Polymer Nanocomposites: With
Focus
on
Simple
Melt-Compounding
Based
Approach
without
Surface
Modification
of
Nanofillers. Materials, 2010, no. 3, pp. 1593–1619. DOI:10.3390/ma3031593 15. Hashimoto Yu., Yamamoto T. Solid state
direct bonding of polymers by vacuum ultraviolet
light below 160 nm. Applied Surface Science, 2017,
no. 419,
pp. 319–327. DOI:
10.1016/j.apsusc.2017.04.032 22. Skurat
V.E.,
DorofeevYu.I. The
transformations of organic polymers during the
illumination by 147.0 and 123.6 nm light. Macromolecular Materials and Engineering,1994,
no. 216,
pp. 205–224. DOI:
10.1002/apmc.1994.052160114 j p
16. Chapiro A. Radiation Effects in Polymers In
book: Encyclopedia of Materials: Science and
Technology (Second Edition) Elsevier, Amsterdam,
2004,
1120
p. DOI:
10.1016/B0-08-043152-
6/01918-5. 17. Pavlenko V.I., Cherkashina N.I., Demkina
L.N. Influence of Hydrothermal Treatment on
Crystalline Form of SiO2 Synthesized by Sol-Gel
Method. IOP Conf. Series: Materials Science and
Engineering, 2018, no. 327, 052026. p
23. Fozza A.C., Klemberg-Sapieha J.E.,
Wertheimer M.R. Vacuum Ultraviolet Irradiation of
Polymers. Received in August 2018 Для цитирования:
Павленко В.И., Черкашина Н.И., Манаев В.А., Сидельников Р.В. Изменение морфологии и
термооптических характеристик композита с кристаллическим диоксидом кремния при вакуумно-
тепловом
воздействии
//
Вестник
БГТУ
им. В.Г. Шухова. 2018. №11. С. 83–90. DOI: 10.12737/article_5bf7e356b23403.63058557 Information about the authors Information about the authors
Pavlenko, Vyacheslav I. DSc, Professor. Е-mail: belpavlenko@mail.ru. Belgorod State Technological University named
after V.G.Shukhov. Russia, 308012, Belgorod, st.Kostyukova, 46. Cherkashina, Natalia I. PhD, Assistant professor. Е-mail: cherkashina.ni@bstu.ru. Belgorod State Technological Uni-
versity named after V.G.Shukhov. Russia, 308012, Belgorod, st. Kostyukova, 46. Manaev, Vladimir A. Researcher. Е-mail: xxi-vek.2015@yandex.ru. Belgorod State Technological University named
after V.G.Shukhov. Russia, 308012, Belgorod, st. Kostyukova, 46. Sidelnikov, Roman V. Bachelor student. Belgorod State Technological University named after V.G.Shukhov. Russia,
308012, Belgorod, st. Kostyukova, 46. REFERENCES Plasmas Polymers, 1999, no. 4. pp. 183-
206. DOI: 10.1023/A:1021853026619 For citation:
Pavlenko V.I., Cherkashina N.I., Manaev V.A., Sidelnikov R.V. The changes in the morphology and thermo-
optical characteristics of the composite with crystalline silica under vacuum-thermal effect. Bulletin of BSTU
named after V.G. Shukhov, 2018, no. 11, pp. 83–90. DOI: 10.12737/article_5bf7e356b23403.63058557 For citation: Pavlenko V.I., Cherkashina N.I., Manaev V.A., Sidelnikov R.V. The changes in the morphology and thermo-
optical characteristics of the composite with crystalline silica under vacuum-thermal effect. Bulletin of BSTU
named after V.G. Shukhov, 2018, no. 11, pp. 83–90. DOI: 10.12737/article_5bf7e356b23403.63058557 90
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On the scale locality and vortex stretching in homogeneous shear turbulence
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RESEARCH ARTICLE | JULY 12 2023
On the scale locality and vortex stretching in homogeneous
shear turbulence
Zhou Yu-Xuan (周雨轩)
Lu Zhi-Ming (卢志明)
; Wu Jian-Zhao (吴建钊)
; Shen Jie (沈杰)
; Dong Si-Wei (董思卫)
;
Physics of Fluids 35, 075126 (2023)
https://doi.org/10.1063/5.0156179
View
Online
Export
Citation
20 May 2024 16:54:43
Physics of Fluids
ARTICLE
pubs.aip.org/aip/pof
On the scale locality and vortex stretching
in homogeneous shear turbulence
Cite as: Phys. Fluids 35, 075126 (2023); doi: 10.1063/5.0156179
Submitted: 27 April 2023 . Accepted: 24 June 2023 .
Published Online: 12 July 2023
Yu-Xuan Zhou (周雨轩),1
Jian-Zhao Wu (吴建钊),1,2,a)
1,2
and Zhi-Ming Lu (卢志明)
Jie Shen (沈杰),1
Si-Wei Dong (董思卫),3
AFFILIATIONS
1
Shanghai Key Laboratory of Mechanics in Energy Engineering, Shanghai Institute of Applied Mathematics and Mechanics,
School of Mechanics and Engineering Science, Shanghai University, Shanghai 200072, China
2
Shanghai Institute of Aircraft Mechanics and Control, Zhangwu Road, Shanghai 200092, China
3
State Key Laboratory of Aerodynamics, Mianyang 621000, China
a)
Author to whom correspondence should be addressed: jianzhao_wu@shu.edu.cn
ABSTRACT
C 2023 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://
V
creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0156179
I. INTRODUCTION
Turbulent flows are commonly characterized by hierarchical
structures of various scales. Richardson1 and Kolmogorov2,3 proposed
the energy cascade picture that the kinetic energy introduced at large
scales cascades through the interaction between multiscale structures
to smaller scales until it reaches the Kolmogorov scale, where it is dissipated by fluid viscosity.
For the cascade model, one key assumption is the scale locality
that flow structures primarily transfer energy to structures of adjacent
smaller scales. In previous studies, turbulence was commonly described
in spectral space, and scale locality was defined as the localness of
energy transfer for triad interaction between wavenumbers.4,5 Recently,
spatial filtering techniques are proposed to study scale interactions in
physical space.6–8 Some studies support scale locality,7,9,10 while others
suggest that kinetic energy can jump directly from large eddies to
much smaller ones.11,12 Doan et al.6 used bandpass filtering to extract
Phys. Fluids 35, 075126 (2023); doi: 10.1063/5.0156179
C Author(s) 2023
V
turbulent structures at various scales in homogeneous isotropic turbulence (HIT) and calculated the transfer fluxes between these scales.
Their direct numerical simulation (DNS) results show that eddies with
scale L mainly transfer kinetic energy to eddies with scale 0:3L.
Cardesa et al.10 studied the temporal evolution of energy flux across
scales in HIT and homogeneous shear turbulence (HST). They
reported that disturbances in energy flux propagate independently of
forcing. Their results show that cascades are scale-local processes where
energy is continuously transferred from one scale to the next one in
decreasing size order. Furthermore, they found fluid lumps of energy at
scale L appear within lumps at 2 L and die within those at L=2 by tracking flow regions containing energy at different scales.7 Additionally,
some researchers have studied scale locality for the interscale transfer
of other physical quantities such as helicity8 and magnetic energy.13
Another important question in energy cascade phenomenology is what physical mechanism drives the cascade process.
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The interscale transfer of kinetic energy and Reynolds stress in homogeneous shear turbulence (HST) is numerically investigated using
three-dimensional bandpass filtering technique. The flow fields of a statistically steady HST are obtained using direct numerical simulation at
three Reynolds numbers 2000, 5000, and 12 500 based on box depth. Visualizations of typical flow structures of bandpass filtered fields show
that the small-scale structures are nearly isotropic, whereas the large-scale ones show the preferential alignment with the direction of mean
shear. Quantitative results of both kinetic energy and Reynolds stress fluxes between two specific scales show the existence of scale locality.
In specific, the eddies of a length scale L mostly transfer their energy or Reynolds stress to eddies of size 0.3L to 0.4L, which seems to be independent in the limit of the high Reynolds number. Furthermore, through the analysis on the vortex stretching, it is shown that the smallscale structures of scale Lx are stretched mostly by straining structures of size about 2 Lx , while large-scale structures are stretched mostly by
mean shear. Finally, the evaluation of alignment between vortical structures and strain rate shows that small-scale structures are more likely
to align with the strain structures of five times their size, and large-scale ones are mainly align with the mean shear strain. These findings can
help enhance our comprehension of the interscale transfer and scale interaction of vortical structures in turbulence.
Physics of Fluids
II. METHODS
A. Numerical experiment
We consider the turbulent flow of incompressible fluids in a doubly periodic box subject to a homogeneous mean shear rate S, as
shown in Fig. 1. The governing equations of HST are given by
Phys. Fluids 35, 075126 (2023); doi: 10.1063/5.0156179
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FIG. 1. Sketch of homogeneous shear turbulence. The shear is introduced in x
direction. Periodic condition is adopted in x and z directions. Specially, the shearperiodic condition is applied in y direction. The colors represent the magnitude of
streamwise fluctuating velocity, u1, in the range ½1; 1 from blue to red.
r u ¼ 0;
(1)
ð@t þ Sy@x Þu þ u ru ¼ Su2 e1 rp þ r u;
2
(2)
where u ¼ ðu1 ; u2 ; u3 Þ denotes the fluctuating velocity, p is the modified pressure normalized by the fluid density, is the kinematic viscosity, and e1 is the unit vector in x direction. Taking the box depth Lz
and the characteristic velocity SLz for the normalization, there are
three dimensionless control parameters in the HST flow: two aspect
ratios of the numerical domain Axz ¼ Lx =Lz and Ayz ¼ Ly =Lz , and
the Reynolds number Rez ¼ SL2z =, where Lx is the box width and Ly
is the box height. For the statistically stable state, the HST is usually
described by the Reynolds number based on Taylor microscale,
Rek ¼ q2 ð5=ð3eÞÞ1=2 , where q2 ¼ hui ui i is the twice kinetic energy
and e ¼ h@j ui @j ui i is the dissipation rate, respectively. The integral
scale, Le ¼ ðq2 =3Þ3=2 =hei, is the scale of the largest eddies. The
Corrsin length is defined as Lc ¼ ðe=S3 Þ1=2 ,37 below which eddies are
not influenced by mean shear.
In this work, HST flows are numerically simulated using the inhouse code, which was detailedly described and has been well validated
in previous works.27,35,36 The computational domain is periodic in x
and z, with boundary conditions in y that enforce periodicity between
uniformly shifting points at the upper and lower boundaries. The spatial
discretization is dealiased Fourier spectral in the two periodic directions
(x and z) and compact finite differences with spectral-like resolution in
y. Time stepping is third-order explicit Runge–Kutta modified by an
integrating factor for the mean-flow advection. The details of our
numerical simulation are described in the Table I, and basic flow statistics are compiled in Table II. Note that the aspect ratios Axz and Ayz
are chosen following the acceptable range (2 < Axz < 5; Axz < 2Ayz )
proposed by Sekimoto et al.,35 in which the flow is found to be as free
as possible from the domain effects. The flow statistics obtained in
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Since Taylor,14,15 many researchers have focused on how large-scale
strain-rate fields stretch small-scale vortices in HIT and found that
the vortex stretching is closely related to the energy transfer between
different scales.6,7,16–20 Leung et al.19 reported that vortices at a
given scale are stretched by somewhat larger structures, and the
energy was also transferred from these larger structures to the small
eddies, which was consistent with classical energy cascade theory.
By using volume fraction analysis, they showed that enstrophy
structures tend to cluster around larger structures. Goto et al.20
quantitatively studied the energy cascade in forced HIT and found
that a hierarchy of anti-parallel vortex tube pairs were generated by
being stretched by straining fields with scale around 2–8 times
larger and were weakened by strain fields with scale around half
time smaller. Doan et al.6 showed that the stretching is dominated
by the maximum principal strain rate of straining structures. More
recently, Carbon and Bragg21 suggested that strain-rate self-amplification is the main contributor to energy transfer. Johnson22 showed
that amplification of single-scale and multi-scale strain rates
accounts for roughly 5/8 of the energy cascade rate, with the
remaining 3/8 due to vorticity stretching.
In the past decades, energy cascade has been extensively studied
in HIT. However, in practical situations, turbulence is often accompanied by mean shear. This deviates from isotropic turbulence due to
more complex energy transfer mechanism.23–26 Recently, Dong et al.27
studied the energy cascade in HST from the perspective of coherent
structure and pointed out that the characteristic flow structure related
to energy transfer is a vortex with an inverted hairpin shape, followed
by an upright hairpin shape. However, they did not elucidate the locality of the cascade and the role of vortex stretching in shear turbulence.
In addition, the spatial/scale transport of Reynolds stress at different
scales is also closely related to the energy cascade in shear turbulence.28–33 Kawata and Alfredsson28 showed that the inverse interscale
transport of Reynolds stress supports the production of large-scale turbulent kinetic energy.
In this paper, we consider the simplest type of shear turbulence,
known as HST. HST can be considered as a bridge between the idealized HIT and more realistic turbulent shear flows, as it shares natural
energy-generation mechanisms with shear flows while retaining
homogeneity simplicity.34–36 This paper aims to (i) reveal energy cascade and Reynolds stress interscale transport and its locality in physical space; (ii) investigate vortex stretching mechanisms between
various scales; and (iii) study multi-scale vortex structure morphology
in shear turbulence. The DNS dataset is analyzed using a bandpass filtering technique.19 The remainder of this paper is organized as follows:
Section II introduces numerical schemes for DNS and the spatial
bandpass filtering approach. Section III presents results focusing on (i)
morphological characteristics of vortex structures in Sec. III A, (ii)
energy and Reynolds stress transfer across scales in Sec. III B, and (iii)
quantitative analysis of vortex stretching mechanisms in Secs. III C
and III D. Conclusions are given in Sec. IV.
ARTICLE
Physics of Fluids
ARTICLE
TABLE I. Simulation parameters for the DNS of HST used in this paper. Rez is the
Reynolds number based on the box depth; Axz ¼ Lx =Lz and Ayz ¼ Ly =Lz are the
box aspect ratios of the computational domain; St is the no-dimension time of simulation running; Dx=g; Dy=g, and Dz=g are the grid resolutions in terms of the average Kolmogorov length scale g, with their standard deviations due to intermittency.
The values of Dx and Dz are computed from the number of Fourier modes before
dealiasing.
Rez
Nx ; Ny ; Nz
Axz Ayz St
2000 188, 384, 96 3
5000 432, 576, 144 3
12 500 766, 512, 255 3
Dx=g
Dy=g
Dz=g
4 714 1.6 6 0.07 1.1 6 0.05 1.1 6 0.05
4 633 1.3 6 0.06 1.3 6 0.06 1.3 6 0.06
2 565 1.4 6 0.10 1.4 6 0.10 1.4 6 0.10
TABLE II. Basic flow statistics for HST simulations in Table I. Rek ¼ q2 ð5=3eÞ1=2
is the Reynolds number based on the Taylor microscale (q2 and e are the twice
kinetic energy and dissipation rate, respectively); k ¼ qð5=eÞ1=2 is the Taylor
microscale; Lc ¼ ðe=S3 Þ1=2 is the Corrsin length;37 Le ¼ ðq2 =3Þ3=2 =hei is the integral scale; S ¼ Sq2 =hei is the Corrsin shear parameter;37 x0 ¼ hxi xi i1=2 ; n and
f are the Lumley invariants with f2 ¼ bij bji =6 and n3 ¼ bij bjk bki =6, where
bij ¼ hui uj i=hui ui i dij =3.
Rez
Rek
k=g
12
20
34
…
43
75
137
…
…
…
S
x0 =S
n
6.98 5.30 0.090
7.35 7.16 0.096
7.66 10.62 0.103
6.97 5.30 0.095
7.53 10.9
f
0.104
0.105
0.109
0.105
0.101 0.108
the present simulations are compared with the results reported in the
Ref. 35. A good agreement between them validates our numerical
simulations.
B. Bandpass filtering technique
In this work, the bandpass filtering technique is used to extract
structures at a chosen length scale.6,19 The bandpass filtered velocity
uL ðx; tÞ at a specific scale L can be obtained from the unfiltered field
according to
ð
aL @
uðr; tÞGðL; x rÞdr;
(3)
uL ðx; tÞ ¼ pffiffiffi
L @L V
pffiffiffi
where GðL; rÞ is the Gaussian filter with the filter scale L, and a ¼ 2.
Other physical quantities, such as vorticity xL ¼ r uL , can be constructed from the filtered velocity field uL . As demonstrated by Leung
et al.,19 Eq. (3) can effectively reduce the velocity field containing
information centered around the scale L in physical space. Compared
with other techniques such as subtracting two low-pass filters with different scales,7,8,38 the banding filtering technique does not require
manual specifications. This technique has been applied to the investigation of morphology of multi-scale flow structures19 and scale interaction of kinetic energy transfer6 and helicity13 in HIT. In this study,
we use this technique to study the characteristics of flow structure and
Phys. Fluids 35, 075126 (2023); doi: 10.1063/5.0156179
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examine scale locality of kinetic energy and Reynolds stress interscale
transfer in HST.
III. RESULTS AND DISCUSSION
A. Flow structure and anisotropy in HST
We first examine the flow structures of different scales in HST.
Figure 2 shows the typical structure visualized by the isosurfaces of
QL =QLrms ¼ 2 using the bandpass filtered velocity field with four
different scales L ¼ 100g; 50g; 20g; and 5g at Rez ¼ 12 500. Here,
QL ¼ ðXL : XL SL : SL Þ=2 is the second invariant of the velocity
gradient AL ¼ ruL , where XL ¼ ðAL ðAL Þ> Þ=2 is the rotation tenqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
sor and SL ¼ ðAL þ ðAL Þ> Þ=2 is the strain tensor, QLrms ¼ hðQL Þ2 i
is the root mean square of QL with hi denoting the ensemble averaging. It is shown that at the largest scale L ¼ 100g, large structures are
observed and found to be preferentially aligned with the shear direction as shown in Fig. 2(a). With decreasing the filter scale, the structures become more difficult to discern, which tend to be homogeneous
and isotropic as shown in Figs. 2(b)–2(d).
To further assess the anisotropy of flow structures at different
scales, the Lumley triangle based on the second 3f2 and third 2n3
invariants of the Reynolds stress anisotropy tensor bij ¼ hui uj i=q
dij =3 is used.40 Here, f ¼ ðbij bji =6Þ1=2 represents the degree of
anisotropy and n ¼ ðbij bjk bki =6Þ1=3 signifies the nature of anisotropy.
Figure 3 plots the anisotropic invariant map of f-n obtained from the
bandpass filtered field at different scales. It is shown that with decreasing the filter scale L, the value of f decreases to zero, and thus, the
anisotropy degree is reduced, indicating the return to the isotropy
from large to small scales. It is also found that not only the n value but
also the n sign changes with decreasing L. This implies the occurrence
of the change on the nature of anisotropy, i.e., a transition from the
cigar-shaped turbulence anisotropy to the pancake-shaped turbulence
anisotropy. This transition phenomenon is also observed in the temporal evolution of anisotropy for the shear rapid distortion.39
B. Scale locality of interscale transfer
Next, we focus on interscale transfer mechanism of kinetic energy
and Reynolds stress, and then examine the scale locality of the energy
and Reynolds stress cascade. We
the velocity u into parts
P decompose
Li
u
,
with
the
conservation of kinetic
with a series
of
scales,
i.e.,
u
¼
L
i
P
energy, Li uLi uLi =2 ¼ u u=2. We choose two specific scales L and
S with L > S. Taking the dot product of uL ; uS with Eq. (2) and ensemble averaging, assuming that the cross terms are zero, and using the
homogeneity, one obtains the kinetic energy transport equations of
EL ¼ hðuL uL Þ=2i and ES ¼ hðuS uS Þ=2i,
X
dEL
huL ðuruLi ÞihxL xL i;
¼ ShuL1 uL2 ihuL ðuruS Þi
dt
L 6¼S
i
(4)
X
dES
huS ðu ruLi Þi hxS xS i:
¼ ShuS1 uS2 i þ huL ðu ruS Þi
dt
L 6¼L
i
(5)
Note that in Eq. (5), we use the equality hu ðu ru Þi
¼ huL ðu ruS Þi. From Eqs. (4) and (5), it readily knows that
S
L
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2000
48 13.78
5000
68 15.86
12 500
104 19.71
2000 from
48 …
Ref. 35
12 500 from 105 …
Ref. 35
Lc =g Le =g
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FIG. 2. Isosurfaces of the second invariant QL ¼ ðXL : XL SL : SL Þ=2 of the velocity gradient tensor from bandpass filtered field at the same instant at Rez ¼ 12 500,
where XL is the rotational tensor and SL is the rate-of-strain tensor. (a) L ¼ 100g, (b) L ¼ 50g, (c) L ¼ 20g, and (d) L ¼ 5g. The threshold is set as QL =QLrms ¼ 2. The colors represent the magnitude of streamwise fluctuating velocity, uL1 , in the range ½1; 1 from blue to red.
PL!S ¼ huL ðu ruS Þi can be regarded as the energy transfer flux
from scale L to S.
Similarly, for the interscale transfer of Reynolds stress, one can
obtain the transport equation of ERL ¼ huL1 uL2 i and ERS ¼ huS1 uS2 i as
follows:
X L!L
dERL
PR i þ 2hAL1j AL2j i; (6)
¼ ShuL2 uL2 i þ 2hpL SL12 i PL!S
R
dt
L 6¼S
i
X S!L
dERS
PR i þ 2hAS1j AS2j i; (7)
¼ ShuS2 uS2 i þ 2hpS SS12 i þ PL!S
R
dt
L 6¼L
i
where PL!S
¼ huL1 ðu ruS2 Þi huL2 ðu ruS1 Þi is the interscale
R
transfer flux of Reynolds stress from scale L to S.
To examine the scale locality of interscale transfer of both energy
and Reynolds stress, the bandpass filtering technique is applied to
extract uL and uS , and then, we calculate the transfer flux PL!S and
PL!S
R . Figure 4 shows the variation of normalized energy transfer flux
^ L!S with the ratio S/L for various scales L at different
P
^ L!S is the transfer flux normalized
Rez ¼ 2000; 5000; 12 500. Here, P
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^
by its maximum value, i.e., P
¼ PL!S =maxðPL!S Þ. It is found in
L!S
^
Fig. 4 that the trend of P
is basically similar for both different L
^ L!S is almost
and Rez. For all Rez studied, the peak position of P
located around S=L 0:3 for different filtering scales L. This indicates
that turbulent eddies of scale L primarily transfer energy to eddies of a
smaller scale approximately 0:3L.
of
A similar analysis is carried out for the transfer flux PL!S
R
Reynolds stress between two scales. Figure 5 shows the normalized
^ L!S as a function of the scale ratio S/L for the same cases conflux P
L!S
R
^
sidered in Fig. 4. It is found that the behavior of P
is very similar
R
L!S
^
to that of energy transfer. The P R peaks are robustly located at
S=L 0:4. This indicates that the Reynolds stress of scale L is mainly
transferred to that at a scale of approximately 0:4L. This supports the
Reynolds stress interscale transfer picture, in which the Reynolds stress
is almost transferred from one scale to its neighboring smaller scales,
as the kinetic energy cascade. Moreover, it is interesting to find that at
^ L!S is negative at small S/L. This
low Rez and small L, the value of P
R
indicates an inverse interscale transfer in Reynolds stress in HST. This
phenomenon has also been observed in wall turbulence.28,29
L!S
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PL!S and PL!S
attains. It is shown that for all cases, the values of
R
E
ðS=LÞmax lie in the interval of ð0:25; 0:5Þ as depicted in Fig. 6(a), and
the ðS=LÞRmax values in the interval of ð0:35; 0:6Þ. As L and Rez
increase, the value of ðS=LÞEmax tends toward 0.3 and the ðS=LÞRmax
value is about 0.4. This strongly supports the locality of kinetic energy
cascades and interscale transfer of Reynolds stresses in HST. Similar
results on the scale locality of energy cascade in HIT are reported in
Ref. 6 This implies that the shear effect does not influence the localness
of energy cascade in turbulence. The similar results in both HIT and
HST indicate that the underlying interscale energy mechanism is
mainly driven by inertial force in homogeneous turbulence, which
strongly support the classic picture of energy cascade proposed by
Kolmogorov and Richardson.1,2
C. Scale-dependent contributions to the stretching
FIG. 3. Lumley invariants of the Reynolds stress anisotropy tensor. n and f are the
Lumley invariants with f2 ¼ bij bji =6 and n3 ¼ bij bjk bki =6, where bij ¼ hui uj i=
huk uk i dij =3. The dash curves are the realizability limits. The open symbol represents the turbulent field without filtering, and solid symbol with colors represents the filtered turbulent field with filter scale L. The symbol $ denotes the time evolution of the
Reynolds stress invariants for the shear rapid distortion. Each $-data are obtained
after a time interval St ¼ 0:5 starting from the origin (corresponding to isotropy).39
where hiQLx >QLrmsx is the conditional averaging over the rotation-
x
dominated region of QLx > QLrms
. If GLf S !Lx is positive (or negative), it
means that the vorticity of scale Lx is stretched (or contracted) by the
^ L!S ¼ PL!S =maxðPL!S Þ, as a function of S/L for different Reynolds numbers: (a) Rez ¼ 2000, (b) Rez ¼ 5000, and (c)
FIG. 4. Normalized energy transfer function, P
L!S
L
S
¼ hu ðu ru Þi and maxðPL!S Þ is the maximum of each curve.
Rez ¼ 12 500. Here, P
^ L!S ¼ PL!S =maxðPL!S Þ, as a function of S/L for different Reynolds numbers: (a) Rez ¼ 2000, (b)
FIG. 5. Normalized Reynolds stress u1 u2 transfer function, P
R
R
R
L!S
Rez ¼ 5000, and (c) Rez ¼ 12 500. Here, PR ¼ huL1 ðu ruS2 Þi huL2 ðu ruS1 Þi and maxðPL!S
R Þ is the maximum of each curve.
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To further quantify the locality of kinetic energy and Reynolds
stress transfer flux, we plot the values of ðS=LÞEmax and ðS=LÞRmax as a
function of the scale L for different Rez in Fig. 6. Here, ðS=LÞEmax and
ðS=LÞRmax , respectively, denote the position at which the maximum of
We further examine the scale locality of interscale transfer from
the view of point of vortex stretching, which is considered as one of the
main physical mechanisms of cascade process.6,16–20 The vortex stretching is be expressed as xSx. In this work, to analyze the vortex stretching
between two specific scales, we define GLf S !Lx as the stretching rate of
the vorticity xLx at scale Lx by the strain-rate tensor at scale LS,
Lx LS Lx
x S x
GLf S !Lx ¼
;
(8)
jxLx j2
x
QLx >QLrms
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FIG. 6. Ratio ðS=LÞmax yielding the maximum (a) kinetic energy and (b) Reynolds stress transfer flux from eddies of a large-scale L to ones of a small-scale S. The error bars
denote the standard deviations.
strain rate of scale LS. Similarly, we also consider the stretching rate of
the vorticity at scale at Lx by mean strain rate S,
Lx Lx
x Sx
x
GM!L
¼
:
(9)
m
jxLx j2 QLx >QLrms
x
FIG. 7. The contribution GLf S !Lx of the strain rate filtered at LS to the stretching of vortices at Lx for different Reynolds number: ðaÞ Rez ¼ 2000; ðbÞ Rez
¼ 5000; and ðcÞ Rez ¼ 12 500. The solid symbols correspond to the twice-scale contribution (LS ¼ 2Lx ).
Phys. Fluids 35, 075126 (2023); doi: 10.1063/5.0156179
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Note the symmetry and mean shear in HST, only S 12 and S 21 are nonzero (¼ S=2).
Figure 7 shows GLf S !Lx as a function of LS at three different scales
Lx ¼ 5g; 10g; 20g for different Rez. It is observed in Fig. 7(a) that
when LS < Lx (e.g., Lx ¼ 10g; 20g), GLf S !Lx is negative, whereas
when LS > Lx ; GLf S !Lx is positive. This indicates that vortices are
stretched by strain-rate structures larger than their own scale and contracted by strain-rate structures smaller than their own scale. Specially,
one finds that the value of GLf S !Lx is maximal when LS 2Lx .
This means that strain-rate structures at 2Lx have the greatest contribution on the stretching of vortices at scale Lx . When LS =Lx is very
large, the contribution of vortex stretching tends toward almost zero.
It is shown in Figs. 7(a)–7(c) that these results are robust for all
Reynolds numbers, with only the absolute value of GLf S differing. At
the same filter scale, as the Reynolds number Rez increases, the value
of GLf S also increases accordingly.
To compare the contribution of vortex stretching by large-scale
x !Lx
by strain
strain rates with that by mean strain rate, we plot G2L
f
M!Lx
by mean strain rate in Fig. 8(a). It is found that
rate at 2Lx and Gm
x !Lx
with increasing Lx , the value of G2L
decreases, while the value of
f
x
increases.
For
small
scales
(e.g.,
Lx ¼ 5g; 10g; 20g), the value
GM!L
m
x !Lx
x
is much larger than that of GM!L
, indicating that the
of G2L
m
f
contribution of strain rate at 2Lx is significant, whereas for large scales
(e.g., Lx ¼ 40g at Rez ¼ 2000; 5000; Lx ¼ 80g at Rez ¼ 12 500),
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x !Lx
x
FIG. 8. (a) The contribution of mean strain rate, GM!L
(dotted curve), and turbulent strain rate filtered at scale 2Lx to the stretching of vortices filtered at scale Lx ; G2L
m
f
(solid curve) at different Reynolds number Rez. (b) Corrsin length Lc (blue line) and critical scale Lcr (red line) as a function of Reynolds number based on Taylor microscale
cr !Lcr
cr
Rek , respectively. The critical scale, Lcr, is defined as the point where GM!L
¼ G2L
.
m
f
D. Alignment of vorticity and stretching directions
Furthermore, we analyze the vortex stretching mechanism from
the alignment statistics between the vorticity and the straining structures. The vortex stretching term GLf S !Lx can be rewritten as
GLf S !Lx ¼ haLS cos2 ha þ bLS cos2 hb þ cLS cos2 hc iQLx >QLrms
x ;
(10)
where aLS ; bLS , and cLS are eigenvalues of strain-rate tensor SLS at scale
LS, with aLS bLS cLS , and hi (i ¼ a; b; c) are the angle between the
filtered vorticity vector xLx at scale Lx and the corresponding eigenvector ei of SLS . ea corresponds to the direction of maximum stretching,
and ec corresponds to the direction of maximum compression. Due to
the incompressibility constraint, the sum of the eigenvalues of the
strain-rate tensor is zero, i.e., aLS þ bLS þ cLS ¼ 0. Recent studies have
shown that the sign of the intermediate eigenvalue bLS is probably
related to the direction of the energy cascade (forward or backward).27,42
The alignment between vorticity at scale Lx and the principal
directions ei of the strain at scale LS is evaluated by
cos hi ¼
xLx ei
:
jxLx jjei j
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(11)
Similarly, the alignment between vorticity and mean flow is given by
cos hM ¼
xLx e a
;
jxLx jje a j
(12)
where e a ¼ ð1; 1; 0Þ is the direction of stretching of mean strain rate.
Figure 9 shows the probability density function (p.d.f.) of jcos ha j
and jcos hb j between xLx filtered at scale Lx ¼ 5g and the straining
structures filtered at LS ¼ 100g; 25g; 15g; and 5g for all Reynolds
numbers studied. When without filtering, the vorticity tends to align
with the intermediate eigenvector eb of strain rate, which is similar to
the results in HIT reported by Ashurst et al.43 In fact, for a uniformly
sheared laminar flow, x is always aligned with the direction of eb .
When applying the bandpass filtering, it is observed in Figs. 9(a)–9(c)
that the filtered vorticity xLx becomes aligned with the ea corresponding to the most extensional strain rate for LS > Lx . It is seen that all
results are almost on the same curve for different Reynolds numbers.
Only some differences at LS ¼ 100g appear due to the fact that the
alignment characteristics at such large scale are significantly influenced
by mean shear for low Reynolds turbulence.
Figure 10 shows the probability, Pð0:99 jcos ha j 1Þ, of
obtaining a perfect alignment between the filtered vorticity xLx and
the most extensional strain of SLS as a function of LS =Lx . It can be
seen that for all cases studied, the peaks of the probability P occur at
LS =Lx 5, implying that xLx is mostly alignment with the stretching
direction of strain rate filtered at scales LS ¼ 5Lx . This agrees with the
previous results that the vorticity are more likely to align with the
stretching direction of strain rate at 3–5 times the scale in HIT6,19 and
wall turbulence.41
In order to compare the alignment between vorticity and stretching direction of mean flow with that of turbulent strain rate, we plot
the p.d.f. of cos hM for four different filter scales Lx ¼ 5g; 10g; 20g;
and 40g in Fig. 11. It is observed from the Fig. 11(a) that as the scale
Lx increases, the probability of alignment between the filtered vorticity and the stretching direction of mean flow increases. Compared
with cos ha represented by the black line in Fig. 11, we find that
small-scale vorticity (solid lines) tends to align with the stretching
direction of the fluctuating strain rate, whereas large-scale vorticity
(dashed lines) tends to align with the stretching direction of the
mean flow.
35, 075126-7
20 May 2024 16:54:43
x !Lx
x
GM!L
is larger than G2L
, indicating that the contribution of
m
f
mean strain rate is significant. This shows that small-scale vortices are
mainly stretched by strain-rate structures at twice of their scale, while
the large-scale vortices are stretched by mean strain rate. This is consistent with previous results reported by Motoori and Goto in the logregion of turbulent channel flow or turbulent boundary layer.38,41 To
well distinguish the large scales from the small scales, we define the critx !Lx
x
¼ G2L
. When
ical scale Lcr, i.e., when Lx ¼ Lcr , we have GM!L
m
f
Lx < Lcr , vortices at Lx are mainly stretched by strain-rate structures
at twice of their scale; otherwise, they are mainly stretched by mean
strain rate. This signifies that Lcr indicates the division between vortex
stretching dominant by mean strain rate and that by the strain rate
structures at twice of their scale. It is noteworthy that Lcr only depends
on the Reynolds number. Figure 8(b) shows the variation of Lcr and the
Corrsin length Lc with respect to Rek . It is seen that Lcr linearly
increases with increasing Rek , i.e., Lcr 0:8Rek 14. It is also interesting to note that the critical scale Lcr is proportional to Corrsin length
Lc, i.e., Lcr 2Lc .
Physics of Fluids
ARTICLE
pubs.aip.org/aip/pof
FIG. 10. Probability of perfect alignment (0:99 j cos ha j 1) between the vorticity
filtered at Lx and the stretching direction of strain rate filtered at scale LS as a function
of the ratio LS =Lx for different Rez. Here, Lx ¼ 5g is fixed and LS is varied.
IV. CONCLUSION
To reveal the interscale transfer mechanism of kinetic energy and
Reynolds stress, we have conducted DNS of statistically stationary HST
at three Reynolds numbers Rez ¼ 2000, 5000, and 12 500. We decompose
the velocity fields into different scales by using the three-dimensional
Phys. Fluids 35, 075126 (2023); doi: 10.1063/5.0156179
C Author(s) 2023
V
bandpass filtering technique. A detailed study of kinetic energy and
shear Reynolds stresses cascade is carried out in real space, especially
the scale locality of cascade. Our results show that flow structures with
scale L transfer energy mainly to structures with scale 0:3L. For
Reynolds stress, this value is about 0:4L. Through the analysis of the
vortex stretching mechanism, we found that (i) small-scale vortices are
stretched by strain-rate structures twice their scale and more likely to
align with the stretching direction at 5 times their scale, which is consistent with the scale locality of the energy cascade; (ii) large-scale vortices
are directly stretched and amplified by mean shear, and more likely to
align with the stretching direction of the mean flow. These findings are
consistent with our visualization of the isosurfaces of Q; i.e., the smallscale structures look chaotic and disorganized (more isotropic), while
the large-scale structures preferentially align with the streamwise direction. In addition, we found that the Corrsin length can effectively distinguish between large and small scales. Considering the similarity
between these findings, a close link between the vortex stretching
mechanism and energy transfer in HST can be summarized. First,
kinetic energy is generated by mean shear and injected into large-scale
structures. This causes large-scale structures to align with mean shear
and be stretched by it. Through the vortex stretching mechanism,
large-scale vortices transfer energy to small-scale vortices. At a given
scale Lx , small-scale vortices are mostly stretched by structures of a
scale of about twice their scale and more likely to align with the stretching direction at 5 times their scale. This gives rise to a transfer of energy
from the scale Lx to the scale ð1=5; 1=2ÞLx . This process continues
until to the dissipation scales. Finally, the results of this paper are
35, 075126-8
20 May 2024 16:54:43
FIG. 9. P.d.f. of alignment between the vorticity filtered at scale Lx ¼ 5g and the principal directions of the strain rate filtered at different scales LS: (a) LS ¼ 100g, (b)
LS ¼ 25g, (c) LS ¼ 15g, and (d) LS ¼ 5g. Solid symbols denote a and open symbols denotes b. The black solid line (denotes a) and dashed line (denotes b) are computed
using unfiltered fields for the case Rez ¼ 12 500.
Physics of Fluids
ARTICLE
pubs.aip.org/aip/pof
qualitatively similar for three Reynolds numbers, which provides a
strong support for the classical picture of the energy cascade and its
locality.
ACKNOWLEDGMENTS
This work was sponsored by the Natural Science Foundation
of China under Grant Nos. 11988102, 92052201, 12032016,
11972220, 11825204, 12072185, and 12102246 and the Shanghai
Science and Technology Program under Project Nos. 20ZR1419800
and 21PJ1404400.
AUTHOR DECLARATIONS
Conflict of Interest
The authors have no conflicts to disclose.
Author Contributions
Yuxuan zhou: Data curation (equal); Formal analysis (equal);
Investigation (equal); Methodology (equal); Validation (equal);
Visualization (equal); Writing – original draft (equal). Jianzhao Wu:
Conceptualization (equal); Formal analysis (equal); Funding acquisition (equal); Methodology (equal); Supervision (equal); Writing –
original draft (equal); Writing – review & editing (equal). Jie Shen:
Formal analysis (equal); Methodology (equal); Visualization (equal);
Writing – review & editing (equal). Siwei Dong: Formal analysis
(equal); Supervision (equal). Zhiming Lu: Conceptualization (equal);
Phys. Fluids 35, 075126 (2023); doi: 10.1063/5.0156179
C Author(s) 2023
V
Formal analysis (equal); Funding acquisition (equal); Methodology
(equal); Supervision (equal); Writing – review & editing (equal).
DATA AVAILABILITY
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
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ARTICLE
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https://openalex.org/W4386389096
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https://sdc-journal.com/index.php/journal/article/download/508/409
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Ukrainian
| null |
РОЗВИТОК ФОТОГРАФІЇ В УКРАЇНІ У ХХ СТ. ТА ЇЇ ІНСТИТУАЛІЗАЦІЯ ЯК АРХІВНОГО ДОКУМЕНТА
|
Socìum, dokument, komunìkacìâ
| 2,023
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cc-by
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ISSN (print) 2518-7600 ISSN (online) 2524-1060
https://sdc-journal.com РозвитоК фотогРафІї в УКРаїнІ У хх ст. та її
ІнститУалІзацІя яК аРхІвного ДоКУмента Ігор Довжук,
доктор історичних наук,
професор кафедри соціальних
комунікацій,
документознавства та
інформаційної діяльності,
E-mail: i.v.dovzhuk@ukr.net
ORCID: https://orcid.org/0000-
0001-6941-6336
Університет Григорія
Сковороди в Переяславі,
вул. Сухомлинського, 30,
м. Переяслав, Київська обл.,
Україна, 08401 Ihor Dovzhuk,
Doctor of Historical Sciences,
Professor of the Department of
Social Communications,
Documentation and Information
Activity
E-mail: i.v.dovzhuk@ukr.net
ORCID: https://orcid.org/0000-
0001-6941-6336
Hryhorii Skovoroda University
in Pereiaslav, 30,
Sukhomlynskoho Str., Pereiaslav,
Kyiv region, Ukraine, 08401 м. Переяслав, Київська обл.,
Україна, 08401 Society. Document. Communication (2023) Ed. 19. 81 – 102 Society. Document. Communication (2023) Ed. 19. 81 – 102 УДК 77(477)«19»(091):930.25
DOI: https://doi.org/10.31470/2518-7600-2023-19-81-102 ABSTRACT The article examines the development of photography, photo
affairs, and the chemical-photographic industry in Ukraine in the
20th century, as well as the institutionalization of photography as
an archival document. Already from the beginning of the 20th century we can talk
about the high level of development of photography in Ukraine, the
formation of its infrastructure, the rapid expansion of development
directions in many Ukrainian regions. The expansion of the
functional and instrumental characteristics of photography, the 81 Розвиток фотографії в Україні у ХХ ст. та її інституалізація як
архівного документа development of photomechanical printing processes became
prerequisites and catalysts for the formation of professional photo
art, photojournalism, the formation and enrichment of the system
of its genres, and the expansion of the circle of photography
specialists. The level of perception of photography and photography is
changing, there is a need for generalization and scientific
understanding of it, creation of a theory of photography. So, since
1914, there were already many photographers-artists and
professional scientists in Kyiv. Active photo activity brought to life
and the emergence of specialized periodicals. During the short time
of the development of photo circles in Kharkiv, specialized
magazines were founded. It is noted that at the beginning of 1930, on the initiative of
Professor Oleksa Balytskyi and doctor Stepan Dmochovskyi, the
Ukrainian Photographic Society was created in Lviv (with branches
in Stanislav, Ternopil, and Rohatyn). The society had more than 200
members, its own premises and a library. In the 1960s and 1980s, a network of specialized archives of
film, photo, and audio documents emerged, including archives of
the Union level and archives of the Union republics, including of
Ukraine. In the state archives, special archival funds are created
for the transfer, storage and research of photo documents, their
institutionalization takes place, which has received a new
development since the beginning of the 1990s. Keywords: Ukraine, photography, photography, photo
education, Ukrainian photographic societies, photo documents. Постановка проблеми. Датою винаходу фотографії, що
стала офіційною, став день 7 січня 1839 р., коли відомий
французький фізик і астроном Франсуа Араго (1786–1853)
зробив доповідь на засіданні Паризької Академії наук, членом
якої він був з 1809 р., про загальні принципи дагеротипії –
способу отримання стійкого зображення в камері-обскурі, що 82 © Довжук Ігор Development of photography in Ukraine in the XX century and its
institutionalization as an archive document була винайдена Луї Дагером1 у 1835 р. Ф. 1 Дагер луї Жак манде (1787–1851) – французький художник, хімік і винахідник,
один із творців фотографії. © Dovzhuk Ihor ABSTRACT Араго ознайомив
членів Академії з першим фотографічним процесом, а також
дав пояснення принципу утворення прихованого зображення. У серпні 1839 р. опубліковано технічний опис дагеротипії, і з
цього часу фотографічний процес став надбанням людства, а
7 січня почали відзначати як день народження фотографії. Одразу після офіційного оприлюднення методу дагеротипії
фотографія стала поширюватися в Європі, особливо активно у
ХХ ст. Ці процеси стали базовими для розповсюдження
фотографії і фотографування в Україні. аналіз останніх досліджень і публікацій. Цією
проблемою в різний час займалися вчені та професіонали-
практики, серед яких можна назвати таких, як С. Горевалов,
Н. Зикун, І. Грушицька, Т. Ємельянова, О. Федотова та ін. метою дослідження є розглянути розвиток вітчизняної
фотографії, фотографічної техніки, діяльність фотографічних
студій і товариств, а також інституалізацію фотографії як
архівного джерела у ХХ ст. виклад основного матеріалу. Оскільки українські землі
були поділені між двома імперіями, фотографування тут
розвивалося різними шляхами. Це обумовило певну різницю у
завданнях фотографічних товариств, у технологіях та
суспільній ролі фотографії в Україні. Улітку 1839 р. фотографія з’явилася в Західній Україні. Професор Львівського університету, анатом Йозеф Беррес
(1796–1844) проводив численні досліди з друком відбитків із
дагеротипів за методикою офорту. У квітні 1840 р. він описав
свій винахід у «Віденській газеті», а пізніше, у липні цього
року, видав у Відні окрему брошуру (Groushitska, 2021). У Наддніпрянщину фотографію принесли в 1840-х рр. іноземні фотографи, учасники щорічних міжнародних
київських ярмарків («контрактів»), які часто залишалися в
місті. Першими фотографами-поселенцями стали французи 83 © Dovzhuk Ihor Розвиток фотографії в Україні у ХХ ст. та її інституалізація як
архівного документа Жак і Шарль-Поль Гербсти, останній відкрив у Києві
дагеротипну студію. Згодом ініціативу перехопили місцеві
поціновувачі нових технологій. У першій половині 40-х років
перші фотографи з’явилися в Одесі, у 1851 р. – в Харкові та
інших українських містах. Перше фотоательє в Харкові відкрив
у 1858 р. купець Василь Досєкін (1829–1900), а у 1880–1890-
х рр. у місті діяло вже понад 30 фотоательє. Жак і Шарль-Поль Гербсти, останній відкрив у Києві
дагеротипну студію. Згодом ініціативу перехопили місцеві
поціновувачі нових технологій. У першій половині 40-х років
перші фотографи з’явилися в Одесі, у 1851 р. – в Харкові та
інших українських містах. Перше фотоательє в Харкові відкрив
у 1858 р. купець Василь Досєкін (1829–1900), а у 1880–1890-
х рр. у місті діяло вже понад 30 фотоательє. Серед відомих харківських фотографів можна назвати
О. Іваницького2, М. Лещинського3 та інших, які отримали
визнання не лише в Україні, а й представляли український
світлопис на фотовиставках у Європі. 2 Іваницький олексій михайлович (1854–1920) – фотохудожник, дійсний член
Російського фотографічного товариства (з 1905 р.), член музейної комісії
Харківського художньо-промислового музею (1892–1895 рр.). Широку популярність
отримав завдяки серії фотографій з місця аварії імператорського поїзда Олександра
ІІІ 17 (29) жовтня 1888 р. біля станції Борки під Харковом. 3 лещинський михайло якович (1872–1927) – харківський фотограф, засновник
фотоательє «Русская Светопись» (після 1917 р. «Красная Светопись»). 4 Бекетов микола миколайович (1827–1911) – український і російський фізико-
хімік, член Петербурзької академії наук (з 1886 р.). © Довжук Ігор ABSTRACT Перші фотостудії у
Миколаєві виникли у 1868 р., власниками яких були Фрідріх
Дюрнбек та Микола Короєдов (Groushitska, 2021). Зазначимо,
що
створення
вітчизняної
мережі
професійних фотоустанов відбувалося ще у другій половині
ХІХ ст. Окрім звичайної побутової зйомки портрету та пейзажу,
з’явилися паростки документальної та наукової фотографії. Підтвердженням цього є праці чернігівського митця
А. Муренка (1837–1875) «Від Оренбурга через Хиву до
Бухари» та «Світлопис підпоручника А. Муренка». Почала
виходити й вітчизняна фотолітература. Так, одеський майстер
Й. Мигурський у 1859 р. видав перший в Україні «Практичний
підручник з фотографії та новітніх удосконалень і
застосувань», а в 1865 р. побачила світ книга харківського
професора М. Бекетова4
«Розвиток та сучасний стан
світлопису». Набула актуальності й фотоосвіта – ще 1863 р. Й. Мигурський почав викладати теорію і практику мистецтва, 84 © Довжук Ігор Development of photography in Ukraine in the XX century and its
institutionalization as an archive document намагався створити фотоінститут, але одержав відмову від
губернатора. Піонерами української фотографії на території
західноукраїнських земель були А. Карпюк, В. Шухевич – у
Львові, С. Дмоховський, Є. Любович у Перемишлі, Ф. Величко
у Станіславові (сучасний Івано-Франківськ) (Федотова, 2020:
435–436). У 1895–1897 рр. відкрито фотокурси у Києві та Львові, а
1903 р. – художньо-ремісничу майстерню друкарської справи
в Києві, де викладали фотографічні дисципліни. За словами
українського
вченого
в
галузі
фотографії
Миколи
Олександровича Петрова (1875–1940), це була «єдина в Росії
школа фотографічної справи». Важливу роль у розвитку
фотоосвіти відіграв ІІ з’їзд російських діячів з фотографічної
справи, що відбувся в Києві 1908 р. З 1895 р. у Львові
розпочали видавати перший фотожурнал (Введенський, 2004). М. Петров після закінчення сільськогосподарського
відділення Ризького політехнічного інституту, з 1900 до 1903
року «вдосконалювався в науках» у Німеччині, причому не
лише за здобутим там фахом, а й у галузі фотографії. У 1904 р. він отримав звання члена-кореспондента Фотографічного
товариства в Карлсруе, а 1905 р., відразу після влаштування на
роботу в Київський політехнічний інститут (КПІ), почав читати
лекції та вести практичні заняття з фотографії – першим у
вищих навчальних закладах Російської імперії. Пізніше цей
перший загальний курс з фотосправи розвинувся в кілька
спеціалізованих: у різні роки в різних навчальних закладах
М. Петров викладав «Історію фотографії», «Енциклопедію
фотографії»,
«Практичну
фотографію»,
«Технічну
фотографію» тощо. Крім того, у перший же рік своєї роботи в
КПІ він узявся створювати при Механічних майстернях
інституту спеціальну фотографічну лабораторію, яку невдовзі
було визнано однією з найкращих у країні. © Dovzhuk Ihor © Довжук Ігор ABSTRACT З часом тут не лише
отримували практичні навички студенти, а й проводилася
науково-дослідна робота, результатом якої стало розроблення
нових методів дослідження речових доказів за допомогою їх 85 Розвиток фотографії в Україні у ХХ ст. та її інституалізація як
архівного документа фотографування
для
потреб
судової
експертизи
в
інфрачервоних, ультрафіолетових та рентгенівських променях;
технології прочитання вицвілих, стертих або витравлених
написів тощо. Таким чином завдяки М. Петрову більш ніж на
два десятиліття КПІ став центром розвитку фотографічних
технологій в Україні (Грушицька, 2014, 41: 288) Варто зазначити, що в 1906 р. М. Петрова було обрано
головою Київського товариства фотографів-аматорів «Дагер». Цю посаду він обіймав до 1912 р. Товариство об’єднувало
спочатку любителів фотографії лише Києва, а згодом –
Київської та інших українських губерній. За його головування
Товариство «Дагер» стало відомим в усьому світі, адже
фотороботи
його
членів
неодноразово
визнавалися
найкращими на виставках, що проводилися не лише в
Російській імперії, а й далеко за її межами. М. Петров був добре знаний не лише як фотохудожник,
дослідник і викладач – він мав високий авторитет і як теоретик
фотомистецтва. Його роботи друкувалися регулярно. Писав і
видавався він, до речі, кількома мовами і всіляко пропагував
вітчизняну фотографію за кордоном. Так, у 1912 році в
Німеччині видрукував книжку «Художня фотографія в Росії»,
у 1913-му – в Англії «Пікторіальна фотографія в Росії». У
1916 р. М. Петрова відрядили в розпорядження Кабінету
науково-судової експертизи при прокурорі Київської судової
палати. Після 1917 і до 1934 р. він працював у Науково-
дослідному інституті судових експертиз, де очолював спочатку
секцію, а згодом відділ фотографічних досліджень. Проте не
полишав і викладацької роботи: до 1920 р. читав спеціальні
курси в КПІ, з 1921-го по 1923-й – у новоствореному
Київському фотографічному інституті, потім до 1930 р. – у
Київському художньому інституті, а з 1930 р. – у Київському
державному інституті кінематографії, отримавши звання
професора (Янковий & Стефанович, 2010). На
західноукраїнських
землях
перші
організації
фотолюбителів були створені при Пресовій Квартирі УСС 86 © Довжук Ігор Development of photography in Ukraine in the XX century and its
institutionalization as an archive document (Українського Січового Стрілецтва) наприкінці 1914 р. у
Карпатах, у містечку Ворохта. Перші фотографії «в полі»
робили І. Іванець5, Ю. Буцманюк6, Б. Гнатевич7, М. Угрин-
Безгрішний8, та інші (організували 1916 р. виставку у Відні,
повторили її 1934 р. у Львові). У 1915 р. при тій же Пресовій
Квартирі УСС В. Бобинський9 та Л. 5 Іванець Іван Йосипович (1893–1946) – український художник, редактор,
видавець, фотограф. 6
Буцманюк Юліан (1885–1967) – український і канадський художник-
монументаліст. © Dovzhuk Ihor ABSTRACT Гец10 уклали перший
збірник «Стрілецька Антологія», який містив 36 творів,
проілюстрованих 144 роботами військових фотографів (Трачун,
2014: 72). Змінюється
рівень
сприйняття
фотографії
і
фотодіяльності, з’являється потреба її узагальнення і наукового
осмислення, створення теорії фотосправи. Так, з 1914 р. у Києві
було вже чимало фотографів-митців і професіоналів-науковців:
М. Бобир,
І. Гаас,
А. Губчевський,
І. Єзерський,
К. Парчевський; уже названий нами М. Петров і дослідник
мікрофотографії В. Фаворський11 (Трачун, 2014: 19). Активна фотодіяльність викликала до життя і виникнення
спеціалізованих періодичних видань. За короткий час розвитку
фотогуртків у Харкові засновуються журнали «Фото-Кіно»,
«Фотожурнал» (1924 р.), «Фото для всіх» (1928–1930 рр.) і Активна фотодіяльність викликала до життя і виникнення
спеціалізованих періодичних видань. За короткий час розвитку
фотогуртків у Харкові засновуються журнали «Фото-Кіно»,
«Фотожурнал» (1924 р.), «Фото для всіх» (1928–1930 рр.) і 5 Іванець Іван Йосипович (1893–1946) – український художник, редактор,
видавець, фотограф. 6
Буцманюк Юліан (1885–1967) – український і канадський художник-
монументаліст. 7 гнатевич Богдан Петрович (1894–1968) – український військовий і політичний
діяч, командант УВО, член ОУН, історик, редактор. 8 Угрин-Безгрішний микола (1883–1960) – український прозаїк, поет, драматург,
видавець, фотограф, педагог. 9 Бобинський василь Петрович (1898–1938) – український поет, журналіст,
перекладач. Член літературної організації «Західна Україна» та ВУСПП. Один із
представників розстріляного відродження. 9 Бобинський василь Петрович (1898–1938) – український поет, журналіст,
перекладач. Член літературної організації «Західна Україна» та ВУСПП. Один із
представників розстріляного відродження. 10 гец лев (леон) львович (1896–1971) – український графік і живописець, педагог,
громадський діяч. 10 гец лев (леон) львович (1896–1971) – український графік і живописець, педагог,
громадський діяч. 11 фаворський василь Іванович (1880–1935) – український криміналіст, професор
(з 1934 р.). Навчався в Київському університеті (фізико-математичний факультет,
природознавче відділення). Від 1911 р – приват-доцент Київського університету,
читав курси лекцій з ботаніки, загальної біології, а згодом і «наукової фотографії». 87 © Dovzhuk Ihor Розвиток фотографії в Україні у ХХ ст. та її інституалізація як
архівного документа «Фото – соцбудівництву» (1932–1934 рр.) (Горевалов & Зикун,
2015: 39). Слід зазначити, що до 1917 р. фотографічна та
кінематографічна промисловість у Російській імперії була
відсутня, а все фотографічне обладнання, фотоматеріали й
фотохімікати ввозилися з-за кордону. Тому основною
перешкодою в розвитку фотографічної науки в країні була
відсутність належної матеріальної бази. У серпні 1919 р. РНК РСФРР прийнято декрет «Про
перехід фотографічної і кінематографічної торгівлі та
промисловості у відання Народного комісаріату з освіти». Відповідно до цього декрету була ліквідована приватна
власність на фотопідприємства. Для забезпечення керівництва
націоналізованими підприємствами у вересні 1919 р. при
Наркомосі організовано Всеросійський фотокіновідділ. © Довжук Ігор 14 Українське фотографічне товариство (УФОТО або UFT) – спілка українських
фотоаматорів Західної України, створена у Львові з метою об’єднати українських
фотографів і пропагувати мистецтво та техніку фотографії серед українського
населення. Існувало з 1930 до 1939 року. Діяльність товариства відновлено 9
листопада 2021 р. 13 Кирилов Єлпідіфор анемподистович (1883–1964) – фізик, доктор фізико-мате-
матичних наук, професор. 12 З 1933 р. – Науково-дослідний інститут фізики Одеського університету. © Dovzhuk Ihor 12 З 1933 р. – Науково-дослідний інститут фізики Одеського університету.
13 Кирилов Єлпідіфор анемподистович (1883–1964) – фізик, доктор фізико-мате-
матичних наук, професор.
14 Українське фотографічне товариство (УФОТО або UFT) – спілка українських
фотоаматорів Західної України, створена у Львові з метою об’єднати українських
фотографів і пропагувати мистецтво та техніку фотографії серед українського
населення. Існувало з 1930 до 1939 року. Діяльність товариства відновлено 9
листопада 2021 р. ABSTRACT У грудні 1925 р ХІV з’їзд ВКП (б). проголосив курс на
індустріалізацію
країни. Одним
із
завдань
політики
індустріалізації було забезпечення економічної незалежності
держави та зміцнення її обороноздатності. Наприкінці 1920-х –
на початку 1930-х рр. у СРСР розпочато створення вітчизняної
хіміко-фотографічної промисловості. У цей період радянська
промисловість не володіла технологією виробництва ні основи,
ні світлочутливих шарів фотоплівок. Найбільші фірми Eastman
Kodak (США) і Agfa (Німеччина), що володіли тоді майже всім
світовим ринком кінофотоматеріалів і суворо охороняли
секрети своєї технології, відмовили Радянському Союзу у
технічній допомозі. Тому перед радянською фотографічною
наукою постало завдання створення технологічної бази молодої
хіміко-фотографічної промисловості. Ще з 1918 р. в Москві почав дослідження в галузі
фотографії науково-дослідний відділ Аерофотопарка. У
Петрограді (з 1924 р. – Ленінград) засновано Вищий інститут
фотографії і фототехніки, який проіснував через фінансові
труднощі тільки до 1923 р. й був перетворений у
Ленінградський кінофототехнікум. 1931 р. на базі цього 88 Development of photography in Ukraine in the XX century and its
institutionalization as an archive document технікуму організовано Ленінградський інститут кіноінженерів
(ЛІКІ). 1926 р. створено Науково-дослідний інститут фізики в
Одесі12. 1929 р. почав
діяти
Науково-дослідний
кінофотоінстітут
у
Москві
(НДКФІ,
пізніше
ДержНДІХІМФОТОПРОЕКТ), в якому були об’єднані
дослідження як хіміко-фотографічних процесів, так і
кінотехнічних проблем. Цього ж року розпочато роботи в галузі
фотографії у Науково-дослідному фізико-хімічному інституті
ім. Л. Я. Карпова (Москва). У зв’язку зі збільшенням науково-
фотографічних завдань і необхідністю підготовки фахівців у
цій галузі 1927 р. заснована кафедра фотохімічної технології в
Московському вищому технічному училищі (МВТУ). У цих
установах стали проводитися роботи для потреб хіміко-
фотографічної промисловості. Роботи в галузі вивчення дії світла на фотографічні шари
й на окремі кристали галогенідів срібла, механізму утворення
прихованого зображення в СРСР були зосереджені головним
чином у трьох наукових центрах – ленінградському,
московському та одеському. У НДІ фізики Одеського
університету дослідження з наукової фотографії проводились
під керівництвом професора Є. А. Кирилова13, який у 1920-х рр. поклав початок роботам в Україні з вивчення оптичних
властивостей галогенідо-срібних кристалів і фотографічних
шарів і став засновником одеської школи наукової фотографії
(Грушицька, 2014, 39: 286). На початку 1930 р. з ініціативи професора Олекси
Балицького і лікаря Степана Дмоховського було створене
Українське фотографічне товариство14 у Львові (з філіями у 89 © Dovzhuk Ihor Розвиток фотографії в Україні у ХХ ст. та її інституалізація як
архівного документа Станіславі, Тернополі й Рогатині). Товариство мало понад 200
членів, власне приміщення і бібліотеку. У 1933-1939 рр. видавало журнал «Світло й Тінь», головними редакторами
якого були Олександр Мох15, Степан Щурат16. © Довжук Ігор ABSTRACT УФОТО
влаштовувало щомісячні внутрішні виставки і щорічні
репрезентативи на Всеслов’янській мистецькій фотовиставці17,
брало участь у фотоконкурсах «Кодака»18, «Філіпса»19 та ін. Станіславі, Тернополі й Рогатині). Товариство мало понад 200
членів, власне приміщення і бібліотеку. У 1933-1939 рр. видавало журнал «Світло й Тінь», головними редакторами
якого були Олександр Мох15, Степан Щурат16. УФОТО
влаштовувало щомісячні внутрішні виставки і щорічні
репрезентативи на Всеслов’янській мистецькій фотовиставці17,
брало участь у фотоконкурсах «Кодака»18, «Філіпса»19 та ін. р
у
у ф
ур
У середині 1930-х рр. А. І. Кіпріанов20 у Харкові поряд з
педагогічною діяльністю в університеті активно проводив
наукові дослідження, одночасно працюючи в Українському
інституті прикладної хімії (УІПХ) (1924–1936 рр.), де він
організував та очолював лабораторію органічного синтезу й
почав
займатися
барвниками,
що
мали
властивості
фотосенсибілізаторів. Найефективнішим з них виявився
барвник
з
порядковим
номером
11у
(3,3/-диетил-9-
метилтіакарбоціанін-йодид)
–
перший
сенсибілізатор,
використаний ще 1934 р. у вітчизняній промисловості для
виробництва чутливих кіно- й фотоплівок. Подібні роботи
майже одночасно проводилися у Всесоюзному НДКФІ у
Москві. З 1936 по 1941 р. дослідження ціанінових барвників
15 мох олександр Іванович (1900–1975) – український поет, публіцист, перекладач,
журналіст, видавець. 16
Щурат
степан
васильович
(1909–1990)
–
український
учений-
літературознавець. 17 У 1935 р. відбулася Перша Всеслов’янська виставка мистецької фотографії у
Загребі, Хорватія. 18 Eastman Kodak Company – американська компанія, всесвітньо відомий виробник
фототехніки та фото- і кінотоварів. Штаб-квартира – у місті Рочестер, штат Нью-
Йорк (США). 19 Концерн «філіпс» засновано в 1891 р. братами-інженерами Герардом і Антоном
Філіпсами, які відкрили у Ейндговені (місто на півдні Нідерландів) невелику
фабрику з виробництва електроламп. Через декілька років «Електролампові заводи
Філіпс» стали відомими в країні та вже у 1916 р. королева Нідерландів Вільгельміна
надала підприємству Філіпсів право називатися королівським – з приставкою
«Конінклек». У 1999 р. у Нідерландах брати посмертно отримали звання «найкращі
підприємці XX століття». 20 Кіпріанов андрій Іванович (1896–1972) – український радянський хімік,
академік АН УРСР (з 1945 р.). 90 © Довжук Ігор Development of photography in Ukraine in the XX century and its
institutionalization as an archive document А. І. Кіпріанов проводив в Інституті хімії при Харківському
університеті. У 1945 р. А. І. Кіпріанов був призначений
директором Інституту органічної хімії АН України в Києві,
керував організованою тут лабораторією органічного синтезу
(Грушицька, 2014, 41: 289). Суспільний інтерес і зацікавленість сприяли формуванню
в Україні системи навчання фахівців із фотосправи. У 1920–
1930-х pp. до підготовки фототехніків-лаборантів і фотографів
долучалися
мистецько-промислові
школи. Мистецтво
фотографії викладали й у Київському художньому інституті та
Київському інституті кінематографії21. 21 Сьогодні – Київський національний університет театру, кіно і телебачення імені
І. К. Карпенка-Карого. 22 Байконур – космодром у Казахстані, перший і найбільший у світі. Збудований у
СРСР 1955 р.
23 леонов олексій архипович (1934–2019) – радянський космонавт, перша людина,
яка вийшла у відкритий космос.
24 Береговий георгій тимофійович (1921–1995) – український радянський
льотчик-космонавт, генерал-лейтенант авіації, двічі Герой Радянського Союзу. 22 Байконур – космодром у Казахстані, перший і найбільший у світі. Збудований у
СРСР 1955 р. ABSTRACT А вже після Другої
світової війни фотографію було включено до навчальних планів
підготовки кінооператорів Київського театрального інституту
ім. Карпенка-Карого; техніку фотографії – у Львівському
поліграфічному інституті, а для побутових потреб у великих
містах періодично влаштовували курси фотографії, які
випускали фотографів-техніків. При Міністерстві культури в
Києві створили експериментальну фотостудію. Цей час на українських землях ознаменувався і розвитком
фототехніки. Українські підприємства стали флагманами цієї
справи. До перших плівкових портативних фотоапаратів, які
вироблялися на території України, належать фотоапарати серії
«ФЕД», що випускалися Харківським машинобудівним
заводом «ФЕД», заснованим у 1927 р. У повоєнні роки на
заводі тривав випуск фотоапаратів серії «ФЕД», припинений
1990 р. (Горевалов & Зикун, 2015: 54). Особливою сторінкою в історії розвитку фотосправи є
діяльність легендарного українського заводу спеціального
приладобудування «Арсенал» у м. Київ. Усі космічні старти
колишнього СРСР і Росії забезпечувалися з використанням
оптично-електронних систем орієнтування виробництва
«Арсеналу». Значним досягненням став створений у 1970-х
роках комплекс імітаторів зовнішньої візуальної обстановки, 91 © Dovzhuk Ihor Розвиток фотографії в Україні у ХХ ст. та її інституалізація як
архівного документа які формують повну ілюзію маневрування і керування
космічним кораблем при наземній підготовці космонавтів. Імітаторами, виготовленими на заводі, укомплектовані
моделювальні стенди та тренажери, центри підготовки
космонавтів та космодром «Байконур»22. Фототехніка, створена арсенальцями, використовувалась
при фотозйомці з борту космічних кораблів серії «Восток»,
«Союз», місячних кораблів серій «Луна» та «Зонд», орбітальної
станції «Салют», а також у відкритому космосі. Унікальні
зйомки з космосу, зроблені арсенальськими фотокамерами
«Київ-10», космонавтами Олексієм Леоновим23 та Георгієм
Береговим24, обійшли всю світову пресу (Трачун, 2014: 146). Після війни на «Арсенал» було доставлено обладнання
заводу відомої фірми «Carl Zeiss Jena» (виробник фототехніки
та оптичних приладів), вивезене з Німеччини в рахунок
репарацій. Це визначило подальший профіль виробництва. З
1946 р. завод перепрофілювався на випуск оптичних, оптико-
механічних і оптико-електронних приладів військового
призначення. «Арсенал» став режимним підприємством
військово-промислового комплексу СРСР. Зазначимо, що одним з найвідоміших видів цивільної
продукції заводу була фототехніка, історія якої почалась з
першої масової фотокамери «Киев-2» (1949 р.). Далекомірні
фотоапарати «Киев» – перша фототехніка, яку почав
виготовляти «Арсенал». Моделі «Киев-2» і «Киев-3» були
точними копіями німецьких камер Contax-II та Contax-III. До
налагодження лінії виробництва на заводі «Арсенал»,
фотоапарати «Киев» виготовлялися на заводі Цейсса в Йєні
(НДР). Пізніше всю документацію та обладнання вивезли до 92 © Довжук Ігор © Довжук Ігор Development of photography in Ukraine in the XX century and its
institutionalization as an archive document Києва та почалося виробництво фотокамер на «Арсеналі». © Dovzhuk Ihor ABSTRACT Далекомірні фотоапарати «Киев» (35 мм) – моделі «Киев-2» і
«Киев-3» відрізнялися один від одного наявністю експонометра
в останньому. У 1956 р. з’явилися нові модифікації «Киев-2А»
і «Киев-3А», до яких додали X-синхроконтакт для фотоспалаху. На більшості апаратів назва писалася російською («Киев»), але
були також випуски з написанням назви українською («Київ»)
(Этапы развития отечественного фотоаппаратостроения. «Киев-2»). Києва та почалося виробництво фотокамер на «Арсеналі». Далекомірні фотоапарати «Киев» (35 мм) – моделі «Киев-2» і
«Киев-3» відрізнялися один від одного наявністю експонометра
в останньому. У 1956 р. з’явилися нові модифікації «Киев-2А»
і «Киев-3А», до яких додали X-синхроконтакт для фотоспалаху. На більшості апаратів назва писалася російською («Киев»), але
були також випуски з написанням назви українською («Київ»)
(Этапы развития отечественного фотоаппаратостроения. «Киев-2»). У 1957–1958 рр. «Арсенал» модернізував «Киев-2» і
«Киев-3», замінивши їх на «Киев-4А» та «Киев-4» відповідно. У нових моделях була оновлена задня стінка, дещо змінений
корпус та експонометр. Ці моделі вироблялися на заводі доволі
довго: «Киев-4» – до 1979 р., «Киев-4А» – до 1980. Існували
також модифікації «Киев-4М» (1976 р.) та «Киев-4АМ»
(1980 р.), в яких доданий центральний контакт, модифікована
голівка взводу затвора та перероблено механізм перемотування
плівки на «рулетку». Ці модифікації виготовлялися на заводі
до 1985 р. Останнім новим далекомірним фотоапаратом був
«Киев-5». За технічними даними він подібний до «Киева-4»,
але зовнішній вигляд камери був перероблений, зокрема верхня
частина «Киев-5» мала курковий взвод затвора, автоматичний
компенсатор паралакса, рулетку для перемотування плівки. Далекомірні
камери
«Киев»
комплектувалися
такими
штатними об’єктивами: «Юпитер-8» 2/50 (всі моделі), «Гелиос-
94» 1,8/50 (Киев-5), «Гелиос-103» 1,8/50 (Киев-4М, Киев-4АМ). Далекомірні фотоапарати «Киев» мали свій байонет – Contax-
Киев (або Contax RF). Перші дзеркальні камери, які почав серійно виготовляти
«Арсенал», мали оригінальний байонет «Автомат» власної
розробки. До цієї лінійки входять такі фотокамери: «Киев-10»
(1965–1974 рр.); «Киев-15TEE» (1974–1980 рр.); «Киев-15TTL»
(1980-ті рр.)
(Этапы
развития
отечественного
фотоаппаратостроения. «Киев-10»). 93 © Dovzhuk Ihor Розвиток фотографії в Україні у ХХ ст. та її інституалізація як
архівного документа Розвиток фотографії в Україні у ХХ ст. та її інституалізація як
архівного документа У 1970-х рр. на заводі «Арсенал» для заміни попередніх
моделей була розроблена нова лінійка дзеркальних фотокамер
із байонетом Н, що фактично являв собою байонет F фірми
Nikon. До цієї лінійки входять такі фотокамери: «Киев-17»
(1975–1984 рр.); «Киев-18» (1978–1988 рр., серійно не
випускалася); «Киев-19» (1985–1991 рр.); «Киев-19М»; «Киев-
20» (1983–1986 рр.). У 1980-х рр. завод «Арсенал» почав виробляти шкальну
фотокамеру «Киев-35», прототипом якої є німецький
фотоапарат «Minox 35». Камера «Киев-35» існувала у двох
модифікаціях:
«Киев-35А»;
«Киев-35АМ». До
середньоформатних камер належали: «Киев-6С»; «Киев-6С
TTL»; «Киев-60». Модульні середньоформатні камери
представлені такими моделями: «Салют»; «Салют-C»; «Киев-
88»; «Киев-88СМ»; «Киев-90». Користувалися популярністю в
Україні й за кордоном камери «Киев-19М» і середньоформатні
камери «Киев-60 ТТL» та «Киев-88 СМ» (Этапы развития
отечественного фотоаппаратостроения. «Киев-35А»). У 1980-х рр. завод «Арсенал» почав виробляти шкальну
фотокамеру «Киев-35», прототипом якої є німецький
фотоапарат «Minox 35». Камера «Киев-35» існувала у двох
модифікаціях:
«Киев-35А»;
«Киев-35АМ». До Виробництво фотоматеріалів було організовано в Україні
на ВАТ «АК «Свема» (колишнє ВО «Свема») – українському
підприємстві з виробництва кіно-, фото- та рентгеноплівки,
фотопаперу, магнітофонних стрічок та касет, заснованому 1
жовтня 1931 р. та розташованому в місті Шостка (Сумська
область). У 1934 р. на фабриці почали виготовляти плівку для
звукозапису та рентген-плівку. Вже у 1937 р. на плівку,
виготовлену на цьому підприємстві, був знятий фільм
«Великий громадянин» – перший радянський художній фільм
повністю знятий на власну кіноплівку. 1938 р. почали
виготовляти кольорову плівку. Також суттєвий внесок у галузь
внесло підприємство «Фотон» у м. Київ. Воно випускало понад
400 найменувань фотопаперу, серед яких універсальний
фотопапір «Унібром», «Фотобром», «Бромпортрет» та ін. (Горевалов & Зикун, 2015: 54–55). Усе це забезпечувало популярність фотосправи в Україні,
якість фотозображень, розширення діапазону функцій прес-
фотографії. 94 © Довжук Ігор © Довжук Ігор Development of photography in Ukraine in the XX century and its
institutionalization as an archive document Зазначимо, що в Україні питання організації архівного
зберігання фотодокументів на державному рівні було порушено
у 1925 р. Так, 16 грудня 1925 р. ВУЦВК та РНК УСРР прийняли
постанову «Про єдиний державний архівний фонд УСРР»
(Нариси історії архівної справи в Україні), до якого вперше
було включено кіно- і фотодокументи. Наступним кроком стало
створення 20 червня 1932 р. Всеукраїнського центрального
фотокіноархіву в м. Києві25. Упродовж багатьох років він був і
залишається найбільшим в Україні центром, що забезпечує
збирання, збереження та запровадження в науковий і
культурний обіг аудіовізуальних документів. 13 серпня 1958 р. постановою Ради Міністрів СРСР
затверджено «Положення Про Державний архівний фонд
СРСР», згідно з яким у кожній установі, організації та
підприємстві, що займалося виробництвом чи експлуатацією
25 З 1943 р. – Центральний державний архів кінофотофонодокументів УРСР, нині –
Центральний державний кінофотофоноархів України ім. Г. С. Пшеничного. © Dovzhuk Ihor 25 З 1943 р. – Центральний державний архів кінофотофонодокументів УРСР, нині –
Центральний державний кінофотофоноархів України ім. Г. С. Пшеничного. Розвиток фотографії в Україні у ХХ ст. та її інституалізація як
архівного документа Серйозні зрушення у джерелознавчих дослідженнях
фотодокументів відбулися в 1960–1980-х рр. У цьому контексті
слід відзначити праці М. Тихомирова, М. Черноморського,
Н. Єрмакової,
І. Ковальченка,
С. Шмідта,
Ф. Шевченка,
А. Санцевича та ін. Про необхідність спеціальних досліджень
інформаційних можливостей фотодокументів та введення їх до
наукового обігу писала О. Медушевська. Важливим чинником
у розвитку вітчизняного фотодокументознавства стала
розгорнута в 1960–1970-х рр. на сторінках радянських фахових
часописів «Вопросы архивоведения» і «Советские архивы»
дискусія щодо джерелознавчих проблем фотодокументів. Її
учасники Л. Крюкова, І. Фесуненко, Л. Рошаль, Л. Пушкарьов,
В. Магідов та ін., порушуючи питання про роль і значення
фотодокументів як історичних джерел, насамперед намагалися
розвіяти скептицизм істориків щодо цих документів як джерел
узагалі та зламати домінуючий стереотип щодо можливостей
їхнього використання лише як ілюстративного матеріалу до
історичних фактів. 13 серпня 1958 р. постановою Ради Міністрів СРСР
затверджено «Положення Про Державний архівний фонд
СРСР», згідно з яким у кожній установі, організації та
підприємстві, що займалося виробництвом чи експлуатацією
25 З 1943 р. – Центральний державний архів кінофотофонодокументів УРСР, нині –
Центральний державний кінофотофоноархів України ім. Г. С. Пшеничного. 95 © Dovzhuk Ihor Розвиток фотографії в Україні у ХХ ст. та її інституалізація як
архівного документа кінофотофонодокументів, повинні створюватися відповідні
архіви. Після
п’яти
років
з
моменту
виготовлення
кінофотофонодокументи мали передаватися на зберігання до
архіву: союзні – до державного архіву КФФД СРСР,
республіканські – до республіканського архіву КФФД, а
місцеві – до державних обласних архівів. кінофотофонодокументів, повинні створюватися відповідні
архіви. Після
п’яти
років
з
моменту
виготовлення
кінофотофонодокументи мали передаватися на зберігання до
архіву: союзні – до державного архіву КФФД СРСР,
республіканські – до республіканського архіву КФФД, а
місцеві – до державних обласних архівів. У
1967 р. Центральний
державний
архів
кінофотофонодокументів СРСР було поділено на два архіви –
Центральний
державний
архів
кінофотодокументів
і
Центральний державний архів звукозаписів СРСР. Протягом
60–70-х років завершилося формування кінофотофоноархівів
республіканського рівня. Кінофотофоноархіви були створені в
Азербайджані, Естонії, Латвії, Литві, Молдові. Наприкінці
70-х років почали діяли кінофотофоноархіви у Москві й
Ленінграді. У
1967 р. Центральний
державний
архів
кінофотофонодокументів СРСР було поділено на два архіви –
Центральний
державний
архів
кінофотодокументів
і
Центральний державний архів звукозаписів СРСР. Протягом
60–70-х років завершилося формування кінофотофоноархівів
республіканського рівня. Кінофотофоноархіви були створені в
Азербайджані, Естонії, Латвії, Литві, Молдові. Наприкінці
70-х років почали діяли кінофотофоноархіви у Москві й
Ленінграді. У процесі подальшого архівного будівництва відбувалися
зміни в діяльності архівних установ. Так, у зв’язку з ухвалою
у квітні 1980 р. © Довжук Ігор Розвиток фотографії в Україні у ХХ ст. та її інституалізація як
архівного документа постанови Ради Міністрів СРСР «Про
затвердження Положення про Державний архівний фонд СРСР
і Положення про Головне архівне управління при Раді
Міністрів СРСР» Уряд України 6 червня 1980 р. затвердив нове
положення про Головне архівне управління при Раді Міністрів
УРСР і мережу центральних державних архівів (Нариси історії
архівної справи в Україні). Відповідно до нового «Положення
про
Державний
архівний
фонд
СРСР»
склад
кінофотофонодокументів поповнювався відеозаписами. У процесі подальшого архівного будівництва відбувалися
зміни в діяльності архівних установ. Так, у зв’язку з ухвалою
у квітні 1980 р. постанови Ради Міністрів СРСР «Про
затвердження Положення про Державний архівний фонд СРСР У 1990-х рр. радянська мережа кінофотофоноархівів
фактично перестала існувати. Тепер в Україні функціонує
Центральний державний архів кінофотофонодокументів
ім. Г. С. Пшеничного
та
Центральний
державний
аудіовізуальний та електронний архів, але об’єктами зберігання
кінофотофонодокументів є й інші архіви, зокрема обласні,
міські та відомчі. Активно почали формуватися приватні
архіви, де зберігаються матеріали про сім’ю, родовід тощо. Кінофотофонодокументи створюються в результаті діяльності 96 © Довжук Ігор Development of photography in Ukraine in the XX century and its
institutionalization as an archive document видавництв, преси, громадських організацій, науково-
дослідних установ, радіостанцій, кінофабрик та інших
підприємств й організацій, збирають їх і приватні особи. До
архівів кінофотофонодокументи від приватних осіб надходять
лише за їхнім бажанням у результаті дарунків або продажу. На початку 1990-х років уперше у вітчизняній
історіографії кінофотознавство розглядалось як спеціальна
історична дисципліна, що займається вивченням кіно- і
фотодокументів. Згодом назва дисципліни була замінена більш
точним терміном «фотодокументознавство». На сучасному
етапі розвитку науки фотодокументознавство розглядається як
окремий напрям аудіовізуального документознавства. Завдяки
студіям М. Базанової, Л. Маркітан, Н. Слончак, Н. Топішко та
інших були виявлені, досліджені та введені до наукового обігу
значні комплекси фотодокументів. Дослідження фотоджерел
включає: їхнє виявлення, вивчення часу й обставин створення
фотодокументів; відбір джерел з їхньою подальшою
класифікацією і систематизацією, зовнішньою та внутрішньою
критикою. Критеріями
відбору
джерел
є
значимість
зафіксованих у них подій чи фактів, ступінь атрибутивності цих
документів, їхній технічний стан, наявність супровідної
документації тощо (Ємельянова, 2013: 318). © Dovzhuk Ihor висновки та перспективи подальших розвідок у
даному напрямку. Таким чином, уже з початку ХХ ст. можемо говорити про
високий рівень розвитку в Україні фотосправи, сформованість
її інфраструктури, швидке розширення напрямів розвитку в
багатьох українських регіонах. Розширення
функціонально-інструментальних
характеристик фотографії, розвиток фотомеханічних процесів
друку стали передумовами й каталізаторами становлення
професійного фотомистецтва, фотожурналістики, формування
і збагачення системи її жанрів, розширення кола фахівців
фотосправи. Доведена визначальна роль українських майстрів
у виробництві й удосконаленні фототехніки й існування
фотоіндустрії. 97 Розвиток фотографії в Україні у ХХ ст. та її інституалізація як
архівного документа У 1960–1980-х рр. виникла мережа спеціалізованих
архівів кінофотофонодокументів, що включала архіви як
союзного рівня, так і архіви союзних республік, у т.ч. України. У державних архівах створюються спеціальні архівні фонди
для передачі, зберігання і дослідження фотодокументів,
відбувається їхня інституалізація, що отримала новий розвиток
з початку 1990-х рр. Розглядувана проблематика має перспективи подальшого
дослідження,
передусім
щодо
архівного
зберігання
фотодокументів на початку ХХІ ст. ДЖеРела та лІтеРатУРа Д
Арсенал (завод). URL: https://uk.wikipedia.org/ wiki/
1. Арсенал_(завод) Арсенал (завод). URL: https://uk.wikipedia.org/ wiki/
1. Арсенал_(завод) Введенський О. Історія львівської фотографії. Поступ. 2. 2004. 17 травня. URL: http://portal.lviv.ua/digest/ 2004/05/17/
122648.html Введенський О. Історія львівської фотографії. Поступ. 2. 2004. 17 травня. URL: http://portal.lviv.ua/digest/ 2004/05/17/
122648.html Горевалов С., Зикун Н. Єдність слова і зображення. 3. Фотожурналістика в системі засобів масової комунікації. Київ,
2015. 287 с. Горевалов С., Зикун Н. Єдність слова і зображення. 3. Фотожурналістика в системі засобів масової комунікації. Київ,
2015. 287 с. Грушицька І. Б. Розвиток фотографії в Україні (1839 –
4. I пол. ХХ ст.). Наукові праці історичного факультету
Запорізького національного університету. 2014. Вип. 41. С. 285-291. Грушицька І. Б. Школа
наукової
фотографії
5. Є. А. Кирилова. Наукові праці історичного факультету
Запорізького національного університету. 2014. Вип. 39. С. 283-288. Ємельянова Т. О. Фотодокументознавство. Енциклопедія
6. історії України: у 10 т. / редкол.: В. А. Смолій (голова) та ін. Київ: Наук. думка, 2013. Т. 10 : Т-Я. 2013. 784 с. Нариси історії архівної справи в Україні / за заг. ред. 7. І. Б. Матяш та К. І. Климової. Київ: КМ Академія, 20021. 612 с. Нариси історії архівної справи в Україні / за заг. ред. 7. І. Б. Матяш та К. І. Климової. Київ: КМ Академія, 20021. 612 с. Трачук О. Золотий вік української фотографії. URL:
8. http://primetour.ua/uk/company/articles/1.html Трачук О. Золотий вік української фотографії. URL:
8. http://primetour.ua/uk/company/articles/1.html 98 © Довжук Ігор © Довжук Ігор Development of photography in Ukraine in the XX century and its
institutionalization as an archive document Трачун А. История украинской фотографии XIX-XXI вв. 9. Киев: Балтия-Друк, 2014. 255 с. Федотова О. О. Становлення та розвиток фотографії на
10. українських землях у дорадянський період. Topical issues of the
development of modern science. Abstracts of VII International
Scientific and Practical Conference Sofia, Bulgaria, 11-13 March
2020. С. 435-440. Федотова О. О. Становлення та розвиток фотографії на
10. українських землях у дорадянський період. Topical issues of the
development of modern science. Abstracts of VII International
Scientific and Practical Conference Sofia, Bulgaria, 11-13 March
2020. С. 435-440. Этапы развития отечественного фотоаппаратостроения. 11. «Киев-35А». URL: http://photohistory.ru/1207248177836584.html Этапы развития отечественного фотоаппаратостроения. 12. «Киев-10»,
«Киев-11». URL:
http://photohistory.ru/
1207248179443786.html Этапы развития отечественного фотоаппаратостроения. 13. «Киев-2», 1947-1955, «Киев-2А» и др. URL: http://
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y
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Янковий В., Стефанович Д. Віртуоз фотографії, або ім’я,
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y.kiev.ua/znamenitye-kievlyane/uchyonye/virtuoz-fotografiyi-abo-
imya-povernute-iz-zabuttya [in Ukrainian]. y p
y
Groushitska, I.B. (2021). The development of photography in
15. Ukraine (1839 – I half of XX century). Zaporizhzhia Historical
Review,
1(41), 285-291. URL: https://istznu.org/index.php/
journal/article/view/1252 [in English]. Groushitska, I.B. (2021). The development of photography in
15. Ukraine (1839 – I half of XX century). Zaporizhzhia Historical
Review,
1(41), 285-291. URL: https://istznu.org/index.php/
journal/article/view/1252 [in English]. © Dovzhuk Ihor © Довжук Ігор анотацІя У статті розглядаються питання розвитку фотографії,
фотосправи та хіміко-фотографічної промисловості в Україні
у ХХ ст., а також інституалізація фотографії як архівного
документа. Уже з початку ХХ ст. можемо говорити про високий
рівень розвитку в Україні фотосправи, сформованість її
інфраструктури, швидке розширення напрямів розвитку в
багатьох українських регіонах. Розширення функціонально-
інструментальних характеристик фотографії, розвиток
фотомеханічних процесів друку стали передумовами й
каталізаторами становлення професійного фотомистецтва,
фотожурналістики, формування і збагачення системи її
жанрів, розширення кола фахівців фотосправи. Змінюється
рівень
сприйняття
фотографії
і
фотодіяльності, з’являється потреба узагальнення і наукового 101 © Dovzhuk Ihor Розвиток фотографії в Україні у ХХ ст. та її інституалізація як
архівного документа її осмислення, створення теорії фотосправи. Так, з 1914 р. у
Києві було вже чимало фотографів-митців і професіоналів-
науковців. Активна фотодіяльність викликала до життя і
виникнення спеціалізованих періодичних видань. За короткий
час розвитку фотогуртків у Харкові засновуються профільні
журнали. Зазначено, що на початку 1930 р. з ініціативи професора
Олекси Балицького і лікаря Степана Дмоховського було
створене Українське Фотографічне Товариство у Львові (з
філіями у Станіславі, Тернополі й Рогатині). Товариство мало
понад 200 членів, власне приміщення і бібліотеку. У 1960-1980-х рр. виникла мережа спеціалізованих архівів
кінофотофонодокументів, що включала архіви союзного рівня
та архіви союзних республік, у т.ч. України. У державних
архівах створюються спеціальні архівні фонди для передачі,
зберігання і дослідження фотодокументів, відбувається їхня
інституалізація, яка отримала новий розвиток з початку
1990-х рр. Ключові слова: Україна, фотографія, фотосправа,
фотоосвіта,
українські
фотографічні
товариства,
фотодокументи. 102 © Довжук Ігор © Довжук Ігор
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https://openalex.org/W3169216643
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https://digital.csic.es/bitstream/10261/266349/1/GYA_2021_V72_e404.pdf
|
English
| null |
Effects of virgin olive oil phenolic compounds on health: solid evidence or just another fiasco?
|
Grasas y aceites
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cc-by
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GRASAS Y ACEITES 72 (2)
April-June 2021, e404
ISSN-L: 0017-3495
https://doi.org/10.3989/gya.0217201 GRASAS Y ACEITES 72 (2)
April-June 2021, e404
ISSN-L: 0017-3495
https://doi.org/10.3989/gya.0217201 J.M. Castellanoa and J.S. Peronaa, J.M. Castellanoa and J.S. Peronaa, aInstituto de la Grasa-CSIC. Campus University Pablo de Olavide, Building 46. 41013 Seville (Spain). Corresponding author: perona@ig.csic.es aInstituto de la Grasa-CSIC. Campus University Pablo de Olavide, Building 46. 41013 Seville (Spain). Corresponding author: perona@ig.csic.es ubmitted: 14 February 2020; Accepted: 18 April 2020; Published online: 03 June 2021 Submitted: 14 February 2020; Accepted: 18 April 2020; Published online: 03 June 2021 SUMMARY: Current research suggests that virgin olive oil (VOO) phenolics are potent preventive and therapeutic agents against
metabolic diseases associated with inflammation and oxidative stress. Evidence-based medicine requires these effects be proved in
randomized controlled trials (RCT), which are then assessed in meta-analyses, to ensure that the alleged health benefits really proceed in
humans. The available evidence is limited to the ability of VOO phenolic compounds to protect lipoproteins from oxidation and to reduce
systolic pressure in hypertensive individuals. No RCT assessing the effects of VOO phenolics on diabetes and neurodegenerative diseases
have been performed, and those focused on osteoarthritis and cancer provided very scarce information. Therefore, RCT in extensive and
diverse population groups, with different disorders and phenolic doses adjusted to usual VOO consumptions are necessary to achieve high
quality scientific evidence before nutritional recommendations can be given to the general public. KEYWORDS: Evidence; Health; Hydroxytyrosol; Oleocanthal; Phenolic Compounds; Virgin Olive Oil RESUMEN: Efectos de los compuestos fenólicos del aceite de oliva virgen en la salud: ¿evidencia sólida o simplemente otro fiasco? Las investigaciones actuales indican que los compuestos fenólicos del aceite de oliva virgen (AOV) son potentes agentes preventivos y
terapéuticos contra las enfermedades metabólicas asociadas con la inflamación y el estrés oxidativo. La medicina basada en la evidencia
requiere que estos efectos se prueben en ensayos aleatorizados controlados (RCT), que son después evaluados en meta-análisis, para
garantizar que los supuestos beneficios para la salud realmente se registran en humanos. La evidencia disponible se limita a la capacidad de
los compuestos fenólicos del AOV para proteger las lipoproteínas de la oxidación y reducir la presión sistólica en individuos hipertensos. No se han realizado RCT que evalúen el efecto de estos compuestos sobre la diabetes y las enfermedades neurodegenerativas, y los que
se centraron en la osteoartritis y el cáncer han proporcionado información muy escasa. Por lo tanto, nuevos RCT, en grupos de población
extensos y diversos, con diferentes patologías y con dosis de fenoles ajustadas a los consumos habituales de VOO, deben desarrollarse,
para lograr evidencia científica de alta calidad antes de que se puedan dar recomendaciones nutricionales al público en general. PALABRAS CLAVE: Aceite de Oliva Virgen; Compuestos Fenólicos; Evidencia; Hidroxitirosol; Oleocantal; Salud
Citation/Cómo citar este artículo: Castellano JM, Perona JS. 2021. PALABRAS CLAVE: Aceite de Oliva Virgen; Compuestos Fenólicos; Evidencia; Hidroxitirosol; Oleocantal; Salud
Citation/Cómo citar este artículo: Castellano JM, Perona JS. 2021. Effects of virgin olive oil phenolic compounds on health: solid
evidence or just another fiasco? Grasas Aceites 72 (2), e404. https://doi.org/10.3989/gya.0217201 1. INTRODUCTION More than 30 phenolic compounds that can play
a role in the health promoting qualities of VOO have
been identified, among which there is considerable
variation regarding their concentration (0.02 to
600 mg/kg) (Servili et al., 2009). This variability
depends on the type of phenolic compound, but also
on many other factors such as the olive tree variety,
geographical origin, cultivation techniques, ripening
stage at the time of harvest, processing and storage. Among VOO phenolics, secoiridoids are present in
the greatest amount, but the most interesting ones from
the point of view of health are probably oleuropein
aglycone
and
its
metabolite
hydroxytyrosol. However, the presence of oleocanthal should not be
ignored, as this compound has received a great deal
of attention since it was suggested that it might have
anti-inflammatory activity (Beauchamp et al., 2005). In 2014, Francesco Visioli published an editorial
response to a review by Tang et al., (2014) on the
effects on cardiovascular health of resveratrol,
a natural phenolic compound found in red wine
(Visioli, 2014). Resveratrol was alleged to interact
with multiple targets in a variety of cardiovascular
disease models to exert protective effects or induce
a reduction in cardiovascular risks. However,
Visioli’s article was entitled “The resveratrol fiasco”
and concluded that after more than 20 years of
well-funded research, resveratrol had no proven
human activity. He claimed that there was a lack of
clinical trials and the majority of available evidence
came from cell culture and animal models. He
also suggested that the resveratrol fiasco is not the
only one of that kind in pharma-nutrition research. Considering that the association of virgin olive oil
(VOO) phenolic compounds and health was initiated
in the late 90s (some of the early studies were carried
out by Visioli himself (Visioli et al., 1998)) and that
the bulk of experimental data were obtained from in
vitro and animal models, are VOO phenolics another
example of scientific fiasco? In order to answer
this question, the present review summarizes the
current knowledge on the effects of VOO phenolic
compounds on human health, focusing on the data
obtained from randomized clinical trials (RCT) and
their meta-analyses and systematic reviews, which
provide the highest level of scientific evidence. 1. INTRODUCTION Several experimental studies in in vitro systems
and animal models have shown that the possible
benefits of VOO phenolics are associated with their
anti-inflammatory, antioxidant and vasodilatory
activity, which in theory makes them key preventive
or therapeutic agents for metabolic diseases related
to oxidative stress and inflammation (Covas et al.,
2006). However, most of these investigations used
supraphysiological doses of phenolics (> 10 μM)
(Catalán et al., 2015) and, therefore, it is difficult to
translate their results into physiological relevance for
humans. VOO phenolics are bioavailable in humans,
i.e. they are susceptible to being absorbed and to exert
a bioactive effect on the organism (de la Torre, 2008). It has been proposed that in humans hydroxytyrosol
is dose-dependently absorbed and excreted in urine
after the intake from VOO (Rubió et al., 2012;
Oliveras-López et al., 2014). However, a more
recent study in Sprague-Dawley rats suggested that
different dosages of hydroxytyrosol do not provide
a linear, dose-dependent plasma concentration or
excretion in urine (Domínguez-Perles et al., 2017). Nevertheless, it has been estimated that the amount
of phenolics ingested from VOO consumption does
not exceed 9 mg/day in Mediterranean countries
(Parkinson and Cicerale., 2016). ubmitted: 14 February 2020; Accepted: 18 April 2020; Published online: 03 June 2021 Effects of virgin olive oil phenolic compounds on health: solid
evidence or just another fiasco? Grasas Aceites 72 (2), e404. https://doi.org/10.3989/gya.0217201 PALABRAS CLAVE: Aceite de Oliva Virgen; Compuestos Fenólicos; Evidencia; Hidroxitirosol; Oleocantal; Salud Citation/Cómo citar este artículo: Castellano JM, Perona JS. 2021. Effects of virgin olive oil phenolic compounds on health: solid
evidence or just another fiasco? Grasas Aceites 72 (2), e404. https://doi.org/10.3989/gya.0217201 Copyright: ©2021 CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International
(CC BY 4 0) License Copyright: ©2021 CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International
(CC BY 4.0) License. 2 • J.M. Castellano and J.S. Perona Grasas y Aceites 72 (2), April-June 2021, e404. ISSN-L: 0017-3495. https://doi.org/10.3989/gya.0217201 2. VOO PHENOLIC COMPOUNDS However, this health
claim is focused on the protection provided by
hydroxytyrosol and there are currently no accepted
claims for the other benefits that have been attributed
to VOO or its phenolic compounds. Although a large number of in vitro and
experimental animal studies are available, according
to Evidence-Based Medicine (Woolf et al., 1990), the
healthy properties of a food and/or its components
must be proved in RCT, in order to ensure that the
alleged health benefits really stand out. The highest
degree of scientific evidence is obtained through
meta-analyses and systematic reviews of these RCT. However, at the moment the number and variety of
RCT carried out using VOO phenolic compounds
is very limited and there is only one meta-analysis
and less than five systematic reviews assessing
the healthy effects of VOO phenolic compounds. With regard to RCT, there are currently about 50
published articles that can be classified as such in
which the effect of VOO phenolic compounds on
health has been evaluated. A high number of these
studies were published by the group of Covas and
their collaborators and the rest by three or four
research teams, the absolute majority of which were
Spanish or Italian. The intake of high phenolic VOO only seemed to
produce a modest but significant reduction in oxidized
LDL-cholesterol (Z=1.98, p=0.05) and blood
pressure (Z=4.04, p<0.001). For the rest of parameters
related to cardiovascular disease (malondialdehyde,
LDL-cholesterol, HDL-cholesterol, total cholesterol
and triglycerides), no significant effects were found. Therefore, the conclusion of this meta-analysis was
that a high phenolic VOO provides small beneficial
effects on systolic pressure and plasma oxidative
status. No association between the intake of phenolic
compounds from VOO and cardiovascular risk could
be obtained. In
addition
to
this
meta-analysis,
two
systematic reviews were published, both of them
in 2019. Bahramsoltani et al., (2019) published
a comprehensive review about dietary phenolic
compounds and atherosclerosis and, even more
recently, Schwingshackl et al., (2019) carried out
a systematic review and network meta-analysis to
assess the impact of different types of olive oil on
cardiovascular risk factors. 2. VOO PHENOLIC COMPOUNDS There is ample evidence indicating that VOO
consumption provides benefits in key processes
associated with the development of a number
of diseases and pathophysiological conditions. These include atherosclerosis, diabetes mellitus,
obesity, metabolic syndrome, cancer, arthritis and
neurodegenerative diseases (Covas et al., 2015). Despite its high oleic acid content, VOO is more
than just a monounsaturated fatty acid-rich fat. VOO contains minor compounds with potent
pharmacological activity, which are classified
into two large groups: those that form part of the
unsaponifiable fraction and those with a phenolic
nature. The former are lipophilic and may be
extracted with organic solvents after saponification
of the oil, while the latter are water soluble. Since 2011, the European Food Safety Authority
(EFSA) accepts a claim about the benefits of daily
intake of VOO rich in phenolic compounds over
the oxidation of low density lipoproteins (LDL),
maintenance of normal (fasting) blood levels of
triglycerides, HDL-cholesterol and blood glucose Grasas y Aceites 72 (2), April-June 2021, e404. ISSN-L: 0017-3495. https://doi.org/10.3989/gya.0217201 Effects of virgin olive oil phenolic compounds on health: solid evidence or just another fiasco? • 3 (EFSA Panel on Dietetic Products, Nutrition and
Allergies (NDA), 2011). The acceptance of these
claims by EFSA authorizes their inclusion on the
labels of olive oil bottles. However, this health
claim is focused on the protection provided by
hydroxytyrosol and there are currently no accepted
claims for the other benefits that have been attributed
to VOO or its phenolic compounds. et al., 2015). The sample size of these trials was quite
variable, ranging from 24 participants in the study
published by Moreno-Luna et al., (2012), to 200
individuals in the one carried out by Covas et al.,
(2006). In addition, there was variability in the health
status of the participants. In five of these studies,
the subjects were healthy and in the remaining
three ones, the recruited individuals already had
a cardiovascular event. The doses used in these
tests ranged from 0 to 19.5 mg of total phenolics. Furthermore, in all these trials what was compared
was the intake of a high phenolic VOO with a refined
olive oil. Therefore, only the total phenolic content
was considered and the concentrations in different
phenolic compounds were not distinguished. (EFSA Panel on Dietetic Products, Nutrition and
Allergies (NDA), 2011). The acceptance of these
claims by EFSA authorizes their inclusion on the
labels of olive oil bottles. Grasas y Aceites 72 (2), April-June 2021, e404. ISSN-L: 0017-3495. https://doi.org/10.3989/gya.0217201 3.
VOO
PHENOLIC
COMPOUNDS
AND
CARDIOVASCULAR DISEASE The role of VOO phenolics as cardiovascular
protectors has been a matter of research for a long
time. However, as very recently displayed by Visioli
et al., (2020), most investigations used isolated
compounds in pharmacological in vitro approaches,
rather than more nutritional human trials. Still,
enough data from human studies assessing surrogate
markers of cardiovascular disease have been
generated (Bogani et al., 2007; Visioli et al., 2005;
Covas et al., 2006) to allow for the completion of a
meta-analysis of RCT (Hohmann et al., 2015) and
two systematic reviews (Bahramsoltani et al., 2019;
Schwingshackl et al., 2019). The review by Bahramsoltani et al., (2019)
included only six RCT, while the aforementioned
meta-analysis by Hohmann and colaborators
included eight. Of these 6 trials, only one reached
a score of 3 on the Jadad Scale (Jadad et al., 1996)
which measures the methodological quality of a
clinical trial from 0 (very poor) to 5 (rigurous) points. The others stayed at 2 or below. What was observed
in the best designed study (Covas et al., 2006) was
an effect on the postprandial isoprostane F2 levels
in plasma, which is a biomarker of oxidative stress. In this double-bind trial, 40 mL of three olive oils Indeed, this meta-analysis was aimed at assessing
the effects of high phenolic olive oil on risk factors
for cardiovascular disease. It was published in 2015
and included the results of 8 human RCT (Hohmann Grasas y Aceites 72 (2), April-June 2021, e404. ISSN-L: 0017-3495. https://doi.org/10.3989/gya.0217201 4 • J.M. Castellano and J.S. Perona with different phenolic contents were administered
in a single dose: low (2.7 mg/kg), medium (164 mg/
kg) and high (366 mg/kg). The authors observed
that the concentration in total phenolic compounds
in LDL increased in the postprandial period in a
direct relationship with the phenolic content of the
oils ingested. Moreover, plasma concentrations
of
tyrosol,
hydroxytyrosol,
and
3-O-methyl-
hydroxytyrosol correlated with changes in the total
phenolic compound contents in LDL. The other RCT
included in the review also confirmed these results,
as well as reduced lipid peroxidation (measured
as 8-iso-PGF2α) and inflammation (assessed as
intercellular adhesion molecule-1, ICAM-1). Guallar et al., 2013). Possibly, the story is not as
simple as it was believed. For instance, it has been
proposed that LDL can become atherogenic even
before oxidation. 4.
VOO
PHENOLIC
COMPOUNDS
AND
TYPE 2 DIABETES MELLITUS/METABOLIC
SYNDROME )
The systematic review by Schwingshackl et al.,
(2019) included thirteen RCTs with 611 participants
(mainly healthy) and compared refined olive oil,
commercial olive oil (blend of refined and virgin
olive oils), low-phenolic extra VOO and high-
phenolic VOO. No differences for total cholesterol,
HDL-cholesterol, triglycerides, and diastolic blood
pressure were observed. However, the phenolic
content positively correlated with a slight reduction
in LDL-cholesterol (mean difference -0.14 mmol/L,
95%-CI: -0.28, -0.01) and oxidized LDL-cholesterol
(standardized mean difference: -0.68, 95%-CI: -1.31,
-0.04). Both, high- and low-phenolic VOO reduced
systolic blood pressure compared to refined olive oil
(range of mean difference: -2.99 to -2.87 mmHg). The authors concluded that high phenolic VOO may
improve some cardiovascular risk factors, although
the implications for public health were limited due
to the overall low or moderate level of evidence
provided and also because the duration of these RCTs
was too short (≤12 weeks) and no data could be found
for relevant outcomes such as cardiovascular events. Accumulated data obtained from experimental
models indicate that VOO phenolics have the
potential to normalize metabolic syndrome and
its pathophysiological complications, including
diabetes. Diabetes and metabolic syndrome are
linked to each other through insulin resistance,
and subjects diagnosed with metabolic syndrome
have a high risk of developing T2DM (Shin et al.,
2013). However, there are currently no RCT aimed
at specifically assessing the effect of VOO phenolics
on Type 2 diabetes (T2DM) patients, although two
systematic reviews on the effects of these compounds
on metabolic syndrome have been published (Chiva-
Blanch and Badimon, 2017; Saibandith, 2017). The review by Chiva-Blanch and Badimon
(2017) was focused on human intervention trials
administering phenolic-rich foods to patients
with metabolic syndrome. They included a single
RCT, carried out by Venturini et al., (2015), which
administered extra VOO (10 mL/day). That trial
reported an increase in the total radical-trapping
antioxidant parameter (TRAP)/uric acid ratio, with
no apparent effects on other markers of oxidative
stress. On the other hand, the authors did not find
changes in the lipid profile, plasma glucose, insulin
resistance or blood pressure. From
these
meta-analysis
and
systematic
reviews, it can be concluded that the protective
effect of VOO phenolics is related to blood pressure
and LDL oxidation only. Nonetheless, although the
oxidative hypothesis of atherosclerosis has been
accepted for decades, the true contribution of LDL
oxidation to cardiovascular disease is still unclear
(Arsenault et al., 2017; Visioli et al., 2020). 3.
VOO
PHENOLIC
COMPOUNDS
AND
CARDIOVASCULAR DISEASE Modification of these lipoproteins
begins with a desialylation and it is followed by a
cascade of other physical and chemical alterations
that increase LDL atherogenicity, including particle
size reduction, increase in its density and negative
electrical charge and loss of lipids (Summerhill et
al., 2019). Therefore, the effects of VOO phenolics
on cardiovascular disease via LDL modification are,
as of today, dubious and there is need of more RCT
that directly address the claim. Grasas y Aceites 72 (2), April-June 2021, e404. ISSN-L: 0017-3495. https://doi.org/10.3989/gya.0217201 4.
VOO
PHENOLIC
COMPOUNDS
AND
TYPE 2 DIABETES MELLITUS/METABOLIC
SYNDROME Oleuropein
also enhanced the endogenous antioxidant response
in the CA1 hippocampal area of rats suffering from
colchicine-induced cognitive dysfunction, improving
the redox status of glutathione, and increasing the
activity of antioxidant enzymes, such as superoxide
dismutase or catalase (Pourkhodadad et al., 2016). Similarly, hydroxytyrosol has been related to the
amelioration of insulin resistance in the brain of mice
(Kulas et al., 2020). In APP/PS1 mice, rodent models
of AD, hydroxytyrosol improved cognitive function,
increased the expression of antioxidant enzymes and
phase 2 response genes, and reduced inflammatory
factors in the brain of these animals (Peng et al., 2016). It is noteworthy that the RCT involving patients
with metabolic syndrome and published in 2016 by
D’Amore et al., was not included in both systematic
reviews stated above. In that trial, 12 subjects
with metabolic syndrome and 12 healthy controls
received a single dose of 50 mL VOO from two olive
varieties: coratina, providing 491 ppm of phenolic
compounds, and peranzana, which had 270 ppm of
phenolics. The most interesting result found in this
study was the modification of the transcriptome
of peripheral blood mononuclear cells, switching
them to a less deleterious inflammatory phenotype. However, no relevant changes in the components of
the metabolic syndrome were observed. 4.
VOO
PHENOLIC
COMPOUNDS
AND
TYPE 2 DIABETES MELLITUS/METABOLIC
SYNDROME While
the hypothesis is supported by hundreds of in vitro
and animal studies, it does not explain why human
trials with some antioxidants, such as vitamin E,
did not provide sufficient convincing evidence
for cardiovascular prevention (Sesso et al., 2008; The systematic review by Saibandith et al.,
(2017) did assess the effects of VOO phenolics on
components of the metabolic syndrome (i.e. glucose
levels, blood pressure, central obesity, triglycerides
and HDL-cholesterol). They summarized the current
knowledge obtained from 18 clinical trials that were Grasas y Aceites 72 (2), April-June 2021, e404. ISSN-L: 0017-3495. https://doi.org/10.3989/gya.0217201 Effects of virgin olive oil phenolic compounds on health: solid evidence or just another fiasco? • 5 not specifically aimed at this syndrome. Saibandith and
collaborators confirmed the effects of VOO phenolics
on systolic blood pressure observed in previous
systematic reviews and meta-analyses, but they did not
draw significant outcomes on obesity, triglycerides or
HDL. Still, some reductions in plasma glucose and in
biomarkers associated with glucose homeostasis were
reported, but only in three of those trials. Surprisingly,
the authors did not include the RCT by Venturini et
al., (2015) that had been published a couple of years
before, and therefore, they did not analyzed any
RCTs involving patients with metabolic syndrome. The systematic review could only conclude that there
was good evidence showing that, when consumed at
appropriate doses, VOO phenolics may reduce blood
pressure in hypertensive subjects and improve plasma
glucose in pre-diabetic individuals. contribute to the development of several clinical
entities, including T2DM and Alzheimer’s disease. The close association between cerebral insulin
resistance and Alzheimer’s Disease brought some
authors to propose the name “type 3 diabetes” for
this illness (de la Monte and Wands, 2008). For the moment, there are no RCTs available
in which olive oil phenolics have been tested on
Alzheimer’s disease or other neurodegenerative
disorders. Therefore, no systematic reviews or meta-
analysis of RCT have been performed so far. All
available information refers to observational studies
and those carried out in animal models, which, as
mentioned above, provide a low level of scientific
evidence. Regarding the potential neuroprotective effects
of olive phenolics, it has been reported that dietary
supplementation with oleuropein aglycone reduced
the amount of amyloid-beta oligomers in the brain of
Alzheimer’s model mice (Luccarini et al., 2015), at
the same time that they significantle improved their
cognitive functions, presenting greater learning and
memory capacities (Pantano et al., 2017). 5.
VOO
PHENOLIC
COMPOUNDS
AND
ALZHEIMER’S DISEASE AD is pathologically characterized by substantial
neuronal and synaptic losses, and decreased
cognitive abilities, which are associated with
cerebral deposits of amyloid-beta-enriched plaques
and neurofibrillary aggregates of the Tau protein,
as well as with chronic inflammation and oxidative
stress (Rosales-Corral et al., 2015) elicited by the
pathological activation of glial cells (Scimemi et al.,
2013). Brain has revealed nowadays as an insulin-
sensitive organ, where the hormone regulates
important physiological processes, such as nutrient
homeostasis, reproduction, cognition, and memory,
and also exerts neurotrophic, neuromodulatory,
and neuroprotective effects (Blazquez et al., 2014). Disturbances in insulin signaling in the brain may Oleocanthal has also been associated with a
reduction in amyloid-beta oligomers, as well as an
attenuation of astrocytes activation and a reduction
in systemic inflammation in neurons and astrocytes
cells lines (Batarseh et al., 2017). Likewise,
oleocanthal has demonstrated that it may modulate
the astrocyte activation in TgSwDI mice, a model of
Alzheimer’s disease (Qosa et al., 2015). Grasas y Aceites 72 (2), April-June 2021, e404. ISSN-L: 0017-3495. https://doi.org/10.3989/gya.0217201 8. CONCLUSIONS The benefits of VOO phenolic compounds
on health have been extensively investigated,
and recent studies support the belief that these
components may play a key role in the amelioration
of pathophysiological conditions. In particular,
studies on in vitro systems and animal models have
shown that oleuropein derivatives, hydroxytyrosol
and oleocanthal exert potent pharmacological
activities on markers of cancer, atherosclerosis
and metabolic diseases. More specifically, on
those associated with inflammatory processes and
oxidative stress. However, meta-analyses of RCT,
from which the highest level of scientific evidence
should be obtained, do not support those effects on
humans, except for some markers of cardiovascular
risk. In fact, the strongest piece of evidence available
has been found for the ability of VOO phenolic
compounds to protect lipoproteins from oxidation
and to reduce systolic blood pressure in hypertensive
individuals (Table 1). Unfortunately, there is not enough high-level
evidence at the moment to confirm that the intake
of phenolic compounds isolated or as components
of the VOO can be healthy (Table 2). No RCT have
been carried out to assess the direct effect of VOO
phenolics on diabetes or neurodegenerative diseases
and very scarce information can be collected from the
ones that have focused on osteoarthritis and cancer. Therefore, it is still necessary to develop double-bind
RCT in extensive and diverse population groups,
with different disorders and with doses of phenols
adjusted to usual VOO consumptions, in order to
provide a greater degree of scientific evidence before
nutritional recommendations may be given to the
general population. 7.
VOO
PHENOLIC
COMPOUNDS
AND
ARTHRITIS There is currently only one RCT evaluating the
effect of VOO phenolic compounds on arthritis
(Takeda et al., 2013). This trial was aimed at
determining whether hydroxytyrosol intake could
reduce knee pain in individuals with gonarthrosis
(n=25). An extract of Olea europaea containing
approximately 22% of hydroxytyrosol, at a dose
of 11 mg/day for 4 weeks, was administrated to the
participants. The conclusion of the trial was that 6.
VOO
PHENOLIC
COMPOUNDS
AND
CANCER These trials only reported determinations
related to the oxidation of nucleic acids, in
particular the concentrations of 8-oxo-7,8-dihydro-
2’-deoxyguanosine, which is a marker of DNA
oxidation. Between 10 and 182 subjects participated
in these RCT, and the phenolic doses from VOO
ranged from 10 to 592 mg/kg. Three of the five
human trials showed that VOO phenolics reduced
the 8-oxo-7,8-dihydro-2’-deoxyguanosine levels in
urine, mitochondrial DNA of mononuclear cells and
lymphocyte DNA. The other two trials failed to find
a protective effect on DNA oxidation. Four of these
studies were conducted by the same research group,
and had similar experimental designs (Weinbrenner
et al., 2004; Hillestrøm et al., 2006; Machowetz
et al., 2007; Romeu et al., 2016). Therefore, there
is very little evidence relating the intake of VOO
phenolic compounds with cancer. In fact, the authors
suggested that further investigations are necessary
to clarify the real chemopreventive potential of
these compounds and that intervention studies on
populations at high cancer risk are needed. 6.
VOO
PHENOLIC
COMPOUNDS
AND
CANCER Only one systematic review about the effects
of VOO phenolic compounds on cancer has Grasas y Aceites 72 (2), April-June 2021, e404. ISSN-L: 0017-3495. https://doi.org/10.3989/gya.0217201 6 • J.M. Castellano and J.S. Perona the subjects informed a reduction in knee pain, as
compared to the administration of a placebo. been published, which implies that there are no
meta-analyses available and, in consequence,
the highest level of scientific evidence cannot be
reached. In their review, Fabiani et al., (2016)
included 16 animal studies and 5 RCTs. Most
of the animal studies confirmed the ability of
secoiridoid compounds to inhibit carcinogenesis at
both initiation and promotion/progression phases. However, all human intervention trials included in
this review only investigated the effects of VOO
phenolics on DNA damage and did not evaluate their
effect on the incidence or development of any kind
of cancer. These trials only reported determinations
related to the oxidation of nucleic acids, in
particular the concentrations of 8-oxo-7,8-dihydro-
2’-deoxyguanosine, which is a marker of DNA
oxidation. Between 10 and 182 subjects participated
in these RCT, and the phenolic doses from VOO
ranged from 10 to 592 mg/kg. Three of the five
human trials showed that VOO phenolics reduced
the 8-oxo-7,8-dihydro-2’-deoxyguanosine levels in
urine, mitochondrial DNA of mononuclear cells and
lymphocyte DNA. The other two trials failed to find
a protective effect on DNA oxidation. Four of these
studies were conducted by the same research group,
and had similar experimental designs (Weinbrenner
et al., 2004; Hillestrøm et al., 2006; Machowetz
et al., 2007; Romeu et al., 2016). Therefore, there
is very little evidence relating the intake of VOO
phenolic compounds with cancer. In fact, the authors
suggested that further investigations are necessary
to clarify the real chemopreventive potential of
these compounds and that intervention studies on
populations at high cancer risk are needed. been published, which implies that there are no
meta-analyses available and, in consequence,
the highest level of scientific evidence cannot be
reached. In their review, Fabiani et al., (2016)
included 16 animal studies and 5 RCTs. Most
of the animal studies confirmed the ability of
secoiridoid compounds to inhibit carcinogenesis at
both initiation and promotion/progression phases. However, all human intervention trials included in
this review only investigated the effects of VOO
phenolics on DNA damage and did not evaluate their
effect on the incidence or development of any kind
of cancer. Grasas y Aceites 72 (2), April-June 2021, e404. ISSN-L: 0017-3495. https://doi.org/10.3989/gya.0217201 ACKNOWLEDGMENTS This
review
was
presented
as
an
oral
communication to the VI Conference of the Spanish
Society of Horticultural Sciences “Phenols in olives
and olive oil: origin and evolution”, which was held
in Madrid on the 26-27th June, 2019. Grasas y Aceites 72 (2), April-June 2021, e404. ISSN-L: 0017-3495. https://doi.org/10.3989/gya.0217201 Effects of virgin olive oil phenolic compounds on health: solid evidence or just another fiasco? • 7 Table 1. Availability of studies and degree of evidence for the effect of virgin olive oil phenolics intake on different disorders according
to evidence-based medicine. Disorder
Meta-Analyses
Systematic reviews
RCT
In vivo (animals)
In vitro (cells)
Degree of evidence
Cardiovascular disease
Yes
(1)
Yes
(2)
Yes
(13)
Yes
Yes
Medium
(High por BP and ox
LDL)
Cancer
No
Yes
(1)
Yes
(5)
Yes
Yes
Low
Alzheimer’s disease
No
No
No
Yes
Yes
Very low
Arthritis
No
No
Yes
(1)
Yes
Yes
Low
Diabetes mellitus
No
No
No
Yes
Yes
Low
Metabolic syndrome
No
Yes
(2)
Yes
(2)
Yes
Yes
Low
(High for BP)
aNumbers in parentheses refer to the published number of studies of that kind. RCT, randomized controlled trial; BP, blood pressure; LDL,
low-density lipoprotein. Table 1. Availability of studies and degree of evidence for the effect of virgin olive oil phenolics intake on different disorders according
to evidence-based medicine. aNumbers in parentheses refer to the published number of studies of that kind. RCT, randomized controlled trial; BP, blood pressure; LDL,
low-density lipoprotein. Table 2. Summary of the studies cited in the text. Table 2. Summary of the studies cited in the text. ACKNOWLEDGMENTS Disorder
Study design
n
Intervention
Dose (day)/time
Time
Main markers
References
CVD
RCT
22
2 olive oils
(VOO vs ROO)
40 mL
7 weeks
TXB2, isopros
tanes
Visioli et al.,
2005
RCT
200
3 olive oils
(different PC)
25 mL
3 weeks
TG, HDL, oxLDL
Covas et al.,
2006
RCT
12
EVOO, OO and
CO
50 mL
Postprandial
TXB2, LTB4
Bogani et al.,
2007
RCT
24
2 olive oils
(different PC)
30 mg
8 weeks
BP, CRP, ox-LDL
Moreno-Luna
et al., 2012
Systematic
review
6 RCT
Olive oils (di
fferent PC)
Various
Single-8
weeks
Lipids, oxidation,
inflammation
Bahramsoltani
et al., 2019
Systematic
review
13 RCT
Olive oils (di
fferent PC)
Various
3-12 weeks
TC, LDL, HDL,
TG, ox-LDL, BP
Schwingshackl
et al., 2019
Meta-analy
sis
8 RCT
Olive oils (di
fferent PC)
Various
3-12 weeks
TC, LDL, HDL,
TG, ox-LDL, BP
Hohmann et
al., 2015
Cancer
RCT
12
3 olive oils
(different PC)
25 mL
4 days
8-OHdG
Weinbrenner et
al., 2004
RCT
28
3 olive oils
(different PC)
25 mL
3 weeks
Etheno–DNA
adducts
Hillestrøm et
al., 2006
RCT
58
3 olive oils
(different PC)
25 mL
3 weeks
8-Oxo-guanine
Machowetz et
al., 2007
RCT
33
2 olive oils
(different PC)
25 mL
3 weeks
8-OHdG
Romeu et al.,
2008
Systematic
review
5 RCT
2-3 olive oils
(different PC)
25 mL. 50 g
4 days – 8
weeks
8-OHdG
Fabiani et al.,
2016
Alzheimer’s
Astrocyte
cell line
N/A
Oleocanthal
5 μM
3-7 days
GLT1, GLUT1,
IL-6
Batarseh et al.,
2017
Murine
model (Tg
CRND8)
6/group
Oleuropein
aglycone
50 mg/kg of diet
8 weeks
Aß42, pE3-Aß
aggregation
Luccarini et
al., 2015
Murine
model
(TgSwDI)
6/group
Oleocanthal
6 mg/kg injection
4 weeks
Aß, IL-1ß
Qosa et al.,
2015 8 • J.M. Castellano and J.S. ACKNOWLEDGMENTS bReview focused on phenolics from different sources. 1 RCT from VOO. C Review focused on MetS components, no RCT with MetS diagnosed subject Phytochemistry: ibuprofen-like activity in extra-
virgin olive oil. Nature 437, 45-6. ACKNOWLEDGMENTS Perona Disorder
Study design
n
Intervention
Dose (day)/time
Time
Main markers
References
Rat model
7/group
Oleuropein
10-20 mg/kg diet
10 days
SOD, Catalase,
NO, MDA
Pourkhodadad
et al., 2016
Murine
model (APP/
PS1)
9/group
Hydroxytyrosol
5 mg/kg gavage
6 months
Cognitive, SOD,
inflammation
Peng et al.,
2016
Murine
model (Tg
CRND8)
6/group
Oleuropein
aglycone
12.5 mg/kg of diet
8 weeks
Aß42 , pE3-Aß
aggregation
Pantano et al.,
2017
Arthritis
RCT
25
Hydroxytyrosol
11 mg/day
4 weeks
Knee pain
Takeda et al.,
2013
MetS
RCT
102
Usual diet,
EVOO, fish oil
10 mL/day
12 weeks
TRAP, uric acid
Venturini et al.,
2015
RCT
24
EVOO two
varieties
50 mL
Postprandial
PBMC transcrip
tome
D’Amore et
al., 2016
Systematic
review
1 RCTb
Usual diet,
EVOO, fish oil
10 mL/day
12 weeks
TRAP, uric acid
Chiva-Blanch
and Badimon,
2017
Systematic
review
18 RCTc
EVOO, olive
leaf extract
Various
1 week -1
year
BP, HbA1C, Glu
cose insulin, WC
Saibandith,
2017
a8-OHdG, 8-hydroxy-deoxyguanosine; BP, blood pressure; CO, corn oil; CRP, C-reactive protein; EVOO, extra-virgin olive oil; HDL,
high-density lipoprotein; LDL, low-density lipoprotein; LTB4, leukotriene B4; OO, olive oil; ox-LDL, oxidized LDL; PC, phenolic con
tent; RCT, randomized controlled trial; ROO, refined olive oil; TG, triglycerides; TXB2, thromboxane B2;
aAß, amiloid beta; EVOO, extra-virgin olive oil; GLT1, glutamine transporter 1; GLUT1, glucose transporter 1; HbA1C, glycosylated a8-OHdG, 8-hydroxy-deoxyguanosine; BP, blood pressure; CO, corn oil; CRP, C-reactive protein; EVOO, extra-virgin olive oil; HDL,
high-density lipoprotein; LDL, low-density lipoprotein; LTB4, leukotriene B4; OO, olive oil; ox-LDL, oxidized LDL; PC, phenolic con
tent; RCT, randomized controlled trial; ROO, refined olive oil; TG, triglycerides; TXB2, thromboxane B2; aAß, amiloid beta; EVOO, extra-virgin olive oil; GLT1, glutamine transporter 1; GLUT1, glucose transporter 1; HbA1C, glycosylated
hemoglobin; IL-1ß, interleukin 1 beta; IL-6, interleukin-6; MDA, malodialdehyde; MetS, metabolic syndrome; N/A, not applicable; NO,
nitric oxide; PBMC, Peripheral blood mononuclear cellRCT, randomized controlled trial; SOD, superoxide dismutase; TRAP, peroxyl
radical-trapping antioxidant potential; WC, waist circumference. bReview focused on phenolics from different sources. 1 RCT from VOO. C Review focused on MetS components, no RCT with MetS diagnosed subject aAß, amiloid beta; EVOO, extra-virgin olive oil; GLT1, glutamine transporter 1; GLUT1, glucose transporter 1; HbA1C, glycosylated
hemoglobin; IL-1ß, interleukin 1 beta; IL-6, interleukin-6; MDA, malodialdehyde; MetS, metabolic syndrome; N/A, not applicable; NO,
nitric oxide; PBMC, Peripheral blood mononuclear cellRCT, randomized controlled trial; SOD, superoxide dismutase; TRAP, peroxyl
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https://figshare.com/articles/journal_contribution/International_relevance_of_two_measures_of_awareness_of_age-related_change_AARC_/22995029/1/files/40744988.pdf
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International relevance of Two Measures of Awareness of Age-Related Change (AARC)
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Sabatini et al. BMC Geriatrics (2020) 20:359
https://doi.org/10.1186/s12877-020-01767-6 Sabatini et al. BMC Geriatrics (2020) 20:359
https://doi.org/10.1186/s12877-020-01767-6 Open Access © The Author(s). 2020 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://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: A questionnaire assessing awareness of positive and negative age-related changes (AARC gains and
losses) was developed in the US and Germany. We validated the short form of the measure (AARC-10 SF) and the
cognitive functioning subscale from the 50-item version of the AARC (AARC-50) questionnaire in the UK population
aged 50 and over. Methods: Data from 9410 participants (Mean (SD) age = 65.9 (7.1)) in the PROTECT cohort were used to explore
and confirm the psychometric properties of the AARC measures including: validity of the factor structure; reliability;
measurement invariance across men and women, individuals with and without a university degree, and in middle
age, early old age, and advanced old age; and convergent validity with measures of self-perception of aging and
mental, physical, and cognitive health. We explored the relationship between demographic variables (age, sex,
marital status, employment, and university education) and AARC. Results: We confirmed the two-factor structure (gains and losses) of the AARC-10 SF and the AARC-50 cognitive
functioning subscale. Both scales showed good reliability and good convergent validity for AARC losses, but weak
convergent validity for AARC gains. For both scales metric invariance was held for the two subgroups defined by
education level and age. For the AARC-50 subscale, but not for the AARC-10 SF, strong invariance was also held for
the two subgroups defined by sex. Age, sex, marital status, employment, and university education predicted AARC
gains and losses. Conclusions: The AARC-10 SF and AARC-50 cognitive functioning subscale identify UK individuals who perceive
age-related changes in their mental, physical, and cognitive health. Keywords: Subjective aging, Self-perceptions of aging, AARC-10 SF, Cognitive functioning Keywords: Subjective aging, Self-perceptions of aging, AARC-10 SF, Cognitive functioning her behavior, level of performance, or way of experien-
cing life has changed as a consequence of having grown
older” [5], p. 342. AARC reflects the observation that in-
dividuals’ experiences of aging may vary across five life
and behavioral domains including health and physical
functioning, cognitive functioning, interpersonal rela-
tionships, socio-cognitive and socio-emotional function-
ing,
and
lifestyle/engagement. As
the
association
between cognitive complaints and cognitive performance
is well-reported in the empirical literature (e.g., [6, 7]),
among the five AARC behavioral domains, the cognitive International relevance of two measures of
awareness of age-related change (AARC) Serena Sabatini1*
, Obioha C. Ukoumunne2, Clive Ballard1, Allyson Brothers3, Roman Kaspar4, Rachel Collins1,
Sarang Kim5, Anne Corbett1, Dag Aarsland6, Adam Hampshire7, Helen Brooker1 and Linda Clare1 Background Awareness of age-related change (AARC) is a useful
concept that predicts a variety of health-related out-
comes such as depression and psychological and physical
well-being [1, 2] and could be used to motivate engage-
ment in healthy behaviors such as physical activity [3, 4]. AARC refers to “a person’s state of awareness that his or * Correspondence: ss956@exeter.ac.uk
1College of Medicine and Health, REACH, University of Exeter, South Cloisters,
St Luke’s Campus, Exeter EX12LU, UK
Full list of author information is available at the end of the article * Correspondence: ss956@exeter.ac.uk
1College of Medicine and Health, REACH, University of Exeter, South Cloisters,
St Luke’s Campus, Exeter EX12LU, UK
Full list of author information is available at the end of the article The 10-item version of the AARC questionnaire is
made up of selected items from the AARC 50-item ver-
sion. Reliability of the AARC-10 SF is good among US
and German residents aged 40 and over (Cronbach’s α
coefficients ranging from .49 to .75 across subscales) [1]. A 20-item modified version of the AARC questionnaire
adapted for daily use also exists [9]. The 20 items have
been selected from the 50-item version of the AARC
questionnaire. However, each item stem, instead of ask-
ing participants to reflect on their increasing age (“With
my increasing age…”), invites participants to reflect on
their awareness of aging in that specific day (“With my
awareness of aging today…”). Psychometric properties of
the AARC 20-item version have never been explored. Moreover, as the AARC-10 SF covers awareness of
changes in several behaviors and life domains including
socio-emotional, physical, and cognitive functioning, in-
vestigating the associations of the AARC-10 SF with in-
dicators
of
mental,
physical,
and
cognitive
health
provides information about the construct validity of the
AARC-10 SF. As part of the US and German validation
of the AARC-10 SF it has been found that AARC is as-
sociated with indicators of mental and physical health
including psychological well-being, satisfaction with life,
depressive symptoms, and functional and perceived
health [1]. However, despite age playing a role in levels
of AARC [12, 25], construct validity of the AARC-10 SF
has not been explored in individuals younger than 70
years. Moreover, research shows that higher levels of
AARC losses (measured with a 20-item version of the
AARC questionnaire) are associated with more negative
affect [9, 25] which is a key component of anxiety. As
common difficulties among older individuals, such as
poverty and diminished life expectations, are risk factors
for anxiety [26], the association between anxiety and
AARC should also be considered when exploring con-
vergent validity of the AARC-10 SF. Finally, construct
validity of the AARC-50 cognitive functioning subscale
[8] in relation to objective or subjective cognitive assess-
ments has never been explored. AARC may be associated with cognitive functioning
(e.g., [10]). Page 2 of 20 Sabatini et al. BMC Geriatrics (2020) 20:359 Sabatini et al. BMC Geriatrics (2020) 20:359 Page 2 of 20 functioning domain is potentially useful for detecting
early stages of cognitive decline. AARC captures aware-
ness of both positive (AARC gains) and negative (AARC
losses)
age-related
changes
and
acknowledges
that
AARC gains and losses can coexist, even in the same be-
havioral domain [5]. participants [12]. Studying individuals aged 50 years and
above is considered appropriate as people in this age-
group are old enough to be likely to experience AARC. Individuals aged 50 years have previously reported ex-
periencing many age-related changes (e.g., [13–15]) and
shown concern about their physical health [16]. A questionnaire assessing AARC exists in three pub-
lished versions of differing length and across two lan-
guages (English and German). The 50-item version [8]
and short 10-item version (AARC-10 SF; [1]) are avail-
able in English. In the full 50-item version, out of the 50
items, half represent perceived gains and half perceived
losses. There are five gain- and loss-related items repre-
senting each of the five theorized domains. The AARC-
50 questionnaire has been shown to have good reliability
in a sample of US residents aged between 42 and 98
years old [8], with Cronbach’s alpha (α) coefficients ran-
ging from .73 to .89 across all ten subscales. Amongst psychometric properties, it is important to
test measurement invariance to explore whether the
AARC concept is interpreted consistently in the same
way across different population groups (for example, de-
fined by sex, level of education, or age) [12, 17]. Esti-
mated reliable comparisons of AARC scores among
groups can, therefore, be potentially calculated [18–21]. Regarding the AARC questionnaires, measurement in-
variance has so far been tested only for the AARC-10 SF
in relation to different age groups [1]. Other measures of the subjective experience of aging
such as felt age, which reflects how old individuals feel
they are [22, 23], and attitudes towards own aging
(ATOA), which capture individuals’ evaluations of the
changes taking place in their lives as they age [24], are
suitable measures to capture the way in which individ-
uals experience aging, albeit in a more holistic manner
compared to AARC [3]. These constructs, therefore,
were used as part of the exploration of convergent valid-
ity of existing AARC questionnaires. Among these, 9410 participants completed the
AARC questionnaires and were therefore included in the
present study (mean (SD; range) age: 65.9 (7.1; 51–95)
years). Only 0.4% of participants reported having been
diagnosed with mild cognitive impairment. We esti-
mated that a further 1.2% of participants had mild cogni-
tive impairment (as they scored 1.5 SDs below the mean
study sample score in two or more cognitive tasks). Those participants that we identified as having mild cog-
nitive impairment were kept in the analyses. However,
participants with higher levels of AARC losses on the
AARC-10 SF and on the AARC-50 cognitive functioning
subscale had poorer scores on the four objective cogni-
tive tasks, indicating that participants were aware of
their cognitive abilities (see supplementary material
Table 1) and hence their answers to the AARC-10 SF
and the AARC-50 cognitive functioning subscale can be
deemed accurate. y
p
p
g g
This study aims to: (a) confirm the two-factor struc-
ture (one factor for each of gains and losses) and in-
ternal consistency of the AARC-10 SF [1] and the
AARC-50 cognitive functioning subscale [8]; (b) explore
measurement invariance for the AARC-10 SF and for
the AARC-50 cognitive functioning subscale among sub-
groups defined by sex, education level, and age; (c) ex-
plore construct validity of the AARC-10 SF and the
AARC-50 cognitive functioning subscale by quantifying
the associations of the AARC-10 SF with assessments of
subjective aging experiences, physical, mental, and cog-
nitive health and of the AARC-50 cognitive functioning
subscale with assessments of subjective aging experi-
ences and cognitive health; and (d) explore whether
demographic variables predict scores on the AARC-10
SF and AARC-50 cognitive subscale gains and losses. The majority of study participants was of white ethnicity
(98.5% of participants), married (79.1% of participants),
completed a university education (75.8% of participants)
and not retired (42.6% of participants). Demographic char-
acteristics for the study sample are reported in Table 1. Means and standard deviations stratified by age, sex, and
education level for AARC gains and losses assessed both
with the AARC-10 SF and the AARC-50 cognitive func-
tioning subscale are reported in Tables 2 and 3. A high proportion of participants perceived their health
as good (54.1%) or excellent (30.8%). On average partici-
pants did not report functional difficulties (IADL mean
(SD) score = 0.16 (0.77)). As the AARC-10 SF includes only two items
assessing AARC gains and AARC losses respectively in
the cognitive domain, the full 10-item subscale assessing
AARC gains taken from the AARC 50-item version of
the questionnaire makes it possible to more accurately
explore the potential associations of AARC in the cogni-
tive domain with other indicators of cognitive function-
ing. The AARC-10 SF and the AARC-50 cognitive
functioning subscale may be particularly important when
thinking about new ways of preventing poor mental and
physical health and cognitive decline. The AARC-10 SF [1] and the AARC-50 cognitive
functioning subscale [8] are suitable to be used in long
surveys or as screening tools to identify those people at
greater risk of poor mental and physical health and/or
cognitive decline [1, 10]. In order to use these measures
in the UK, due to potential cross-cultural differences in
AARC, it is important to first explore their psychometric
properties in the UK population aged 50 and over [11]. German participants, for example, report fewer AARC
gains,
assessed
with
the
AARC-10
SF,
than
US Existing research in the US and Germany, conducted
by using the 50-item and 20-item versions of the AARC Page 3 of 20 Sabatini et al. BMC Geriatrics (2020) 20:359 ethics committee at the University of Exeter, School of
Psychology (Application ID: eCLESPsy000603 v1.0). ethics committee at the University of Exeter, School of
Psychology (Application ID: eCLESPsy000603 v1.0). questionnaire, suggests that on average individuals who
are older, less well-educated, and/or female have higher
levels of both AARC gains and AARC losses [12, 17]. These findings are in line with research conducted on
other constructs that, similarly to AARC, also capture
individuals’ self-perceptions of aging [23, 27, 28]. Indi-
viduals who report a higher socioeconomic status tend to
experience more AARC gains and fewer AARC losses
(assessed with the 20-item version of the AARC question-
naire) than those with a lower socioeconomic status [17]. However, the role of demographic variables in the UK
population is unexplored. Moreover, other demographic
variables such as marital status and employment status
have never been explored in relation to AARC gains and
losses even though existing literature suggests that they
may influence individuals’ perceptions of aging [29]. y
gy
pp
y
Between 1st January 2019 and 31st March 2019, 14,
797 participants took part in the PROTECT annual as-
sessment. Study design and participants The study was based on analyses of cross-sectional data
collected through the ongoing PROTECT [30] study in
2019. PROTECT is a 25-year longitudinal study launched
in 2014 that assesses participants every year on measures
of physical, mental, and cognitive health, lifestyle, and per-
ceptions of aging through an online platform. Compared to those who did not complete the AARC
questionnaires (N = 5387), the study sample included a
larger proportion of women (79.9% versus 71.3%) and
participants who were better educated (75.8% versus
70.8%), and a lower proportion of individuals who were
employed (42.6% versus 54.7%). Individuals are eligible to participate in the PROTECT
study if they are UK residents, English speakers, aged 50
years and over, have access to a computer and internet,
and do not have a clinical diagnosis of dementia at the
point
of
recruitment. Participants
were
recruited
through national publicity and via existing cohorts of
older adults. Potential participants enrolled through the
PROTECT study website, downloaded the study infor-
mation sheet, and provided consent online. Participants had minimal levels
of current depressive (mean (SD) = 11.5 (3.0)) and anxiety
symptoms (mean (SD) = 9.3 (8.5)), and low levels of both
lifetime depressive symptoms (mean (SD) = 2.7 (3.3)) and
lifetime anxiety symptoms (mean (SD) = 1.0 (2.1)). N = 9410 University education was operationalized as a dichotomous variable. No
university education included those participants that concluded secondary
education or post-secondary education. University education included those
participants that concluded vocational qualification, undergraduate degree,
post-graduate degree, or doctorate. Secondary education = GCSE or O-levels. Post-secondary education = College, A-levels, NVQ3 or below, or similar. Vocational qualification = Diploma, certificate, BTEC, NVQ 4 and above, or
similar. Undergraduate degree = BA or BSc, or similar. Post-graduate degree =
MA, MSc, or similar. Doctorate = PhD Total sample size (N) = 9410 Demographic variables Demographic variables
Participants provided demographic information through
the PROTECT platform at baseline through an online
assessment adapted from Office of National Statistics
measures, which included data on age, sex, ethnic origin,
marital status, employment, and university education. Ethnicity included the following categories: white, mixed
(included white and black Carribean, white and black
African, white and Asian, any other mixed multiple eth-
nic background), Asian, black, or other ethnic groups. Marital status was used as a dichotomous variable (indi-
viduals who were married, in a civil partnership, or co-
habiting were grouped together versus individuals who
were unmarried, divorced, separated, or widowed). Em-
ployment status was used as a dichotomous variable
(employed versus not employed). University education
was used as a dichotomous variable (university educa-
tion versus no university education). Individuals without
a university education were those participants that had
completed secondary education (GCSE/O levels) or
post-secondary education (college, A-levels, NVQ3, or
below). Individuals with a university education were
those participants that had completed vocational qualifi-
cations (diploma, certificate, BTEC, NVQ4, and above),
undergraduate degrees (e.g., BA, BSc), post-graduate de-
grees (e.g., MA, MSc), or doctorates (PhD). Instruments Measures assessing felt age, ATOA, mental and physical
health, and objective cognitive functioning were used to
explore construct validity for the AARC-10 SF. Measures
assessing felt age, ATOA, and objective, self-reported,
and informant-reported assessments of cognitive func-
tioning were used to explore construct validity for the The PROTECT study has ethical approval from the
London Bridge NHS Research Ethics Committee and
Health Research Authority (Ref: 13/LO/1578). Ethical
approval for the data analyses was sought through the Page 4 of 20 Page 4 of 20 Page 4 of 20 Sabatini et al. BMC Geriatrics (2020) 20:359 Sabatini et al. BMC Geriatrics Table 1 Demographic characteristics of the study sample (N =
9410)
Characteristics
Statistic
Age (years), M (SD)
65.9 (7.1)
Range
51–95
Sex (Women %)
79.9
Ethnicity (%)
White
98.5
Mixed
0.5
Asian
0.6
Black
0.1
Other ethnic groups
0.3
Marital status (%)
Married/ civil partnership/ co-habiting
79.1
Widowed/ separated/ divorced/ single
20.9
University education (Yes %)
75.8
Current employment (Yes %)
42.6
N = 9410 Awareness of age-related change (AARC)
AARC-10 SF AARC-50 cognitive functioning subscale. Demographic
variables (age, sex, marital status, employment, and uni-
versity education) were assessed to explore their rela-
tionships with levels of AARC gains and losses assessed
both with the AARC-10 SF and with the AARC-50 cog-
nitive functioning subscale. The AARC-10 SF [1] is a brief tool for capturing per-
ceived age-related gains (AARC gains) and losses (AARC
losses). It contains ten items, five assessing AARC gains
and five assessing AARC losses. Each of these five items
assesses a different AARC behavioral domain (health
and
physical
functioning,
cognitive
functioning, AARC-50 cognitive functioning subscale. Demographic
variables (age, sex, marital status, employment, and uni-
versity education) were assessed to explore their rela-
tionships with levels of AARC gains and losses assessed
both with the AARC-10 SF and with the AARC-50 cog-
nitive functioning subscale. Table 2 Levels of AARC gains and losses stratified by age
Age
Class 1:
50 to 65
(N = 4929)
Class 2:
66 to 75
(N = 3758)
Class 3:
76 and over
(N = 723)
Class 1 vs 2
Class 1 vs 3
Class 2 vs 3
F statistic
(df)
p-value
Mean (SD)
Mean (SD)
Mean (SD)
Mean
difference
[95% CI]
Mean
difference
[95% CI]
Mean
difference
[95% CI]
AARC-10 SF
gains
18.1 (3.9)
17.8 (3.9)
17.4 (3.7)
−0.3
[−0.5, 0.1] −0.7
[−1.0, −0.3] −0.4
[−0.8, −0.1] 12.1 (2)
<.0001
AARC-10 SF
losses
9.4 (3.2)
10.2 (3.1)
12.1 (3.8)
0.8
[0.6, 0.9]
2.7
[2.4, 3.0]
1.9
[1.7, 2.2]
243.4 (2)
<.0001
AARC-50
cognitive
functioning
gains
14.3 (4.4)
13.6 (4.4)
13.3 (4.4)
−0.7
[−0.9, 0.5] −1.0
[−1.4, −0.6] −0.3
[−0.7, 0.1]
34.5 (2)
<.0001
AARC-50
cognitive
functioning
losses
9.8 (3.7)
10.4 (3.5)
12.0 (4.1)
0.6
[0.4, 0.8]
2.2
[1.9, 2.5]
1.6
[1.2, 1.9]
123.0 (2)
<.0001
Total sample size (N) = 9410 Table 2 Levels of AARC gains and losses stratified by age Total sample size (N) = 9410 Page 5 of 20 Sabatini et al. Awareness of age-related change (AARC)
AARC-10 SF BMC Geriatrics (2020) 20:359 Table 3 Levels of AARC gains and losses stratified by sex and educational level Table 3 Levels of AARC gains and losses stratified by sex and educational level
Sex Table 3 Levels of AARC gains and losses stratified by sex and educational level
Sex
Women (N = 7334)
Men (N = 2076)
Mean (SD)
Mean (SD)
t-statistics (df)
p-value
AARC-10 SF gains
18.2 (3.8)
16.9 (4.0)
−14.2203 (9408)
<.0001
AARC-10 SF losses
9.7 (3.2)
10.5 (3.5)
9.2481 (9408)
<.0001
AARC-50 cognitive functioning gains
14.2 (4.4)
13.0 (4.4)
−11.6155 (9408)
<.0001
AARC-50 cognitive functioning losses
10.0 (3.6)
10.9 (3.9)
9.6811 (9408)
<.0001
University education
No university education (N = 2369)
Completed university education (N = 7041)
Mean (SD)
Mean (SD)
t-statistics (df)
p-value
AARC-10 SF gains
18.1 (3.9)
17.9 (3.9)
2.5758 (9408)
=.01
AARC-10 SF losses
10.2 (3.5)
9.8 (3.2)
5.7887 (9408)
<.0001
AARC-50 cognitive functioning gains
14.4 (4.5)
13.8 (4.4)
5.3472 (9408)
<.0001
AARC-50 cognitive functioning losses
10.5 (3.8)
10.1 (3.6)
6.0893 (9408)
<.0001
Total sample size (N) = 9410
University education was operationalized as a dichotomous variable. No university education included those participants that concluded secondary education or
post-secondary education. University education included those participants that concluded vocational qualification, undergraduate degree, post-graduate degree,
or doctorate. Secondary education = GCSE or O-levels. Post-secondary education = College, A-levels, NVQ3 or below, or similar. Vocational qualification = Diploma,
certificate, BTEC, NVQ 4 and above, or similar. Undergraduate degree = BA or BSc, or similar. Post-graduate degree = MA, MSc, or similar. Doctorate = PhD p
University education was operationalized as a dichotomous variable. No university education included those participants that concluded secondary education or
post-secondary education. University education included those participants that concluded vocational qualification, undergraduate degree, post-graduate degree,
or doctorate. Secondary education = GCSE or O-levels. Post-secondary education = College, A-levels, NVQ3 or below, or similar. Vocational qualification = Diploma,
certificate, BTEC, NVQ 4 and above, or similar. Undergraduate degree = BA or BSc, or similar. Post-graduate degree = MA, MSc, or similar. Doctorate = PhD higher levels of awareness of age-related change in the
cognitive domain. higher levels of awareness of age-related change in the
cognitive domain. interpersonal relationships, socio-cognitive and socio-
emotional functioning, and lifestyle/engagement). All ten
items start with the same stem “With my increasing age,
I realize that…”. AARC-50 cognitive functioning subscale AARC-50 cognitive functioning subscale
The cognitive functioning subscale of the AARC-50
questionnaire [8] includes ten items, five assessing
AARC gains and five assessing AARC losses. An ex-
ample item capturing AARC gains in the cognitive do-
main is “With my increasing age, I realize that I have
become wiser”, while an item capturing losses is “With
my increasing age, I realize that I am more forgetful”. Respondents rate how much each item applies to them
on a five-point Likert scale (1 = “not at all”, 2 = “a little
bit”, 3 = “moderately”, 4 = “quite a bit”, and 5 = “very
much”). Scores on the AARC- cognitive functioning
gains and AARC- cognitive functioning losses subscales
are obtained by summing items that fall into the respect-
ive subscales. Subscales scores range from a minimum of
five to a maximum of 25 and higher scores indicate Awareness of age-related change (AARC)
AARC-10 SF An example of an item capturing AARC
gains is “…I appreciate relationships and people much
more”, while an example of an item capturing AARC
losses is “…I have less energy”. Respondents rate how
much each item applies to them on a five-point Likert
scale (1 = “not at all”, 2 = “a little bit”, 3 = “moderately”,
4 = “quite a bit”, and 5 = “very much”). Scores can be ob-
tained for the AARC gains and AARC losses subscales
by summing items that fall into the respective scales. Scales scores range from a minimum of five to a max-
imum of 25 with higher scores indicating higher levels
of awareness of age-related change. Attitudes toward own aging (ATOA) g
g (
)
The ATOA scale is a valid and reliable five-item scale
assessing participants’ attitudes toward their own aging
taken from the Philadelphia Geriatric Center Morale
Scale [24]. For each statement respondents are asked to
make temporal comparisons about changes in energy
level, perceived usefulness, happiness, and quality of life
and to respond on a binary response set (better versus
worse, yes versus no). An example item is “Things keep
getting worse as I get older”. A proportion-based score
can be obtained by summing the participant’s item
scores and by dividing it by the number of responses,
with a score of one indicating that positive attitudes are
implied in all answers and a score of zero indicating that
a negative response is implied in all answers. Instrumental activities of daily living Lawton’s Instrumental Activities of Daily Living Scale
(IADL [44]) is a reliable instrument to assess everyday
functional status. It describes seven activities including
preparing meals, managing medications, and using the
telephone. For each activity respondents have to rate
how difficult they find performing the activity (0 = “no
difficulty”, 1 = “some difficulty”, and 2 = “great diffi-
culty”). The total score ranges from a possible 0 to 14. Perceived health We assessed perceived health with a single-item ques-
tion (taken from the SF-36 [45]) asking participants to
rate their own health on a four-point scale ranging from
excellent to poor (“excellent”, “good”, “fair”, and “poor”). Cognitive functioning – objective assessment assessing lifetime symptoms of depression and anxiety. Nine items assess depressive symptoms and eight items
assess anxiety symptoms. An example of a depressive
symptom question is “Did you lose interest in most
things?”. For each item, participants can answer “yes” if
they have the symptom or “no” if they do not have the
symptom. For both depression and anxiety a total score
can be calculated by summing the items where the par-
ticipants answer yes. For depression and anxiety the total
score can range from zero to nine and from zero to
eight, respectively. Cognitive functioning was measured with the PROTECT
Cognitive Test Battery [31–33] which includes four tests: (1)
the Grammatical Reasoning task assesses verbal reasoning
[34]; (2) the Digit Span task [35] assesses verbal working
memory; (3) the Self-ordered Search task measures spatial
working memory [36]; and (4) the Paired Associate Learning
task [37] assesses visual episodic memory. For each task a summary score can be obtained by sub-
tracting the number of errors from the number of correct
answers. Hence for each task a higher score indicates a
better performance. For digit span the summary score can
range from 0 to 20. For paired associate learning the sum-
mary score can range from 0 to 16. For verbal reasoning
the summary score is also obtained by subtracting the
number of errors from the number of correct answers, but
the score has no set upper or lower limit as the partici-
pants can attempt as many trials as they can manage
within a specific timeframe. Finally, the summary score
for the self-ordered search task can range from 0 to 20. Analyses The Patient Health Questionnaire-9 (PHQ-9 [40]) is a
valid and reliable nine-item scale capturing depressive
symptoms over the previous 2 weeks. It is based directly
on the diagnostic criteria for major depressive disorder
described in the Diagnostic and Statistical Manual
Fourth Edition (DSM IV [41]). Respondents are asked to
indicate how frequently they experience each symptom
on a four-point Likert scale (1 = “not at all”, 2 = “several
days”, 3 = “more than half the days”, and 4 = “nearly
every day”). The total score is the sum of the item scores
and can range from 9 to 36. As the validation of the AARC-10 SF [1] in US and Ger-
man samples supported a two-factor structure (one fac-
tor for each of AARC gains and AARC losses), we used
confirmatory factor analysis (CFA) to confirm this struc-
ture in the UK population. We tested whether the five
items assessing gains and the five items assessing losses
(of the AARC-10 SF) are related to the respective hy-
pothesized underlying factors of AARC gains and AARC
losses. The two factors AARC gains and AARC losses
were allowed to correlate in the CFA model. Error terms
were allowed to correlate for the pair of gains and losses
items for the same AARC behavioral domain (Fig. 1). Anxiety symptoms
h
G
l
d The Generalized Anxiety Disorder-7 (GAD-7 [43]) is a
valid and reliable seven-item measure assessing symp-
toms of generalized anxiety disorder. Respondents are
asked to indicate the frequency of occurrence of a list of
symptoms over the past 2 weeks on a four-point scale (1
= “not at all”, 2 = “several days”, 3 = “more than half the
days”, and 4 = “nearly every day”). The overall score is
the sum of the item scores and ranges from 7 to 28. Cognitive functioning - informant rating and self-rating
The Informant Questionnaire on Cognitive Decline in
the Elderly short form (IQCODE [38, 39]) was adminis-
tered to an informant close to the participant. The
IQCODE is a valid and reliable 16-item questionnaire
that asks respondents to rate the cognitive change of
someone close to them over the last 10 years. Items de-
scribe both cognitive improvement and cognitive decline
(an example item is “Remembering things that have hap-
pened recently”) and can be answered on a five-point
scale (1 = “much improved”, 2 = “a bit improved”, 3 =
“not much change”, 4 = “a bit worse”, and 5 = “much
worse”). The final score is the mean of the item scores. A parallel version of the IQCODE was administered to
the participant (IQCODE - Self [38]). Felt age Felt age was assessed with a single-item question
(adapted from the National Survey of Midlife develop-
ment in the United States; MIDUS [23]) asking partici-
pants to write the age (in years) that they feel most of
the time. A proportional discrepancy score was calcu-
lated by subtracting the participants’ felt age from their
chronological age, and by dividing this difference score
by participants’ chronological age. A positive value indi-
cates a youthful felt age, whereas a negative value indi-
cates an older felt age. Sabatini et al. BMC Geriatrics (2020) 20:359 Page 6 of 20 Page 6 of 20 Cognitive functioning – objective assessment Composite international diagnostic interview-short form CFA was also conducted to confirm the two-factor
structure of the AARC-50 cognitive functioning subscale
[8] (Fig. 1b). For both the AARC-10 SF and the AARC- The Composite International Diagnostic Interview-Short
Form (CIDI-SF [42]) is a reliable and valid measure for Sabatini et al. BMC Geriatrics (2020) 20:359 Page 7 of 20 Sabatini et al. BMC Geriatrics Fig. 1 a Two-factor model of the AARC-10 SF. Measurement model of Awareness of Age-Related Changes (AARC) for the AARC-10 SF. Fully
standardized coefficients are reported. AARC Domain abbreviations: PHY = Health and physical functioning; COG = Cognitive functioning; INT =
Interpersonal relations; SCSE = Social-cognitive and social-emotional functioning; LIFE = Lifestyle and engagement; “+” = Positive domains; “-” =
Negative domains. b Two-factor model of the AARC-50 cognitive functioning subscale. Measurement model of Awareness of Age-Related
Changes (AARC) for the AARC-50 cognitive functioning subscale. Fully standardized coefficients are reported. COG = Cognition, “+” = Positive
domains; “-” = Negative domains Fig. 1 a Two-factor model of the AARC-10 SF. Measurement model of Awareness of Age-Related Changes (AARC) for the AARC-10 SF. Fully
standardized coefficients are reported. AARC Domain abbreviations: PHY = Health and physical functioning; COG = Cognitive functioning; INT =
Interpersonal relations; SCSE = Social-cognitive and social-emotional functioning; LIFE = Lifestyle and engagement; “+” = Positive domains; “-” =
Negative domains. b Two-factor model of the AARC-50 cognitive functioning subscale. Measurement model of Awareness of Age-Related
Changes (AARC) for the AARC-50 cognitive functioning subscale. Fully standardized coefficients are reported. COG = Cognition, “+” = Positive
domains; “-” = Negative domains 50 cognitive functioning subscale, to confirm the need
for a two-factor model (above described), we also fitted
a model in which a single factor loaded on all ten items. For both the AARC-10 SF and the AARC-50 cognitive
functioning subscale, we compared goodness of fit indi-
ces (GOF) of the two-factor model with those of one-
factor model. Because the Chi-squared statistic is often
significant for well-fitting models in large samples [46] al-
ternative goodness of fit measures including the Compara-
tive Fit index (CFI), the Tucker-Lewis index (TLI), the
Root Mean Square Error of Approximation (RMSEA), and
the Standardized Root Mean Square Residual (SRMR)
were examined. Criteria for acceptable model fit were CFI
and TLI > .90, RMSEA < .08 (90% CI: between 0 and .08),
and SRMR < .06 [47]. Results Self-perceptions of aging among the study sample
On the AARC-10 SF the majority of participants re-
ported moderate (20.7%), quite a bit of (47%), or a great
deal of (27.8%) AARC gains and little (60.4%) or moder-
ate (29%) AARC losses. On the AARC-50 cognitive
functioning subscale the majority of participants re-
ported moderate (39.6%) or quite a bit (28.0%) of AARC
gains in their cognition and little (56.6%) or moderate
(28.6%) AARC losses in their cognition. Further details
about proportions of gains and losses perceived by par-
ticipants on the AARC-10 SF and the AARC-50 cogni-
tive functioning subscale are reported in Table 4. Seventeen percent of participants felt younger than their
chronological age. Participants’ mean (SD) score on the
ATOA scale was .52 (0.16) indicating that participants Composite international diagnostic interview-short form BMC Geriatrics (2020) 20:359 Sabatini et al. BMC Geriatrics (2020) 20:359 Page 8 of 20 reported positive ATOA in some items but negative
ATOA in others. health, and objective assessments of cognitive function-
ing (Grammatical Reasoning, Digit Span, Self-ordered
Search, and Paired Associate Learning). Construct valid-
ity for the AARC-50 cognitive functioning subscale was
explored by estimating correlations between the AARC-
50 cognitive functioning subscale and each of felt age,
ATOA, and objective (Grammatical Reasoning, Digit
Span, Self-ordered Search, and Paired Associate Learn-
ing), self-reported, and informant-reported assessments
of cognitive functioning. We used Pearson’s r to quantify
correlations [55]. Correlation coefficients under .10 were
considered negligible, between .10 to .29 were consid-
ered small, between .30 to .49 were considered moder-
ate, and .50 or above were considered large [56]. Psychometric properties of the AARC-10 SF and AARC-50
cognitive functioning scale
Confirmatory factor analysis Psychometric properties of the AARC-10 SF and AARC-50
cognitive functioning scale
Confirmatory factor analysis
For the AARC-10 SF, compared to a one-factor model
(RMSEA = .21; 95% CI: .00, .00; CFI = .48; TLI = .33;
SRMR = .18) the hypothesized two-factor model was a
better fit as indicated by GOF indices (RMSEA = .07;
95% CI: .07, .07; CFI = .94; TLI = .92; SRMR = .05). For the AARC-10 SF, compared to a one-factor model
(RMSEA = .21; 95% CI: .00, .00; CFI = .48; TLI = .33;
SRMR = .18) the hypothesized two-factor model was a
better fit as indicated by GOF indices (RMSEA = .07;
95% CI: .07, .07; CFI = .94; TLI = .92; SRMR = .05). Item characteristics for the ten items of the AARC-10
SF are displayed in Table 5. The associations between
factors and indicators were reasonably strong for all
items (Fig. 1). Factor loadings for the individual domain
items on the gains factor reflect greater heterogeneity of
aging experiences in the gains compared to the losses
factor. To explore whether age, sex, marital status, employ-
ment status, and university education explain variability
in levels of AARC gains and/or AARC losses, we fitted
multiple linear regression models for each of the AARC-
10 SF and the AARC-50 cognitive functioning gains and
losses. We also conducted simple regressions in which
the predictive role of each demographic variable (age,
sex, marital status, employment status, and university
education) on levels of AARC gains/losses was explored
without
controlling
for
the
predictive
role
of
the
remaining demographic variables. For the AARC-50 cognitive functioning subscale, com-
pared to a one-factor model (RMSEA = .29; 95% CI = .00,
.00; CFI = .42; TLI = .26; SRMR = .24) the hypothesized
two-factor model was a better fit as indicated by GOF
indices (RMSEA = .12; 95% CI: .12, .12; CFI = .90; TLI =
.87; SRMR = .05). We calculated modification indices for
the AARC-50 two-factor model; however modification
indices did not suggest any pattern that would have sig-
nificantly improved the model. Item characteristics for
the 10 items of the AARC-50 cognitive functioning sub-
scale are displayed in Table 5. The associations between
construct and indicators were strong for all items
(Fig. 1b). Composite international diagnostic interview-short form The CFA models were fitted using
the sem command in Stata. Analyses included only partici-
pants that provided complete data on all items. over). To explore measurement invariance, we fitted
three CFA models: (a) Model 1 placed no equality con-
straints across groups on factor loadings, item intercepts,
the error variances, the variances of the latent variables,
or the covariances of the latent variables (assumes con-
figural invariance); (b) Model 2 constrained the factor
loadings to be identical across subgroups (assumes
metric invariance); (c) Model 3 constrained the factor
loadings and item intercepts to be identical across sub-
groups (assumes strong invariance). To evaluate the fit of a model compared to a less re-
strictive one, the traditional approach involves assessing
the differences in the Chi-squared fit statistics of the two
examined CFA models by conducting likelihood ratio
tests (LRT). However, as LRTs often result in statistically
significant differences in large samples for models that
are not markedly different in fit [46] and alternative fit
indices are less sensitive to sample size [51], we explored
model differences using alternative GOF indices includ-
ing the Comparative Fit index (CFI), the Root Mean
Square Error of Approximation (RMSEA), and the Stan-
dardised Root Mean Square Residual (SRMR). We con-
cluded that a model had a worse fit than a less
constrained model when the difference in CFI (ΔCFI)
was larger than −.01 [52, 53], the difference in RMSEA
(ΔRMSEA) was larger than .015 [54], and the difference
in SRMR (ΔSRMR) was larger than .03 [54]. We used Cronbach’s alpha (α) to quantify reliability for
the gains and losses subscales of the AARC-10 SF and the
AARC-50 cognitive functioning subscale [48]. We consid-
ered α values between .65 and .95 to be satisfactory. For both the AARC-10 SF and the AARC-50 cognitive
functioning subscale, we used CFA to test measurement
invariance [18, 49, 50] between men and women, be-
tween two groups characterized by university education
(vocational qualification, undegraduate degree, post-
graduate degree, or doctorate) and no university educa-
tion (secondary or post-secondary education, and among
three age groups (middle age = 50 to 65 years; early old
age = 66 to 75 years, advanced old age = 76 years and Construct validity for the AARC-10 SF was explored
by estimating correlations between the AARC-10 SF and
each of felt age, ATOA, measures of mental and physical Sabatini et al. Reliability For the AARC-10 SF item-to-total score correlations
had values between .67 and .78; hence all items reached
satisfactory αs (Table 5). Cronbach’s α value was .77 for
the AARC-10 SF gains scale and .80 for the AARC-10
SF losses scale. For the AARC-50 cognitive functioning
subscale all item-to-total score correlations reached sat-
isfactory values, ranging between .82 and .87 (Fig. 1b). Cronbach’s α value was .86 for the AARC-50 cognitive
functioning subscale gains and .88 for the AARC-50 cog-
nitive functioning subscale losses. gains and losses reported by participants on the AARC-10 SF and the AARC-50 cognitive functioning Table 4 Proportions of gains and losses reported by participants on the AARC-10 SF and the AARC-50 cognitive functioning
subscale
AARC-10 SF
AARC-50 cognitive functioning subscale
Gains
Losses
Gains
Losses
Not being aware of age-related changes
0.1%
3.8%
1.1%
5.7%
Little awareness of age-related changes
4.4%
60.4%
23.4%
56.6%
Moderate awareness of age-related changes
20.7%
29.0%
39.6%
28.6%
Quite a bit of awareness of age-related changes
47.0%
6.0%
28.0%
7.4%
A great deal of awareness of age-related changes
27.8%
0.8%
7.9%
1.9% Page 9 of 20 Sabatini et al. Reliability BMC Geriatrics (2020) 20:359 Table 5 Item characteristics and Cronbach’s αs for the two subscales of the AARC-10 SF and the AARC-50 cognitive functioning
subscale
With my increasing age, I realize that…
AARC-10 SF domaina
Basic item
characteristics
Item-total correlation
Mean
SD
PHYS-
… I have less energy
2.8
1.1
.75
COG-
…my mental capacity is declining
2.1
.9
.77
INT-
…I feel more dependent on the help of others
1.5
.7
.76
SCSE-
…I find it harder to motivate myself
1.7
.8
.78
LIFE-
…I have to limit my activities
1.9
.9
.74
PHYS+
…I pay more attention to my health
3.1
1.1
.78
COG+
…I have more experience and knowledge to evaluate things and people 3.5
1.0
.71
INT+
…I appreciate relationship and people much more
3.8
1.1
.69
SCSE+
…I have a better sense of what is important for me
3.9
1.0
.67
LIFE+
…I have more freedom to live my days the way I want
3.7
1.2
.76
AARC-50 cognitive functioning
Basic item
characteristics
Item-total correlation
Mean
SD
COG1 -
…my mental capacity is declining
2.1
.9
.84
COG2 -
...I am slower in my thinking
1.8
.8
.84
COG3 -
…I have a harder time concentrating
1.7
.8
.85
COG4 -
…learning new things takes more time and effort
2.4
1.0
.87
COG5 -
…I am more forgetful
2.2
1.0
.85
COG1 +
…I have more experience and knowledge to evaluate things and people 3.5
1.0
.86
COG2 +
…I have more foresight
2.7
1.1
.83
COG3 +
…I have become wiser
2.6
1.2
.82
COG4 +
…I think things through more carefully
2.6
1.1
.82
COG5 +
…I gather more information before I make decisions
2.6
1.1
.85
Note: aAARC domain abbreviations: PHY Health and physical functioning, COG Cognitive functioning, INT Interpersonal relations, SCSE Social-cognitive and social-
emotional functioning, LIFE Lifestyle and engagement; “+” = Positive domains; “-” = Negative domains Table 5 Item characteristics and Cronbach’s αs for the two subscales of the AARC-10 SF and the AARC-50 cognitive functioning Mean
SD
COG1 -
…my mental capacity is declining
2.1
.9
.84
COG2 -
...I am slower in my thinking
1.8
.8
.84
COG3 -
…I have a harder time concentrating
1.7
.8
.85
COG4 -
…learning new things takes more time and effort
2.4
1.0
.87
COG5 -
…I am more forgetful
2.2
1.0
.85
COG1 +
…I have more experience and knowledge to evaluate things and people 3.5
1.0
.86
COG2 +
…I have more foresight
2.7
1.1
.83
COG3 +
…I have become wiser
2.6
1.2
.82
COG4 +
…I think things through more carefully
2.6
1.1
.82
COG5 +
…I gather more information before I make decisions
2.6
1.1
.85
Note: aAARC domain abbreviations: PHY Health and physical functioning, COG Cognitive functioning, INT Interpersonal relations, SCSE Social-cognitive and social-
emotional functioning, LIFE Lifestyle and engagement; “+” = Positive domains; “-” = Negative domains Note: aAARC domain abbreviations: PHY Health and physical functioning, COG Cognitive functioning, INT
emotional functioning, LIFE Lifestyle and engagement; “+” = Positive domains; “-” = Negative domains tions: PHY Health and physical functioning, COG Cognitive functioning, INT Interpersonal relations, SCSE Social-cognitive and social-
estyle and engagement; “+” = Positive domains; “-” = Negative domains Measurement invariance between sex groups for the AARC-
10 SF and the AARC-50 cognitive functioning subscale women systematically interpreted at least some items
differently, responses for men and women should not be
compared without taking this sex bias into account. Reliability With respect to measurement invariance for the AARC-
10 SF between sex groups, compared to the model with
all parameters freely estimated (assuming configural in-
variance), the model that restricted factor loadings to be
the same across groups (assuming metric invariance) did
not substantially reduce the GOF (Table 6). Hence, the
meaning of the concepts of AARC-10 SF gains and
AARC-10 SF losses appeared to be the same for men
and women, allowing for valid representation of AARC
gains and losses in correlational studies across men and
women. Restricting item intercepts to be the same for
men and women (assuming strong invariance) substan-
tially decreased model fit as indicated by GOF indices,
meaning that men and women interpret some items of
the AARC-10 SF gains and the AARC-10 SF losses sub-
scales differently; hence scores on single items cannot be
compared among men and women. Since men and With respect to measurement invariance between sex
groups for the AARC-50 cognitive functioning subscale,
compared to the model with all parameters freely esti-
mated in the men and women groups (assuming config-
ural invariance), restricting factor loadings to be the
same across groups (assuming metric invariance) did not
substantially decrease model fit (Table 6). Hence, the
meaning of the concept of AARC as captured by the
AARC-50 cognitive functioning subscale appeared to be
the same for men and women. Restricting item inter-
cepts and factor loadings (assuming strong invariance)
to be equal among men and women did not substantially
decrease GOF indices (RMSEA, CFI, and SRMR) (Table
6), meaning that men and women interpret items of the
AARC-50 cognitive functioning gains and losses sub-
scales in the same way. Hence comparison of both Sabatini et al. BMC Geriatrics (2020) 20:359 Page 10 of 20 Sabatini et al. Reliability Hence, the
meaning of the concepts of AARC gains and AARC
losses in the cognitive domain as captured by the
AARC-50 cognitive functioning subscale appeared to be
the same across people with a university education and
without a university education. Restricting item inter-
cepts (strong invariance) to be equal among participants
with a university education and without a university
education did not substantially decrease GOF indices
(RMSEA, CFI, and SRMR) (Table 7), meaning that
people with a university education and people without a
university education interpret items of the AARC gains
and AARC losses subscales captured by the AARC-50
cognitive functioning subscale in the same way. Hence
for the AARC-50 cognitive functioning subscale com-
parison of both estimated factor means and observed
total
scores
across
items
between
education-based
groups are possible. Reliability BMC Geriatrics Table 6 Summary of the measurement invariance models for
the AARC-10 SF and the AARC-50 cognitive functioning
subscale-sex groups
AARC-10 SF
Models
RMSEA [95% CI]
CFI
SRMR
Model 1: Configural invariance
.07 [.06, .07]
.95
.05
Model 2: Metric invariance
.06 [.06, .07]
.95
.05
Model 3: Strong invariance
.07 [.07, .08]
.92
.71
AARC-50 cognitive functioning subscale
Models
RMSEA [95% CI]
CFI
SRMR
Model 1: Configural invariance
.12 [.12, .12]
.90
.05
Model 2: Metric invariance
.11 [.11, .12]
.90
.05
Model 3: Strong invariance
.11 [.11, .11]
.90
.10
CONFIGURAL INVARIANCE = This model places no equality constraints across
groups on factor loadings, the error variances, the variances of the latent
variables, or the covariances of the latent variables
METRIC INVARIACNE = This model places the factor loadings to be equal
across groups
STRONG INVARIANCE = This model constrains the factor loadings and the item
intercepts to be equal across groups
RMSEA = Root mean square error of approximation. CFI = Comparative fit
index. SRMR = Standard root mean square residual Table 7 Summary of the measurement invariance models for
the AARC-10 SF and the AARC-50 cognitive functioning
subscale-education level
AARC-10 SF
Models
RMSEA [95% CI]
CFI
SRMR
Model 1: Configural invariance
.07 [.07, .07]
.95
.05
Model 2: Metric invariance
.07 [.06, .07]
.95
.05
Model 3: Strong invariance
.06 [.06, .07]
.94
.06
AARC-50 cognitive functioning subscale
Models
RMSEA [95% CI]
CFI
SRMR
Model 1: Configural invariance
.12 [.12, .12]
.91
.05
Model 2: Metric invariance
.11 [.11, .12]
.90
.99
Model 3: Strong invariance
.11 [.11, .11]
.90
.06
CONFIGURAL INVARIANCE = This model places no equality constraints across
groups on factor loadings, the error variances, the variances of the latent
variables, or the covariances of the latent variables
METRIC INVARIACNE = This model places the factor loadings to be equal
across groups
STRONG INVARIANCE = This model constrains item loadings, error-variances of
the items, variances
RMSEA Root mean square error of approximation, CFI Comparative fit index,
SRMR Standard root mean square residual observed total scores across items and estimated factor
means between sex groups are possible. invariance), the model restricting factor loadings to be
the same (assuming metric invariance) across groups did
not markedly decrease model fit (Table 7). Measurement invariance between groups defined by
education level for the AARC-10 SF and the AARC-50
cognitive functioning subscale With respect to measurement invariance for education
level groups (university education versus no university
education) for the AARC-10 SF, compared to the model
with freely estimated parameters in the two groups (as-
suming configural invariance), restricting factor loadings
to be the same across groups (assuming metric invari-
ance) did not decrease model fit substantially (Table 7). Hence, the meaning of the concepts of AARC gains and
AARC losses as captured by the AARC-10 SF appeared
to be the same for people with and without a university
education. Restricting item intercepts (assuming strong
invariance) to be equal among groups with a university
education and without a university education did not
substantially decrease GOF indices (RMSEA, CFI, and
SRMR) (Table 7), meaning that individuals with a uni-
versity education interpret items of the AARC gains and
AARC losses subscales as captured by the AARC-10 SF
similarly to their counterparts without a university edu-
cation. Hence for the AARC-10 SF comparison of both
observed total scores across items and estimated factor
means between education-based groups are possible. Validity of the AARC-10 SF and AARC-50 cognitive
functioning scale in the over 50s UK population functioning scale in the over 50s UK population
Correlational evidence for validity of the AARC-10 SF is
reported in Table 9. As expected, individuals who ex-
perience more AARC gains, assessed with the AARC-10
SF, feel younger (r = .10; 95% CI: .08 to .12) and have
more positive ATOA (r = .12; 95% CI: .10 to .14) com-
pared to individuals who experience fewer AARC gains. People who experience more AARC losses, assessed
with the AARC-10 SF, feel older (r = −.27; 95% CI:
−.29 to −.25) and have more negative ATOA (r =
−.23; 95% CI: −.25 to −.21) compared to individuals
who experience fewer AARC losses. Overall, we found
mixed
and
negligible
correlations
between
AARC
gains, assessed with the AARC-10 SF, and indicators
of mental and physical health. Individuals who experi-
ence higher AARC losses, assessed with the AARC-10
SF, score higher on measures assessing current symp-
toms of depression (r = .21; 95% CI: .19 to .23) and
anxiety (r = .32; 95% CI: .30 to .34), as well as lifetime
symptoms of depression (r = .13; 95% CI: .12 to .16)
and anxiety (r = .16; 95% CI: .14 to .18). With respect to measurement invariance among
the three age groups for the AARC-50 cognitive
functioning subscale, compared to the model that
freely estimated parameters in the three age groups
(assuming configural invariance), the model restrict-
ing factor loadings to be the same (assuming metric
invariance)
across
the
three
age
groups
did
not
markedly decrease model fit (Table 8). Hence, the
meaning of the concepts of AARC gains and AARC
losses in the cognitive domain as captured by the
AARC-50 cognitive functioning subscale appeared to
be the same in middle age, early old age, and ad-
vanced old age. Restricting item intercepts (strong
invariance) to be equal across the three age groups
did not substantially decrease GOF indices (RMSEA,
CFI, and SRMR) (Table 8), meaning that items of
the AARC gains and AARC losses subscales of the
AARC-50 cognitive functioning subscale are inter-
preted in the same way across middle age, early old Participants with better functioning in activities of
daily living and who rate their health more posi-
tively
experience
higher
levels
of
AARC
gains,
assessed with the AARC-10 SF, but these correla-
tions are negligible. Measurement invariance among groups in middle age,
early old age, and advanced old age for the AARC-10 SF
and the AARC-50 cognitive functioning subscale Measurement invariance among groups in middle age,
early old age, and advanced old age for the AARC-10 SF
and the AARC-50 cognitive functioning subscale With respect to measurement invariance among groups
in middle age, early old age, and advanced old age (mid-
dle age = 50 to 65 years; early old age = 66 to 75 years;
advanced old age = 76 years and over) for the AARC-10
SF, compared to the model with freely estimated param-
eters in the three age groups (assuming configural in-
variance), restricting factor loadings to be the same
across groups (assuming metric invariance) did not With respect to measurement invariance for education
level groups (university education versus no university
education) for the AARC-50 cognitive functioning sub-
scale, compared to the model that freely estimated pa-
rameters in the education groups (assuming configural Page 11 of 20 Sabatini et al. BMC Geriatrics (2020) 20:359 Sabatini et al. BMC Geriatrics (2020) 20:359 decrease model fit substantially (Table 8). Hence, the
meaning of the concepts of AARC gains and AARC
losses as captured by the AARC-10 SF appeared to be
the same across middle age, early old age, and advanced
old age. Restricting item intercepts (assuming strong in-
variance) to be equal among age groups did not substan-
tially decrease GOF indices (RMSEA, CFI, and SRMR)
(Table 8), meaning that in middle age, early old age, and
advanced old age individuals interpret items of the
AARC gains and AARC losses subscales of the AARC-
10 SF similarly. Hence for the AARC-10 SF comparisons
of both observed total scores across items and estimated
factor means among age groups are possible. age, and advanced old age. Hence for the AARC-50
cognitive functioning subscale comparisons of both
estimated factor means and observed total scores
across items among age groups are possible. Validity of the AARC-10 SF and AARC-50 cognitive
functioning scale in the over 50s UK population Participants with better func-
tioning in activities of daily living (r = .23; 95% CI:
.21 to .24) and who rate their health more posi-
tively (r = −.44; 95% CI: −.46 to −.43) experience
lower
levels
of
AARC
losses,
assessed
with
the
AARC-10 SF, than participants with worse func-
tional
health
and
who
rate
their
health
more
negatively. Table 8 Summary of the measurement invariance models for
the AARC-10 SF and the AARC-50 cognitive functioning
subscale-age groups
AARC-10 SF
Models
RMSEA [95% CI]
CFI
SRMR
Model 1: Configural invariance
.07 [.07, .07]
.94
.07
Model 2: Metric invariance
.07 [.07, .07]
.94
.07
Model 3: Strong invariance
.08 [.08, .08]
.90
.10
AARC-50 cognitive functioning subscale
Models
RMSEA [95% CI]
CFI
SRMR
Model 1: Configural invariance
.12 [.12, .12]
.91
.06
Model 2: Metric invariance
.11 [.11, .11]
.90
.06
Model 3: Strong invariance
.11 [.11, .11]
.89
.08
CONFIGURAL INVARIANCE = This model places no equality constraints across
groups on factor loadings, the error variances, the variances of the latent
variables, or the covariances of the latent variables
METRIC INVARIACNE = This model places the factor loadings to be equal
across groups
STRONG INVARIANCE = This model constrains item loadings, error-variances of
the items, variances
RMSEA Root mean square error of approximation, CFI Comparative fit index,
SRMR Standard root mean square residual Correlations of the cognitive tasks digit span (r = −.01;
95% CI: −.04 to .01) and paired associate learning (r =
−.01; 95% CI: −.04 to .01) with AARC gains assessed
with the AARC-10 SF were not significant. The cognitive
tasks verbal reasoning (r = −.04; 95% CI: −.07 to −.02)
and self-ordered search (r = −.05; 95% CI: −.08 to −.03)
showed negative and negligible associations with AARC
gains assessed with the AARC-10 SF. The cognitive tasks
digit span (r = −.12; 95% CI: −.14 to −.09), paired associ-
ate learning (r = −.11; 95% CI: −.14 to −.09), verbal rea-
soning (r = −.15; 95% CI: −.18 to −.13), and self-ordered
search (r = −.10; 95% CI: −.12 to −.08) showed negative
small associations with AARC losses assessed with the
AARC-10 SF. Page 12 of 20 Sabatini et al. Validity of the AARC-10 SF and AARC-50 cognitive
functioning scale in the over 50s UK population Perceived health = Participants rated their own health on a four-point scale ranging from excellent to poor “excellent”, “good”, “fair”, and “poor” Validity of the AARC-10 SF and AARC-50 cognitive
functioning scale in the over 50s UK population BMC Geriatrics (2020) 20:359 Table 9 Correlations between AARC-10 SF and measures of self-perceptions of aging, mental and physical health
Correlational evidence of validity of the AARC-10 SF
AARC-10 SF Gains
AARC-10 SF Losses
Pearson’s r
[95% CI]
p-value
Pearson’s r
[95% CI]
p-value
Felt age
.10
[.08, .12]
< .001
−.27
[−.29, −.25]
< .001
CIDI-Lifetime depressive symptoms
.07
[.05, .09]
< .001
.13
[.12, .16]
< .001
CIDI-Lifetime anxiety symptoms
.04
[.02, .06]
< .001
.16
[.14, .18]
< .001
GAD-7
−.03
[−.05, −.01]
.01
.21
[.19, .23]
.01
PHQ-9
−.08
[−.10, −.06]
< .001
.32
[.30, .34]
< .001
IADL
−.03
[−.05, −.01]
< .001
.23
[.21, .24]
< .001
Perceived health
.09
[.08, .12]
< .001
−.44
[−.46, −.43]
< .001
Digit span
−.01
[−.04, .01]
.09
−.12
[−.14, −.09]
< .0001
Paired associate learning
−.01
[−.04, .01]
.32
−.11
[−.14, −.09]
< .0001
Verbal reasoning
−.04
[−.07, −.02]
.001
−.15
[−.18, −.13]
< .0001
Self-ordered search
−.05
[−.08, −.03]
< .0001
−.10
[−.12, −.08]
< .0001
Spearman’s ρ
p-value
Spearman’s ρ
p-value
ATOA
.13
< .0001
−.25
< .0001
AARC-10 SF gains Subscale of the AARC-10 SF assessing AARC gains, AARC-10 SF losses Subscale of the AARC-10 SF assessing AARC losses, Felt age Felt age
discrepancy score between participants’ chronological age and the age they feel they are, ATOA Lawton’s attitudes toward own aging 5-item scale, CIDI-Lifetime
depressive symptoms Composite international diagnostic interview-depressive symptoms, CIDI-Lifetime anxiety symptoms Composite international diagnostic
interview-anxiety symptoms. GAD-7 Generalized anxiety disorder-7, PHQ-9 Patient Health Questionnaire-9, IADL Lawton’s Instrumental activities of daily living
scale. Perceived health = Participants rated their own health on a four-point scale ranging from excellent to poor “excellent”, “good”, “fair”, and “poor” Table 9 Correlations between AARC-10 SF and measures of self-perceptions of aging, mental and physical health
C
l ti
l
id
f
lidit
f th
AARC 10 SF AARC-10 SF gains Subscale of the AARC-10 SF assessing AARC gains, AARC-10 SF losses Subscale of the AARC-10 SF assessing AARC losses, Felt age Felt age
discrepancy score between participants’ chronological age and the age they feel they are, ATOA Lawton’s attitudes toward own aging 5-item scale, CIDI-Lifetime
depressive symptoms Composite international diagnostic interview-depressive symptoms, CIDI-Lifetime anxiety symptoms Composite international diagnostic
interview-anxiety symptoms. GAD-7 Generalized anxiety disorder-7, PHQ-9 Patient Health Questionnaire-9, IADL Lawton’s Instrumental activities of daily living
scale. Validity of the AARC-10 SF and AARC-50 cognitive individuals having lower AARC losses. Regarding the
correlations between the AARC-50 cognitive functioning
subscale and objective cognitive tasks, when compared
to individuals with fewer AARC gains, individuals with
higher levels of awareness of positive changes score
worse in tasks assessing digit span, verbal reasoning, and
self-ordered search; estimate that their cognitive abilities y
g
functioning scale in the over 50s UK population y
g
functioning scale in the over 50s UK population Correlational evidence for validity of the AARC-50 cog-
nitive functioning subscale is reported in Table 10. We
found that those individuals who have higher awareness
of negative changes in their cognitive functioning also
feel older and have more negative ATOA than those Table 10 Correlations between AARC-50 cognitive functioning subscale and measures of self-perceptions of aging, subjective and
objective cognition Table 10 Correlations between AARC-50 cognitive functioning subscale and measures of self-perceptions of aging, subjective and
objective cognition
Correlational evidence of validity of the AARC-50 cognitive functioning subscale
AARC-50 cognitive functioning gains
AARC-50 cognitive functioning losses
Pearson’s r
[95% CI]
p-value
Pearson’s r
[95% CI]
p-value
Felt age
.08
[.06, .10]
< .001
−.19
[−.21, −.17]
< .001
Digit span
−.05
[−.08, −.03]
< .001
−.10
[−.12, −.07]
< .001
Paired associate learning
−.02
[−.05, .00]
.06
−.11
[−.14, −.09]
< .001
Verbal reasoning
−.09
[−.12, −.07]
< .001
−.16
[−.18, −.13]
< .001
Self-ordered search
−.07
[−.10, −.05]
< .001
−.08
[−.11, −.06]
< .001
IQCODE informant
−.05
[−.07, −.03]
< .001
−.01
[−.01, .03]
.51
IQCODE self
−.12
[−.15, −.10]
< .001
.47
[.45, .49]
< .001
Spearman’s ρ
p-value
Spearman’s ρ
p-value
ATOA
.04
< .0001
−.14
< .0001
AARC-50 cognitive functioning gains = Subscale of the AARC 50-item questionnaire assessing gains in the cognitive functioning domain. AARC-50 cognitive
functioning losses = Subscale of the AARC 50-item questionnaire assessing losses in the cognitive functioning domains. Felt age = Felt age discrepancy score
between participants’ chronological age and the age they feel they are. ATOA = Lawton’s attitudes toward own aging 5-item scale. Digit span = Computerized
cognitive task assessing verbal working memory. Paired associate learning = Computerized cognitive task assessing visual episodic memory. Grammatical
reasoning task = Computerized cognitive task assessing verbal reasoning. Self-oriented search = Computerized cognitive task assessing spatial working memory. IQCODE informant = Informant Questionnaire on Cognitive Decline in the Elderly short form asking informants to rate the cognitive change of someone close to
the them over the last 10 years. IQCODE self = Informant Questionnaire on Cognitive Decline in the Elderly short form asking participants to rate their own
cognitive change over the last 10 years AARC-50 cognitive functioning gains = Subscale of the AARC 50-item questionnaire assessing gains in the cognitive functioning domain. AARC-50 cognitive
functioning losses = Subscale of the AARC 50-item questionnaire assessing losses in the cognitive functioning domains. AARC-50 cognitive functioning gains = Subscale of the AARC 50-item questionnaire assessing gains in the cognitive functioning domain. AARC-50 cognitive
functioning losses = Subscale of the AARC 50-item questionnaire assessing losses in the cognitive functioning domains. Felt age = Felt age discrepancy score
between participants’ chronological age and the age they feel they are. ATOA = Lawton’s attitudes toward own aging 5-item scale. Digit span = Computerized
cognitive task assessing verbal working memory. Paired associate learning = Computerized cognitive task assessing visual episodic memory. Grammatical
reasoning task = Computerized cognitive task assessing verbal reasoning. Self-oriented search = Computerized cognitive task assessing spatial working memory.
IQCODE informant = Informant Questionnaire on Cognitive Decline in the Elderly short form asking informants to rate the cognitive change of someone close to
the them over the last 10 years. IQCODE self = Informant Questionnaire on Cognitive Decline in the Elderly short form asking participants to rate their own
cognitive change over the last 10 years y
g
functioning scale in the over 50s UK population Felt age = Felt age discrepancy score
between participants’ chronological age and the age they feel they are. ATOA = Lawton’s attitudes toward own aging 5-item scale. Digit span = Computerized
cognitive task assessing verbal working memory. Paired associate learning = Computerized cognitive task assessing visual episodic memory. Grammatical
reasoning task = Computerized cognitive task assessing verbal reasoning. Self-oriented search = Computerized cognitive task assessing spatial working memory. IQCODE informant = Informant Questionnaire on Cognitive Decline in the Elderly short form asking informants to rate the cognitive change of someone close to
the them over the last 10 years. IQCODE self = Informant Questionnaire on Cognitive Decline in the Elderly short form asking participants to rate their own
cognitive change over the last 10 years Page 13 of 20 Page 13 of 20 Sabatini et al. BMC Geriatrics (2020) 20:359 Sabatini et al. BMC Geriatrics (2020) 20:359 AARC gains; while being a woman significantly predicts
higher levels of AARC gains. We also found that being a
woman, married, in a civil partnership, or co-habiting,
and having a university education significantly predict
fewer AARC losses; while being older significantly pre-
dicts more AARC losses. AARC gains; while being a woman significantly predicts
higher levels of AARC gains. We also found that being a
woman, married, in a civil partnership, or co-habiting,
and having a university education significantly predict
fewer AARC losses; while being older significantly pre-
dicts more AARC losses. have increased over the past ten years; and a person
close to them also estimates that their abilities have in-
creased over the past ten years. However, most of the
above described correlations were of negligible size. Compared to individuals with higher scores on cog-
nitive tests, individuals with lower scores on cogni-
tive tests experience higher levels of negative age-
related changes; correlations among AARC losses
and cognitive tasks were, however, either negligible
or small. Participants who report higher levels of
negative age-related changes in cognition also notice
a decrease in their cognitive abilities over the past
ten years and this correlation was the strongest in
size (r = .47; 95% CI: .45 to .49). However, partici-
pants’ awareness of negative age-related changes is
not associated with the judgment of an informant. Discussion This was the first study exploring psychometric proper-
ties of the AARC-10 SF and the AARC-50 cognitive
functioning subscale in the UK population. We found
that both scales are valid and reliable measures of AARC
gains and AARC losses in the UK population aged 50
and over, that can be used in correlational studies and in
studies comparing AARC across men and women, across
individuals with and without a university degree, and
across middle age, early old age, and advanced old age. However,
some
caution
should be exercised
when y
g
functioning scale in the over 50s UK population From the multiple regressions exploring the ability of
demographic variables to predict gains and losses mea-
sured on the AARC-50 cognitive functioning subscale
(Tables 13 and 14), we found that, overall, being older,
married, in a civil partnership, or co-habiting, and hav-
ing a university education significantly predict fewer
AARC gains; while being a woman and employed signifi-
cantly predict more AARC gains. We also found that be-
ing
a
woman,
employed,
and
having
a
university
education significantly predict fewer AARC losses; while
being older significantly predicts more AARC losses. As the correlation between participants’ perceptions of
negative age-related changes in cognition and percep-
tions of a decrease in cognition over the past ten years
was the only correlation of moderate size, we further ex-
plored whether the size of the correlation varies across
different age-groups (middle age = 50 to 65 years; early
old age = 66 to 75 years, advanced old age = 76 years and
over). We found that the size of the correlation is similar
among participants in middle age (r = .48; 95% CI: .45 to
.51); early old age (r = .47; 95% CI: .44 to .50); and ad-
vanced old age (r = .42; 95% CI: .34 to .91). Tables 12 , 13 and 14 also show the results of simple
regressions with each demographic variable (age, sex,
marital status, current employment status, and university
education) as a predictor of AARC gains and losses mea-
sured with the AARC 10-SF and the AARC-50 cognitive
functioning subscale. Demographic variables as predictors of the AARC-10 SF and
AARC-50 cognitive functioning subscale From the two multiple regressions exploring the ability
of demographic variables to predict gains and losses
measured on the AARC-10 SF (Tables 11 and 12), we
found that, overall, being older, employed, and having a
university education significantly predict lower levels of Table 11 Simple and multiple regressions with demographic variables as predictors of AARC gains scores on the AARC-10 SF
(N = 8639)
Demographic variables as predictors of AARC gains:
Simple regressions
Demographic variables as predictors of AARC gains:
Multiple regression
AARC-10 SF losses
Variables
Coeff. [95% CI]
p-value
Standardized Coeff. Coeff. [95% CI]
p-value
Standardized Coeff. Age
−.02
[−.04, −.01]
< .0001
−.05
−.03
[−.04, −.01]
< .001
−.05
Sex
1.40
[1.21, 1.60]
< .0001
.15
1.31
[1.11, 1.51]
< .001
.14
Marital status
−.39
[−.59, −.19]
< .0001
−.04
−.27
[−.48, −.06]
.01
−.03
Employment
.04
[−.12, .21]
.60
.01
−.23
[−.43, −.03]
.02
−.03
University education −.25
[−.44, −.06]
.01
−.03
−.22
[−.41, −.03]
.02
−.02
Total R2
.03
Adjusted R2
.02
Model F-test
44.61 (5, 8633); p < .001
Note: In the regression models we included only those participants that have no missing data. AARC-10 SF gains = Subscale of the AARC-10 SF assessing AARC
gains. Marital Status was operationalized as a dichotomous variable capturing whether the participant is married/ civil partnership/ co-habiting or widowed/
separated/ divorced/ single. Employment was operationalized as a dichotomous variable capturing whether the participant is working or not. University education
was operationalized as a dichotomous variable. Standardized beta coefficients are calculated by subtracting the mean from the variable and dividing it by its
standard deviation egressions with demographic variables as predictors of AARC gains scores on the AARC-10 SF Note: In the regression models we included only those participants that have no missing data. AARC-10 SF gains = Subscale of the AARC-10 SF assessing AARC
gains. Marital Status was operationalized as a dichotomous variable capturing whether the participant is married/ civil partnership/ co-habiting or widowed/
separated/ divorced/ single. Employment was operationalized as a dichotomous variable capturing whether the participant is working or not. University education
was operationalized as a dichotomous variable. Standardized beta coefficients are calculated by subtracting the mean from the variable and dividing it by its
standard deviation Note: In the regression models we included only those participants that have no missing data. AARC-10 SF gains = Subscale of the AARC-10 SF assessing AARC
gains. Note: In the regression models we included only those participants that have no missing data. AARC-10 SF gains = Subscale of the AARC-10 SF assessing AARC
gains. Marital Status was operationalized as a dichotomous variable capturing whether the participant is married/ civil partnership/ co-habiting or widowed/
separated/ divorced/ single. Employment was operationalized as a dichotomous variable capturing whether the participant is working or not. University education
was operationalized as a dichotomous variable. Standardized beta coefficients are calculated by subtracting the mean from the variable and dividing it by its
standard deviation Demographic variables as predictors of the AARC-10 SF and
AARC-50 cognitive functioning subscale Standardized beta coefficients are calculated by subtracting the mean from the variable and dividing it by its
standard deviation validations of AARC measures, we found small and
moderate
overlap
between
AARC
questionnaires
(AARC-10 SF and AARC-50 cognitive functioning sub-
scale) and measures assessing the way in which individ-
uals experience aging (felt age and ATOA), supporting
the conceptual distinction of AARC from similar con-
cepts [1, 8]. The partial overlap of AARC (assessed with
both the AARC-10 SF and AARC-50 cognitive function-
ing subscale) with felt age and ATOA suggests that
AARC may impact on the way in which individuals feel
older or younger than they are or how changes are re-
ported or appreciated [57, 58]. However, ATOA and felt
age may also be associated with individuals’ perceptions
of AARC, such as perceptions of age-related cognitive validations of AARC measures, we found small and
moderate
overlap
between
AARC
questionnaires
(AARC-10 SF and AARC-50 cognitive functioning sub-
scale) and measures assessing the way in which individ-
uals experience aging (felt age and ATOA), supporting
the conceptual distinction of AARC from similar con-
cepts [1, 8]. The partial overlap of AARC (assessed with
both the AARC-10 SF and AARC-50 cognitive function-
ing subscale) with felt age and ATOA suggests that
AARC may impact on the way in which individuals feel
older or younger than they are or how changes are re-
ported or appreciated [57, 58]. However, ATOA and felt
age may also be associated with individuals’ perceptions
of AARC, such as perceptions of age-related cognitive comparing the scores of men and women on the AARC-
10 SF. Both scales showed good convergent validity for
AARC losses, but weak convergent validity for AARC
gains. Finally, we found that age, sex, marital status, em-
ployment, and university education explained significant
variability in levels of perceived AARC gains and losses
assessed with the AARC-10 SF and the AARC-50 cogni-
tive functioning subscale. Factor loadings for the two-factor model of the
AARC-10 SF and for the two-factor model of the
AARC-50 cognitive functioning subscale were similar to
those found in the US and German validations of the
measures [1, 8], further supporting the use of these
AARC measures in the UK. Demographic variables as predictors of the AARC-10 SF and
AARC-50 cognitive functioning subscale Marital Status was operationalized as a dichotomous variable capturing whether the participant is married/ civil partnership/ co-habiting or widowed/
separated/ divorced/ single. Employment was operationalized as a dichotomous variable capturing whether the participant is working or not. University education
was operationalized as a dichotomous variable. Standardized beta coefficients are calculated by subtracting the mean from the variable and dividing it by its
standard deviation Page 14 of 20 Sabatini et al. BMC Geriatrics (2020) 20:359 Sabatini et al. BMC Geriatrics Table 12 Simple and multiple regressions with demographic variables as predictors of AARC losses scores on the AARC-10 SF
(N = 8639)
Demographic variables as predictors of AARC losses:
Simple regressions
Demographic variables as predictors of AARC losses:
Multiple regression
AARC-10 SF losses
Variables
Coeff. [95% CI]
p-value
Standardized Coeff. Coeff. [95% CI]
p-value
Standardized Coeff. Age
.11
[.10, .12]
< .0001
.24
.09
[.08, .11]
< .001
.20
Sex
−.80
[−.96, −.64]
< .0001
−.10
−.60
[−.76, −.44]
< .001
−.08
Marital status
−.78
[−.95, −.61]
< .0001
−.10
−.52
[−.68, −.35]
< .001
−.06
Employment
−.96
[−1.1, −.82]
< .0001
−.15
−.11
[−.28, .05]
.18
−.02
University education −.45
[−.60, −.29]
< .0001
−.06
−.35
[−.50, −.19]
< .001
−.05
Total R2
.07
Adjusted R2
.07
Model F-test
128.74 (5, 8633); p < .001
Note: In the regression models we included only those participants that have no missing data. AARC-10 SF losses = Subscale of the AARC-10 SF assessing AARC
losses. Marital Status was operationalized as a dichotomous variable capturing whether the participant is married/ civil partnership/ co-habiting or widowed/
separated/ divorced/ single. Employment was operationalized as a dichotomous variable capturing whether the participant is working or not. University education
was operationalized as a dichotomous variable. Standardized beta coefficients are calculated by subtracting the mean from the variable and dividing it by its
standard deviation Note: In the regression models we included only those participants that have no missing data. AARC-10 SF losses = Subscale of the AARC-10 SF assessing AARC
losses. Marital Status was operationalized as a dichotomous variable capturing whether the participant is married/ civil partnership/ co-habiting or widowed/
separated/ divorced/ single. Employment was operationalized as a dichotomous variable capturing whether the participant is working or not. University education
was operationalized as a dichotomous variable. Demographic variables as predictors of the AARC-10 SF and
AARC-50 cognitive functioning subscale Also in line with previous Table 13 Simple and multiple regressions with demographic variables as predictors of gains scores on the AARC-50 cognitive
functioning subscale
(N = 8639)
Demographic variables as predictors of AARC gains:
Simple regressions
Demographic variables as predictors of AARC gains:
Multiple regression
AARC-50 cognitive functioning gains
Variables
Coeff. [95% CI]
p-value
Standardized Coeff. Coeff. [95% CI]
p-value
Standardized Coeff. Age
−.06
[−.07, −.05]
< .0001
−.10
−.04
[−.06, −.03]
< .001
−.07
Sex
1.30
[1.08, 1.52]
< .0001
.12
1.06
[.84, 1.29]
< .001
.10
Marital status
−.51
[−.74, −.28]
< .0001
−.05
−.51
[−.75, −.28]
< .001
−.05
Employment
.78
[.59, .96]
< .0001
.09
.41
[.18, .63]
< .001
.05
University education −.63
[−.84, −.41]
< .0001
−.06
−.63
[−.85, −.42]
< .001
−.06
Total R2
.03
Adjusted R2
.03
Model F-test
51.36 (5, 8633); p < .001
Note: In the regression models we included only those participants that have no missing data. AARC-50 cognitive functioning gains = Subscale of the AARC 50-
item questionnaire assessing gains in the cognitive functioning domain. Marital status was operationalized as a dichotomous variable capturing whether the
participant is married/ civil partnership/ co-habiting or widowed/ separated/ divorced/ single. Employment was operationalized as a dichotomous variable
capturing whether the participant is working or not. University education was operationalized as a dichotomous variable. Standardized beta coefficients are
calculated by subtracting the mean from the variable and dividing it by its standard deviation ultiple regressions with demographic variables as predictors of gains scores on the AARC-50 cognitive Note: In the regression models we included only those participants that have no missing data. AARC-50 cognitive functioning gains = Subscale of the AARC 50-
item questionnaire assessing gains in the cognitive functioning domain. Marital status was operationalized as a dichotomous variable capturing whether the
participant is married/ civil partnership/ co-habiting or widowed/ separated/ divorced/ single. Employment was operationalized as a dichotomous variable
capturing whether the participant is working or not. University education was operationalized as a dichotomous variable. Standardized beta coefficients are
calculated by subtracting the mean from the variable and dividing it by its standard deviation Note: In the regression models we included only those participants that have no missing data. AARC-50 cognitive functioning gains = Subscale of the AARC 50-
item questionnaire assessing gains in the cognitive functioning domain. Demographic variables as predictors of the AARC-10 SF and
AARC-50 cognitive functioning subscale Employment was operationalized as a dichotomous variable
capturing whether the participant is working or not. University education was operationalized as a dichotomous variable.. Standardized beta coefficients are
calculated by subtracting the mean from the variable and dividing it by its standard deviation The positive correlation we found between IADL and
AARC losses is in line with previous studies showing
that individuals with poorer everyday functioning per-
ceive more AARC losses [1]. Hence, individuals’ percep-
tions of AARC losses accurately reflect the negative
changes that individuals experience in their lives. The
finding that individuals with higher AARC gains and/or
lower AARC losses (assessed with the AARC-10 SF) rate
their health more positively is also in line with previous
evidence
[1,
8]. Most
correlations
between
AARC
(assessed with the AARC-10 SF) and indicators of health
were small or moderate, suggesting the presence of mul-
tiple factors alongside AARC gains and AARC losses
that may contribute to experiences of aging and levels of
mental and physical health. The size of the correlations
of AARC (assessed with the AARC-10 SF) with indica-
tors of mental and physical health were stronger for
AARC losses than for AARC gains and this is in line
with existing literature on AARC [2, 65]. It may be that
mental and physical health exert a greater influence on
individuals’ perceptions of AARC losses than on the per-
ceptions of AARC gains [2]. Perceptions of AARC gains
may instead be more influenced by other factors such as
personality traits [66], expectations for the future [12],
and perceived social support [67]. changes. A recent longitudinal study showed that more
negative ATOA predict greater perceived cognitive de-
cline [59]. Similarly, individuals with an older felt age
perceive more age-related losses in the cognitive domain
[60]. Overall, results relating to the construct validity of
the AARC-10 SF and the AARC-50 cognitive functioning
subscale suggest that levels of AARC losses and AARC
gains are informative of individuals’ mental, physical, and
cognitive health. The correlation we found between higher
levels of AARC-10 SF losses and more symptoms of de-
pression is in line with previous evidence describing the
predictive role of higher perceived AARC losses over
higher levels of depressive symptoms (e.g., [1, 8, 9]). Demographic variables as predictors of the AARC-10 SF and
AARC-50 cognitive functioning subscale Marital status was operationalized as a dichotomous variable capturing whether the
participant is married/ civil partnership/ co-habiting or widowed/ separated/ divorced/ single. Employment was operationalized as a dichotomous variable
capturing whether the participant is working or not. University education was operationalized as a dichotomous variable. Standardized beta coefficients are
calculated by subtracting the mean from the variable and dividing it by its standard deviation Page 15 of 20 Sabatini et al. BMC Geriatrics (2020) 20:359 Sabatini et al. BMC Geriatrics Table 14 Simple and multiple regressions with demographic variables as predictors of losses scores on the AARC-50 cognitive
functioning subscale
(N = 8639)
Demographic variables as predictors of AARC losses:
Simple regressions
Demographic variables as predictors of AARC losses:
Multiple regressions
AARC-50 cognitive functioning losses
Variables
Coeff. [95% CI]
p-value
Standardized Coeff. Coeff. [95% CI]
p-value
Standardized Coeff. Age
.08
[.07, .09]
< .001
.17
.07
[.05, .08]
< .001
.13
Sex
−.88
[−1.06, −.70]
< .001
−.11
−.76
[−.95, −.58]
< .001
−.09
Marital status
−.33
[−.51, −.14]
< .001
−.04
−.17
[−.36, .03]
.09
−.02
Employment
−.84
[−.99, −.69]
< .001
−.12
−.24
[−.43, −.06]
.01
−.03
University education −.47
[−.64, −.30]
< .001
−.06
−.41
[−.59, −.24]
< .001
−.05
Total R2
.04
Adjusted R2
.04
Model F-test
70.07 (5, 8633); p < .001
Note: In the regression models we included only those participants that have no missing data. AARC-50 cognitive functioning losses = Subscale of the AARC 50-
item questionnaire assessing losses in the cognitive functioning domains. Marital status was operationalized as a dichotomous variable capturing whether the
participant is married/ civil partnership/ co-habiting or widowed/ separated/ divorced/ single. Employment was operationalized as a dichotomous variable
capturing whether the participant is working or not. University education was operationalized as a dichotomous variable.. Standardized beta coefficients are
calculated by subtracting the mean from the variable and dividing it by its standard deviation Table 14 Simple and multiple regressions with demographic variables as predictors of losses scores on the AARC-50 cognitive
functioning subscale Note: In the regression models we included only those participants that have no missing data. AARC-50 cognitive functioning losses = Subscale of the AARC 50-
item questionnaire assessing losses in the cognitive functioning domains. Marital status was operationalized as a dichotomous variable capturing whether the
participant is married/ civil partnership/ co-habiting or widowed/ separated/ divorced/ single. Demographic variables as predictors of the AARC-10 SF and
AARC-50 cognitive functioning subscale It may therefore be
that cognitively healthy individuals are aware of the sub-
tle cognitive changes they are experiencing but that such
changes are unnoticed by people close to them [69]. between being older and fewer AARC gains is not con-
sistent with previous evidence reporting a positive asso-
ciation between older age and higher levels of AARC
gains [12]; this discrepancy in results may be due to cul-
tural differences as the present study included UK resi-
dents whereas Brothers, Gabrian [12] included US and
German participants. The associations of older age with
lower AARC gains but higher AARC losses may be due
to older individuals having a poorer health status than
younger individuals [73, 74]. We also found that being a man predicts fewer AARC
gains and more AARC losses both in the AARC-10 SF
and in the AARC-50 cognitive functioning subscale. This
finding is also in line with existing evidence on sex dif-
ferences in AARC and in subjective well-being, showing
that men report fewer AARC gains, higher AARC losses,
and lower levels of subjective well-being [17, 25, 75]
than women. This may be due to men being less actively
focused on positive changes compared to women [76,
77]. Indeed, research shows that positive experiences of
aging among women outweigh negative experiences,
despite women being aware of significant changes in
their body due to menopause [78]. g
y p
p
Interestingly, those individuals who performed more
poorly on objective cognitive tasks not only reported
higher levels of AARC losses but also reported higher
levels of AARC gains (assessed with both the AARC-10
SF and AARC-50 cognitive functioning subscale). It may
be that in order to compensate for negative changes in
cognition individuals engage in new cognitively stimulat-
ing
activities
[70,
71],
resulting
in
increased
self-
perception of gains. Alternatively, reporting high levels
of gains alongside high levels of losses may be a strategy
of emotional coping: high levels of losses may cause
mental distress which can be compensated for by direct-
ing thoughts towards positive age-related change [70]. However, the strength of the correlations of AARC gains
with objective cognitive tasks and self-perceived cogni-
tive decline were either of negligible size or small; self-
perceptions of age-related gains in cognition may be in-
fluenced by individuals’ beliefs about aging, more than
by individuals’ actual cognitive functioning. Demographic variables as predictors of the AARC-10 SF and
AARC-50 cognitive functioning subscale Many
of the variables contributing to the aetiology of late-life
onset of depression, such as poor physical health [61], in-
creased dependence on others [62], and having little or no
social support, are normative aspects of older age. This
may explain why the experiences of age-related losses that
are captured in individuals’ perceptions of AARC losses
are associated with current and future levels of depressive
symptoms [63, 64]. This was the first study exploring correlations between
AARC gains and losses and anxiety. Our results have
shown that higher levels of AARC-10 SF losses are corre-
lated with anxiety. Symptoms of anxiety may be expected
in older age and may be a consequence of the negative
changes that people experience in older age [26]. In con-
trast, the correlations we found between AARC-10 SF
gains and symptoms of depression and anxiety were
mixed and negligible. As similar findings were reported in
previous studies [63–65], along with the newly-identified
correlation with anxiety, it may be that when promoting
mental health in older age decreasing AARC losses is
more important than increasing AARC gains. This was the first study exploring correlations of
AARC in the cognitive functioning domain (assessed
with the AARC-50 cognitive functioning subscale) with
objective, subjective, and informant-rated measures of
cognition. We found that higher levels of AARC losses
in the cognitive domain reflect lower objective cognitive
performance and more negative self-evaluations of cog-
nitive changes over 10 years. Moreover, the correlation
between AARC losses in the cognitive domain and Page 16 of 20 Sabatini et al. BMC Geriatrics (2020) 20:359 Sabatini et al. BMC Geriatrics (2020) 20:359 Page 16 of 20 perceived cognitive change over the past ten years
remained consistent across three age groups (aged 50 to
65; aged 66 to 75; and aged 76 and over), suggesting that
the AARC-50 cognitive functioning subscale may detect
across middle age, early old age, and advanced old age
subclinical cognitive decline that is incorporated into in-
dividuals’ ratings of their AARC [1]. This finding is in
line with evidence supporting the value of subjective
cognitive complaints in informing about objective cogni-
tive decline [10, 68]. We also found that participants’ ex-
perience
of
negative
age-related
changes
is
not
correlated with informants’ rating of participants’ change
in cognitive abilities over ten years. Demographic variables as predictors of the AARC-10 SF and
AARC-50 cognitive functioning subscale Overall, as
most of the associations of AARC gains with cognitive
indicators are either small or of negligible size, evidence
for convergent validity for the AARC gains assessed with
the AARC-50 cognitive functioning subscale is weaker
than evidence for AARC losses. In line with existing literature [75, 79, 80], we found
that being married, in a civil relationship, or co-habiting
predicts fewer AARC losses assessed both with the
AARC-10 SF and with the AARC-50 cognitive function-
ing subscale. However we also found that being married,
in a civil partnership, or co-habiting predicts lower levels
of awareness of positive age-related change assessed
both with the AARC-10 SF and with the AARC-50 cog-
nitive functioning subscale. Literature on the role of
marriage in relation to cognitive abilities is heteroge-
neous with some studies reporting lower cognitive abil-
ities among non-married individuals [81] and conversely,
others
report a non-significant association between
marital status and cognition [82]. Our results suggest that working may have distinct ef-
fects on different AARC life domains. Working pre-
dicted fewer AARC losses in cognition (as assessed with
the AARC-50 cognitive functioning subscale) compared
to non-working and this may be due to work stimulating
cognition. Conversely, working predicted fewer AARC
gains in the remaining AARC life domains (assessed
with the AARC-10 SF) and this may be due to non-
working individuals having more leisure time to enjoy
hobbies and friends compared to workers, resulting in
increased likelihood of experiencing age-related gains. A secondary aim of this study was to explore whether
demographic variables (age, sex, marital status, current
employment status, and university education) predict
scores on the AARC-10 SF and AARC-50 cognitive sub-
scale gains and losses. We found that the demographic
variables age, sex, marital status, employment, and uni-
versity education explain some variability in levels of
AARC. We found that being older predicts fewer AARC
gains and more AARC losses both in the AARC-10 SF
and in the AARC-50 cognitive functioning subscale; the
association of higher AARC losses with being older is in
line with previous evidence and with gerontological lit-
erature reporting the greater salience of perceived losses
among
older
individuals
[12,
72]. Demographic variables as predictors of the AARC-10 SF and
AARC-50 cognitive functioning subscale Despite the above limitations this study has a large
sample size including a wide age range of UK partici-
pants. This is the first study testing content validity of
the AARC-50 cognitive functioning subscale with sub-
jective and objective measures of cognitive health. The
quantification of psychometric properties based on a
sample of participants without a diagnosis of dementia is
important because in this study AARC losses in cogni-
tion (assessed with the AARC-50 cognitive functioning
subscale) are associated with objective measures of cog-
nition and therefore could be useful to identify early
cognitive decline, which could in turn support efforts to
prevent dementia. The AARC-50 cognitive functioning
subscale could therefore potentially be used to identify
those segments of the population at greater risk of cog-
nitive decline who require closer cognitive monitoring,
and may benefit from early intervention such as cogni-
tive training programs [1] (pg. 3) or interventions that
help them to accept their age-related changes and to
minimize the negative impact of age-related cognitive
changes [93]. The validation of the AARC-50 cognitive
functioning subscale also makes it possible to conduct
future research to better understand the cross-sectional
relationship of subjective perceptions of cognition with
objective cognitive functioning, as well as the longitu-
dinal
association
with
objective
cognitive
decline. Whereas much research studying perceived cognitive de-
cline as a predictor of objective cognitive decline exists
[10, 94, 95], the AARC-50 cognitive functioning subscale
is particularly useful as it makes it possible to explore
for the first time whether AARC gains convey protection
against cognitive decline. The study has limitations that need to be acknowl-
edged. The sample included mainly white participants,
women, individuals who were married (or in a civil part-
nership or co-habiting) and who had above average edu-
cation and self-reported health. Among the 14,797
participants that took part in the PROTECT annual as-
sessment between 1st January 2019 and 31st March
2019, 9410 participants completed the AARC question-
naires. Compared to those who did not complete the
AARC questionnaires in 2019, the study sample included
a larger proportion of women and participants who were
better educated and a lower proportion of individuals
who were employed. This selection bias may impact on
study results such as on the predictive role that we
found for being female and having a university level edu-
cation over fewer AARC losses. Demographic variables as predictors of the AARC-10 SF and
AARC-50 cognitive functioning subscale The
association We found that people with a university education ex-
perience fewer AARC losses, but at the same time also
experience fewer AARC gains assessed both with the
AARC-10 SF and the AARC-50 cognitive functioning
subscale compared to individuals without a university
education. The fewer AARC losses in the cognitive Page 17 of 20 Page 17 of 20 Sabatini et al. BMC Geriatrics (2020) 20:359 without dementia over 2 months (e.g., [91, 92]). While
cognitive abilities were assessed both through objective
and subjective measures, mental and physical health
were assessed through self-report measures only. Finally,
individuals who completed a vocational qualification
(e.g. diploma or certificate) were considered to have the
same level of education as participants who completed a
undergraduate degree, a master’s degree, or a doctorate. This is a limitation as several types of vocational qualifi-
cations exist, with some vocational qualifications being
comparable to a university level education while others
are not. However, it was not possible to classify partici-
pants’ education in a more detailed manner as PRO-
TECT participants were not asked to specify the type of
vocational qualification they obtained. domain experienced by those with a university education
may be due to such individuals experiencing lower ob-
jective cognitive decline [83]. Indeed education exerts a
protective role against cognitive decline [84, 85]. The
lower score on the AARC-10 SF losses among those
with a university education may be due to more highly
educated people being more likely to engage in healthy
behaviors and therefore to enjoy better physical health
[86–89] and longer life expectancy [90]. g
y
An explanation for the lower levels of both AARC
gains and losses on the AARC-10 SF and on the AARC-
50 cognitive functioning subscale reported by those with
a university education may be that individuals who ex-
perience low levels of age-related losses are less likely to
reflect on age-related changes and as a consequence are
less aware of positive age-related changes. However, we
found that for both the AARC-10 SF and the AARC-50
cognitive
functioning
subscale
correlations
between
AARC gains and AARC losses are negligible, indicating
that there is no overall AARC. The lower levels of
AARC gains reported by individuals with a university
education may be due to more educated individuals at-
tributing positive changes to other causes rather than to
their increased age. Funding
h
k This work was supported by the University of Exeter College of Life and
Environmental Sciences (School of Psychology); University of Exeter College
of Medicine and Health; and the National Health and Medical Research
Council Centre for Research Excellence in Cognitive Health [#1100579 to
Kaarin Anstey]. The above listed funding bodies supported S S throughout
her PhD but did not contribute in the design of the study, data collection,
analysis, interpretation of data, or in writing the manuscript. The AARC-50 cognitive functioning subscale – while
capturing a more narrow facet of the experience of aging
- also proved to be a valid and reliable measure that
could be used to identify those segments of the popula-
tion at greater risk of cognitive decline and that may re-
quire closer cognitive monitoring or may benefit from
early intervention such as cognitive training programs
[1, 96]. Finally, as we found that demographic variables
play a role in the experience of AARC gains and AARC
losses, future studies on AARC should give a more de-
tailed account of the mechanisms that foster the experi-
ence of age-reated gains or losses. Demographic variables as predictors of the AARC-10 SF and
AARC-50 cognitive functioning subscale However, there is no im-
mediate reason to believe that the relationship between
the predictors and AARC losses is different between
those who provided data and those who did not. Data for objective cognitive assessments were not col-
lected on the same day on which participants completed
the AARC questionnaires, but were completed within 2
months of AARC completion. This was because com-
pleting a battery of cognitive tasks is demanding, espe-
cially for older individuals, hence allowing participants
to complete objective cognitive assessments on a separ-
ate day from the remaining measures decreased partici-
pants’ burden and increased the likelihood of collecting
accurate answers. Moreover, cognitive functions do not
deteriorate
or
deteriorate
minimally
in
individuals This is also the first study exploring content validity
for the AARC-10 SF with a measure assessing perceived
symptoms of anxiety, in addition to symptoms of depres-
sion, perceived health, and functional abilities that have
been explored in the US and German validations of the
AARC-10 SF and the AARC-50 cognitive functioning
subscale [1, 8]. Good psychometric properties of the Page 18 of 20 Page 18 of 20 Sabatini et al. BMC Geriatrics (2020) 20:359 Page 18 of 20 Sabatini et al. BMC Geriatrics (2020) 20:359 AARC-10 SF make it possible to use this scale to assess
positive and negative perceptions of age-related changes
in several domains of one’s life in clinical and research
contexts within the UK. AARC-10 SF make it possible to use this scale to assess
positive and negative perceptions of age-related changes
in several domains of one’s life in clinical and research
contexts within the UK. This paper represents independent research funded by the National Institute
for Health Research (NIHR) Biomedical Research Centre at South London and
Maudsley NHS Foundation Trust and King’s College London. Obi
Ukoumunne was supported by the National Institute for Health Research
(NIHR) Applied Research Collaboration (ARC) South West Peninsula. The
views expressed are those of the author(s) and not necessarily those of the
NHS, the NIHR or the Department of Health and Social Care. Ethics approval and consent to participate The PROTECT study received full ethical approval from the London Bridge
NHS Research Ethics Committee and Health Research Authority (Ref: 13/LO/
1578). Ethical approval for the data analyses was sought through the ethics
committee at the University of Exeter, School of Psychology (Application ID:
eCLESPsy000603 v1.0). Availability of data and materials This study was conducted using secondary data collected as part of the
PROTECT ongoing study. PROTECT data are available to investigators outside
the PROTECT team after request and approval by the PROTECT Steering
Committee. Data for the AARC-10 SF questionnaire and for the AARC-50 cog-
nitive functioning subscale will be available from May 2022. References 1. Kaspar R, Gabrian M, Brothers AF, Wahl H-W, Diehl MK. Measuring
awareness of age-related change: development of a 10-item short form for
use in large-scale surveys. Gerontologist. 2019;59(3):e130–e40. 2. Sabatini S, Silarova B, Martyr A, Collins R, Ballard C, Anstey KJ, et al. Associations of awareness of age-related change with emotional and 1. Kaspar R, Gabrian M, Brothers AF, Wahl H-W, Diehl MK. Measuring
awareness of age-related change: development of a 10-item short form for
use in large-scale surveys. Gerontologist. 2019;59(3):e130–e40. 1. Kaspar R, Gabrian M, Brothers AF, Wahl H-W, Diehl MK. Measuring
awareness of age-related change: development of a 10-item short form for
use in large-scale surveys. Gerontologist. 2019;59(3):e130–e40. 2. Sabatini S, Silarova B, Martyr A, Collins R, Ballard C, Anstey KJ, et al. Associations of awareness of age-related change with emotional and Abbreviations e authors declare that they have no competing interests. AARC: Awareness of age-related changes; AARC gains: Awareness of positive
age-related changes; AARC losses: Awareness of negative age-related
changes; AARC-10 SF: 10-item questionnaire assessing awareness of age-
related changes; AARC-50: 50-item questionnaire assessing awareness of age-
related changes; PROTECT: Platform for research online to investigate
genetics and cognition in aging (30); ATOA: Attitudes Toward Own Aging;
IQCODE: The Informant Questionnaire on Cognitive Decline in the Elderly
short form; IQCODE- Self: The Informant Questionnaire on Cognitive Decline
in the Elderly short form, self-version; PHQ-9: The Patient Health
Questionnaire-9; CIDI-SF: The Composite International Diagnostic Interview-
Short Form; GAD-7 : The Generalized Anxiety Disorder-7; IADL: Lawton’s
Instrumental Activities of Daily Living Scale; CFA: Confirmatory factor analysis;
CFI: Comparative fit index; TLI: Tucker-Lewis index; RMSEA: Root mean square
error of approximation; SRMR: Standardised root mean square residual;
GOF: Goodness of fit; LRT: Likelihood ratio tests; ΔRMSEA: Difference in root
mean square error of approximation; ΔSRMR: Difference in standardised root
mean square residual; M: Mean; SD: Standard deviation Authors’ contributions The AARC-10 SF is a valid and reliable measure to iden-
tify segments of the population that experience substan-
tial
change
across
multiple
life
domains
as
a
consequence of their aging process. The brief measure
may also be useful in clinical and counselling settings
within UK to identify those individuals who, because of
higher levels of AARC losses and/or lower levels of
AARC gains, may benefit from interventions helping
them to understand their age-related changes, to adapt
to age-related changes, or to engage in healthy behaviors
counteracting age-related losses [1]. S S served as principal investigator of the research, designed the study,
conducted data analyses, and took the lead in writing the manuscript. L C
contributed to the design of the study, analyses of data, and writing the
manuscript. O U contributed to analyses of data and writing the manuscript. A C, H B, D A, A H, and C B contributed to data collection and design, and
provided feedback on the draft of the manuscript. The remaining co-authors
provided feedback on the draft of the manuscript. The authors have read
and approved the manuscript. Supplementary information Full written consent was obtained for all participants on registration for the
PROTECT study, including consent for re-contact. Participants enrolled
through the PROTECT study website, downloaded the study information
sheet, and provided consent online. Supplementary information accompanies this paper at https://doi.org/10. 1186/s12877-020-01767-6. Additional file 1 Supplementary Table 1 Mean and standard
deviation on the four objective cognitive tasks for five levels of
awareness of negative age-related cognitive changes Author details
1 1College of Medicine and Health, REACH, University of Exeter, South Cloisters,
St Luke’s Campus, Exeter EX12LU, UK. 2NIHR ARC South West Peninsula
(PenARC), University of Exeter, Exeter, UK. 3College of Health and Human
Sciences, Colorado State University, Fort Collins, Colorado, USA. 4Cologne
Center for Ethics, Rights, Economics, and Social Sciences of Health, University
of Cologne, Cologne, Germany. 5Wicking Dementia Research & Education
Centre, University of Tasmania, Hobart, Australia. 6Department of Medicine,
Imperial College London, London, UK. 7Department of Brain Sciences,
Imperial College London, London, UK. Received: 20 March 2020 Accepted: 10 September 2020 Received: 20 March 2020 Accepted: 10 September 2020 Acknowledgments
f l
h g
y
g
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Sensitivity of Surface Fluxes in the ECMWF Land Surface Model to the Remotely Sensed Leaf Area Index and Root Distribution: Evaluation with Tower Flux Data
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Received: 11 November 2020; Accepted: 12 December 2020; Published: 16 December 2020 Received: 11 November 2020; Accepted: 12 December 2020; Published: 16 December 2020 Received: 11 November 2020; Accepted: 12 December 2020; Published: 16 December 2020 Abstract: The surface-atmosphere turbulent exchanges couple the water, energy and carbon budgets
in the Earth system. The biosphere plays an important role in the evaporation process, and vegetation
related parameters such as the leaf area index (LAI), vertical root distribution and stomatal resistance
are poorly constrained due to sparse observations at the spatio-temporal scales at which land surface
models (LSMs) operate. In this study, we use the Carbon Hydrology Tiled European Center for
Medium-Range Weather Forecasts (ECMWF) Scheme for Surface Exchanges over Land (CHTESSEL)
model and investigate the sensitivity of the simulated turbulent fluxes to these vegetation related
parameters. Observed data from 17 FLUXNET towers were used to force and evaluate model
simulations with different vegetation parameter configurations. The replacement of the current
LAI climatology used by CHTESSEL, by a new high-resolution climatology, representative of the
station’s location, has a small impact on the simulated fluxes. Instead, a revision of the root profile
considering a uniform root distribution reduces the underestimation of evaporation during water
stress conditions. Despite the limitations of using only one model and a limited number of stations,
our results highlight the relevance of root distribution in controlling soil moisture stress, which is
likely to be applicable to other LSMs. Keywords: root distribution; leaf area index; evaporation; land surface model atmosphere
Article
Sensitivity of Surface Fluxes in the ECMWF Land
Surface Model to the Remotely Sensed Leaf Area
Index and Root Distribution: Evaluation with
Tower Flux Data Article Article Atmosphere 2020, 11, 1362; doi:10.3390/atmos11121362 Sensitivity of Surface Fluxes in the ECMWF Land
Surface Model to the Remotely Sensed Leaf Area
Index and Root Distribution: Evaluation with
Tower Flux Data David Stevens 1,*
, Pedro M. A. Miranda 1
, René Orth 2
, Souhail Boussetta 3,
Gianpaolo Balsamo 3
and Emanuel Dutra 1,4
1
Instituto Dom Luiz, IDL, Faculty of Sciences, University of Lisbon, 1749-016 Lisbon, Portugal;
pmmiranda@fc.ul.pt (P.M.A.M.); emanuel.dutra@ipma.pt (E.D.)
2
Department for Biogeochemical Integration, Max Planck Institute for Biogeochemistry, 07743 Jena, Germany;
rene.orth@bgc-jena.mpg.de p
1
Instituto Dom Luiz, IDL, Faculty of Sciences, University of Lisbon, 1749-016 Lisbon, Portugal;
pmmiranda@fc.ul.pt (P.M.A.M.); emanuel.dutra@ipma.pt (E.D.) pmmiranda@fc.ul.pt (P.M.A.M.); emanuel.dutra@ipma.pt (E.D.)
2
Department for Biogeochemical Integration, Max Planck Institute for Biogeochemistry, 07743 Jena, Germ
rene.orth@bgc-jena.mpg.de 3
European Centre for Medium-Range Weather Forecasts, Shinfield Park, Reading RG2 9AX, UK;
Souhail.Boussetta@ecmwf.int (S.B.); gianpaolo.balsamo@ecmwf.int (G.B.) g
p
4
Instituto Português do Mar e Atmosfera, IPMA, 1749-077 Lisbon, Portugal g
p
4
Instituto Português do Mar e Atmosfera, IPMA, 1749-077 Lisbon, Portugal
*
Correspondence: sdann@fc.ul.pt 4
Instituto Português do Mar e Atmosfera, IPMA, 1749-077 Lisbon, Portu
*
Correspondence: sdann@fc.ul.pt *
Correspondence: sdann@fc.ul.pt atmosphere 1. Introduction The water, energy,and carbon exchanges between the land surface and the atmosphere are key
components of the Earth system. These exchanges are crucial for the understanding of the Earth
system’ response to the increase in greenhouse gas concentration and climate change. The variety and
complexity of the underlying processes driving these exchanges (from boundary layer turbulence to
plant physiology) are challenging both to observe and to model. From a modelling perspective,
these fluxes provide boundary conditions to numerical weather prediction models (NWP) and
earth system models (ESM). The longer time scales of some of the land surface processes [1,2],
when compared with the atmosphere, are crucial for sub-seasonal to seasonal predictability [3–5] and
to represent climate feedbacks [6,7]. Atmosphere 2020, 11, 1362; doi:10.3390/atmos11121362 www.mdpi.com/journal/atmosphere 2 of 19 Atmosphere 2020, 11, 1362 The turbulent exchanges of latent heat flux (Qle), or evaporation, couples the surface water,
energy and carbon budgets. Evaporation requires available water and energy [8]. Additionally,
a significant part of evaporation is due to plant transpiration [9–11], which is coupled with vegetation
photosynthesis and carbon exchanges, with a key role for soil moisture [12]. From an atmospheric
perspective, the partition of the net surface energy into Qle and sensible heat flux (Qh) modulates
the atmospheric boundary layer evolution with implications ranging from convection and cloud
evolution [13] to the amplification of extremes [14,15]. Vegetation transpiration and carbon uptake for
photosynthesis are mediated by stomatal opening. Therefore, stomatal conductance is an essential
component in the representation of evaporation in land surface models (LSMs). Stomatal conductance
is modulated by environmental factors (e.g., water stress) [16], and the leaf-to-canopy upscaling,
dependent on the vegetation characteristics [17]. p
g
In this study, we focus on the impact of the representation of canopy resistance on the Qle and Qh
fluxes in the European Center for Medium-Range Weather Forecasts (ECMWF) land surface model
(LSM) Carbon Hydrology Tiled ECMWF Scheme for Surface Exchanges over Land (CHTESSEL) [18,19]. This study is motivated by the results of the Land Surface Model Benchmarking Evaluation Project
(PLUMBER [20]), in which CHTESSEL participated, and by Ukkola et al. [21], which focused on
seasonal-scale evaporative droughts. These studies compared the performance of several LSMs
using atmospheric forcing from several FLUXNET towers. Best et al. [20] benchmarked the LSMs
against simple physically based models and empirical relationships. 1. Introduction The striking results showed that
all participating LSMs were outperformed by an out-of-sample linear regression against downward
shortwave radiation for the simulation of sensible heat flux and by a three-variable nonlinear regression
that uses instantaneous atmospheric humidity and temperature in addition to downward shortwave
radiation for the simulation of latent heat flux. Another key finding in PLUMBER was that LSMs had
difficulties in simulating latent heat flux at sites with low annual precipitation, suggesting that this
could be associated with the representation of water stress conditions in the models. The follow-up
study of Ukkola et al. [21] found systematic biases across the LSMs, including CHTESSEL, in the
simulation of energy and water fluxes under water-stressed conditions. Despite these limitations,
Best et al. [20] also found that LSMs performed well, compared with the empirical benchmarks,
when considering metrics for the extremes of the distributions. Therefore, the physical constraints in
the LSMs and their continued development are paramount to simulate conditions outside of training
conditions, as is the case for climate change scenarios [22]. Canopy resistance in CHTESSEL follows an empirical multiplicative formulation describing
the effects of different environmental variables [23], which is a common approach in several LSMs. Another approach is to couple net photosynthesis with canopy resistance [24]. CHTESSEL also has
a module to compute natural land carbon exchanges, but the carbon exchanges are decoupled from
evaporation due to conflicting results in NWP [25], justifying the focus on Qle and Qh in this study. In the resistance approach used by CHTESSEL, vegetation characteristics are considered via the use of
the leaf area index (LAI). The environmental factors account for water stress, which is dependent on soil
moisture content varying linearly between unstressed vegetation and the wilting point. In this study,
we address these two components of the canopy resistance formulation in CHTESSEL: (i) vegetation
characteristics and (ii) water stress. Vegetation characteristics are investigated by testing the use of a high-resolution LAI climatology
based on the Moderate Resolution Imaging Spectroradiometer (MODIS) product. The impact of
a high-resolution albedo climatology is also investigated. The use of this high-resolution data is
motivated by the increased quality and amount of available Earth observations (EO) [26]. Here, we aim
at evaluating the impact of using location specific LAI data when compared with the common LAI data
that have been spatially aggregated to a coarse grid for NWP applications. 2.1.1. Tower Flux Data Observed data from 20 FLUXNET towers were initially considered in this study as in Best et al. [20]. These stations are widely used since they present a variety of vegetation types and climates [20,29,30]. The Spanish stations of ElSaler and ElSaler2 were removed due to the presence of large water bodies
nearby and irrigated agricultural fields. The Canadian station of Merbleue was also problematic and
thus removed since this tower is in the middle of a wetland, which is difficult to correctly model. This corroborates Haverd et al. [31], who also removed these three stations from their study, resulting
in the 17 stations described in Table 1. The data include gap-filled half-hourly driving data for the LSM
(air temperature and humidity, surface pressure, wind speed, precipitation and downward solar and
thermal radiation) and surface sensible and latent heat flux used for model evaluation. Table 1. FLUXNET tower flux stations used in this study. Table 1. FLUXNET tower flux stations used in this study. Station
Country
Latitude
Longitude
Plant Functional Type
Time Period
Amplero
Italy
41.9041
13.6052
Grassland
2003–2006
Blodgett
U.S. 38.8953
−120.633
Evergreen needleleaf
2000–2006
Bugac
Hungary
46.6917
19.6017
Grassland
2002–2006
Espirra
Portugal
38.6394
−8.6018
Evergreen broadleaf
2001–2006
Fort Peck
U.S. 48.3077
−105.102
Grassland
2000–2006
Harvard
U.S. 42.5378
−72.1715
Deciduous broadleaf
1994–2001
Hesse
France
48.6742
7.0656
Deciduous broadleaf
1999–2006
Howard
Australia
−12.4943
131.152
Woody savannah
2002–2005
Howlandm
U.S. 45.2041
−68.7402
Evergreen needleleaf
1996–2004
Hyytiala
Finland
61.8474
24.2948
Evergreen needleleaf
2001–2004
Kruger
South Africa
−25.0197
31.4969
Savannah
2002–2003
Loobos
The Netherlands
52.1679
5.744
Evergreen needleleaf
1997–2006
Mopane
Botswana
−19.9165
23.5603
Woody savannah
1999–2001
Palang
Indonesia
−2.345
114.036
Evergreen broadleaf
2002–2003
Sylvania
U.S. 46.242
−89.3477
Mixed forest
2002–2005
Tumbarumba
Australia
−35.6557
148.152
Evergreen broadleaf
2002–2005
University of Michigan
U.S. 45.5598
−84.7138
Deciduous broadleaf
1999–2003
2.1.2. Satellite Albedo and Leaf Area Index Table 1. FLUXNET tower flux stations used in this study. 2.1.2. Satellite Albedo and Leaf Area Index 2.1.2. Satellite Albedo and Leaf Area Index In this study, we use the combined Moderate Resolution Imaging Spectroradiometer (MODIS)
LAI and fraction of photosynthetically active radiation (FPAR) product (MCD15A3H.006). This is a
4 day composite dataset with a spatial resolution of 500 m available since July 2002 to the present. This MODIS product has been validated in different studies [32,33] reporting uncertainties in the order
of 1 m2/m2. 2. Data and Methods 2.1. Data 1. Introduction The water stress representation
is investigated by testing a uniform root distribution with an associated maximum rooting depth. This is
compared with the current formulation in CHTESSEL that assumes an exponential root distribution [27]. 3 of 19 Atmosphere 2020, 11, 1362 The root distribution defines the maximum amount of water that is reachable by the plants, modulating
the response of transpiration to moisture stress [12,28]. As in Best et al. [20], observed data from 17 stations of the FLUXNET global network of
micrometeorological flux measurements are used to carry out offline simulations with CHTESSEL and
evaluate sensible and latent heat fluxes, with particular emphasis on the representation of evaporation
and its relation with water stressed conditions. The main objective of the study is to evaluate the
representation of canopy resistance by testing (i) the added value of the high-resolution LAI and albedo
to represent local station conditions and (ii) the impact of root distribution on soil moisture stress. The data and methods are described in the next section, followed by the presentation of the results. The results are discussed in Section 4 followed by the main conclusion of this study in Section 5. 2.1.1. Tower Flux Data In addition to the MODIS LAI product, the Copernicus Global Land Service (CGLS)
LAI was also used in this study. The product was obtained from SPOT-VGT and PROBA-V satellite
observations with 1 km resolution using a neural network algorithm [34] and has been available since 4 of 19 Atmosphere 2020, 11, 1362 1999. A comparison between MODIS and CGLS (a new version with a 300 m resolution) LAI products
with ground based observations over North America reported root mean squared differences of 0.57
for the CGLS product and 0.81 to 0.89 for MODIS [35]. In addition to the LAI product, we also use the Combined MODIS bidirectional reflectance
distribution function (BRDF) and Albedo products (MCD43A4.006) [36]. This product has been
available since February 2000 with a 500 m resolution. Wang et al. [37] reported root mean squared
errors ranging between 0.02 for forest regions and 0.03 for agricultural/grassland regions. In this study, we apply the most restrictive quality control (QC) criteria in order to select only the
best quality data. In the case of the albedo product, the model requires snow-free albedo. Therefore,
the MODIS snow products (MOD10A1.006 and MYD10A1.006) were used to mask the albedo data
when snow was present. A less restrictive quality filtering was applied for LAI [32], and good
quality data were also selected (QC < 64—highest and good quality). A similar quality control data
filtering was also applied to the CGLS data (only QC = 0 (land pixel clear observations) or QC = 512
(high latitude (lat) > 55◦and solar zenith angle > 70)). MODIS LAI and albedo, as well as the CGLS LAI were extracted for the 17 FLUXNET stations
(see Table 1) by averaging the data of the central pixel with the four other pixels in the direct vicinity. Monthly means were first computed and the monthly climatology computed excluding months with
more than 50% of missing data, for the full available period until 2018 (since 1999 for the CGLS LAI,
since 2003 for the MODIS LAI and since 2000 for the MODIS albedo). The quality based data screening was too restrictive for the northern latitude stations, especially
during the winter when satellite products are subjected to cloud contamination. In Hyytiala,
Loobos and Palang, the MODIS albedo product has a large amount of cloud contamination, and not
enough data are available to calculate the monthly climatology. 2.1.1. Tower Flux Data The quality control data filtering
was therefore relaxed and changed not only to the best quality data, but also to good quality data
(QC <= 1). After this processing, Hyytiala presented high albedo values in January, associated with
snow contamination. January’s albedo was replaced by a linear interpolation between December and
February. This shows the difficulty in generating snow-free albedo data [38], particularly in regions
and during seasons that are predominantly snow (and cloud) covered. However, this is not expected
to influence the model simulations as during January, the surface albedo has a small impact due to the
reduced amount of available solar radiation in Hyytiala. 2.2. CHTESSEL Model CHTESSEL is the land surface scheme of the ECMWF model [18,25]. In CHTESSEL, each grid-box
is divided into up to 6 land fractions representing vegetation, soil, snow and interception. Surface fluxes
are calculated separately for the different subgrid surface fraction (or “tile”), leading to a separate
solution of the surface energy balance and skin temperature for each of these tiles, which are then
aggregated in each grid-box. In each grid-box, two vegetation types can coexist (high and low
vegetation) controlled by four parameters: dominant high and low vegetation type and the area
fraction for the high and low vegetation. Each vegetation type is characterized by a series of fixed
parameters: (i) minimum canopy resistance rsmin, (ii) vegetation coverage density Cveg, (iii) coefficient
GD, for the dependence of the canopy resistance on water vapour pressure deficit, and (iv) the root
distribution over the soil layers, specified by an exponential profile modulated by the coefficients ar
and br. The numerical values for the parameters (see Table S1 in the Supplementary Material) are
based on the literature [27,39–44] and on expert decisions in the context of the performance of the
model in NWP. For example, the introduction of a seasonally variable LAI was accompanied with a
revision of the minimum canopy resistance [25]. A detailed description of CHTESSEL can be found in
the Integrated Forecasting System (IFS) documentation [45] and here, the details of the evaporation
calculation are presented due to its relevance in the results’ interpretation and discussion. Atmosphere 2020, 11, 1362 5 of 19 For high and low vegetation, the turbulent flux of water is given by: For high and low vegetation, the turbulent flux of water is given by: E =
ρa
ra + rc
(qL −qsat(Tsk)),
ra = (|UL|CH)−1
(1) (1) where ρa is the air density, UL and qL are the wind speed and humidity at the lowest atmospheric
model level, qsat(Tsk) the saturation humidity at skin temperature (Tsk) and CH the turbulent exchange
coefficient (depending on the atmospheric stability). 2.2. CHTESSEL Model In addition to the aerodynamic resistance (ra),
the canopy resistance (rc) [23] is calculated as: rc = rsmin
LAI × f1(Rs) × f2(θ) × f3(Da)
(2) (2) where rsmin is the minimum stomatal resistance (see Table S1), f1 is a function of downward short-wave
radiation (Rs), f3 a function of the atmospheric water vapour deficit (Da) and f2 the soil moisture
resistance given by: 1/ f2(θ) =
0
θ < θpwp
(θ −θpwp)/(θcap −θpwp)
θpwp ≤θ ≤θcap
1
θ > θcap
(3) (3) where θpwp and θcap are the soil moisture at the permanent wilting point and at field capacity,
respectively, and θ is a weighted average of the unfrozen soil water computed using the fraction
of roots in each layer (Rk) [27] using the ar and br coefficients (in Table S1): Rk = 0.5 [exp (−arzk−1/2) + exp (−brzk−1/2) −exp (−arzk+1/2) −exp (−brzk+1/2)]
(4 (4) where zk+1/2 is the depth of the bottom layer k in m and z1/2 is = 0. In CHTESSEL, the soil is
discretized into four layers with thicknesses of 0.07, 0.21, 0.72 and 1.89 m, with lower bounds at 0.07,
0.28, 1 and 2.89 m. In CHTESSEL, the state of vegetation is given by the LAI, entering the canopy resistance
calculation normalizing rsmin (Equation (2)). In the current operational NWP configuration, a satellite
observation based climatology is considered for the representation of LAI. It is based on Collection 5
of MODIS (product MOD15A2). The climatology was derived from 9 years of data (2000 to 2008) and
rescaled to a previous LAI static field used at ECMWF before 2012 [19]. 2.3. Simulation Setup Offline point simulations were performed using CHTESSEL driven by the meteorological data
observed at the towers. The model is initialized with soil moisture at field capacity and runs once for
the full length of the available forcing period for each station. The state at the end of the simulation
is then used to provide initial conditions to start the main simulation. For most sites, this procedure
guarantees enough time for spin-up, in particular for the deeper soil moisture. The exceptions are
Palang and Kruger with only two years. In both cases, the top meter soil moisture in the two years of
simulation does not show a significant drift. In Palang, there is an increase of the deep soil moisture
during the simulation (see Figure S1 in the Supplementary Material), but still within the inter-annual
variability. At each site, only one dominant high or low vegetation type is allowed, defined according to
the plant functional type (PFT). Howard, Kruger and Mopane stations report PFTs that are not available
in CHTESSEL (woody savannah and savannah). These stations were set as tall grass vegetation type
(see Table 2), as this is the closest vegetation type in the vicinity of the stations in the global land cover
dataset used by the model. 6 of 19 Atmosphere 2020, 11, 1362 Table 2. Control (CTR) rsmin and optimal rsmin and RDMAX used in MLAI_RSMINand MLAI_ROOT,
respectively (L defines low and H high vegetation types). Station
Vegetation Type
rsmin CTR
rsmin Optimal
RDMAX Optimal
Amplero
L, short grass
100
100
2
Bugac
L, short grass
100
175
0.5
Fort Peck
L, short grass
100
25
0.5
Howard
L, tall grass
175
125
2
Kruger
L, tall grass
100
300
2
Mopane
L, tall grass
175
300
1
Blodgett
H, Evergreen needleleaf
250
150
3
Espirra
H, evergreen broadleaf
240
75
3
Harvard
H, deciduous broadleaf
175
250
1
Hesse
H, deciduous broadleaf
175
175
2
Howlandm
H, evergreen needleleaf
250
275
0.5
Hyytiala
H, evergreen needleleaf
250
225
0.5
Loobos
H, evergreen needleleaf
250
150
2
Palang
H, evergreen broadleaf
240
275
2
Sylvania
H, interrupted forest
175
400
3
Tumbarumba
H, evergreen broadleaf
240
225
2
University of Michigan
H, deciduous broadleaf
175
400
3 Several model configurations were tested and are resumed in Table 3. 2.3. Simulation Setup These configurations aim at
investigating the role of (i) high-resolution remote sensing LAI and albedo and (ii) model formulation
and parameters. The first configuration, labelled as “CTR” for control, used CHTESSEL original input
data with the ECMWF Integrated Forecasting System (IFS). The monthly climatologies of snow-free
albedo and LAI were extracted from the nearest grid-point of operational NWP fields with a 9 km
resolution. This model configuration serves as a reference against which other model configurations
will be compared and was the same as used in [20]. The second experiment, labelled as “MALB ”,
uses the MODIS extracted high-resolution albedo monthly climatology over the stations. The third
configuration, labelled as “MLAI” uses the MODIS extracted high-resolution LAI monthly climatology
over the station. In both cases, the albedo and LAI data represent the nearest five MODIS pixels from
the station (as described in Section 2.1.2). Table 3. Model simulations acronyms and detailed configuration. CHTESSEL. Simulation
Details
CTR
Control simulation with default CHTESSEL parameters
and input LAI and Albedo
MALB
Same as CTR, but replacing the input albedo climatology
with the new high-resolution MODIS climatology
MLAI
Same as CTR, but replacing the input LAI climatology
with the new high-resolution MODIS climatology
MLAI_NOSMS
Same as MLAI, but removing the soil moisture stress function from the canopy resistance
(setting f2 = 1) when the soil moisture is above the wilting point in Equation (3)
MLAI_RSMIN
Same as MLAI, but selecting the optimal rsmin for each station from a set of
simulations with varying rsmin between 25 and 500. MLAI_ROOT
Same as MLAI, but using a uniform root distribution (Equation (5)) and selecting the optimal
RDMAX for each station from a set of simulations with RDMAX of 0.5, 1, 2, and 3 m. Table 3. Model simulations acronyms and detailed configuration. CHTESSEL. Two additional model configurations were tested to investigate the role of rsmin and root
distribution, both using the MODIS LAI high-resolution data. For rsmin, a set of simulations varying
rsmin between 25 and 500 with a step of 25 was performed for each site. For the root distribution,
a uniform root distribution was adopted up to a maximum rooting depth (RDMAX m) by changing the
computation of the root fraction in each layer in Equation (4) to: Atmosphere 2020, 11, 1362 7 of 19 (5) RDMAX In this configuration, four maximum rooting depths were tested: 0.5, 1, 2 and 3 m. 2.3. Simulation Setup The CTR root
distribution for each vegetation type and for the tested uniform rooting depths are given in Table S1
(in Supplementary Material). The root density distribution and root depth are poorly constrained at
large spatial scales as they vary between individual species and climate factors [46–48]. Therefore,
in this study, a uniform distribution is a simple and straightforward first-order assumption. An optimal model simulation for each site was then selected and labelled MLAI_RSMINfor the
optimal simulations with varying rsmin and MLAI_ROOT for the optimal simulations with varying
the maximum rooting depth RDMAX. The selection of the optimal configuration is described in the
following section. Finally, an idealized experiment removing the soil moisture stress function from
the canopy resistance (MLAI_NOSMS, setting f2 = 1 in Equation (2) when soil moisture is above the
wilting point) was performed. This idealized experiment provides an estimate of evaporation in a
situation of soil moisture at field capacity. 2.4. Evaluation The simulated Qle and Qh are compared with the tower measurements using 4 metrics (i) the mean
bias error (MBE), standard deviation difference (SD), correlation coefficient r , and normalized mean
error (NME). The SD is computed as the absolute difference between 1.0 and the ratio between the
simulated and observed standard deviation. The NME is the mean absolute error normalized by
the mean absolute observed deviations from the mean. These metrics follow Best et al. [20] and are
detailed in Appendix A. The calculations were performed on daily simulated and measured fluxes. For the selection of the optimal simulations with varying rsmin or maximum rooting depth,
a ranking approach considering the four metrics for both Qh and Qle was considered. For each metric
and flux, each simulation was ranked in ascending order from 1 to 20 in the case of rsmin and 1 to 4 in
the case of RDMAX. This resulted in rankings that were then added, and the simulation with the lowest
rank was selected as the optimal simulation. This process was performed independently for each site
and assumed that the metrics for Qle and Qh were of equal weight, and it did not distinguish between
larger or smaller differences between metrics in the ranking. The use of the rankings of each metric
without considering the actual metric value or the differences between simulations is a limitation as
simulations ranked differently can be actually very close. This approach can be interpreted as a simple
optimization strategy to select the optimal rsmin or RDMAX for each site. However, it is not intended
to be a proper model calibration as it is site specific and considers the entire set of observations. The search for the optimal simulations at each site by changing rsmin or RDMAX was designed to
provide an estimate of the upper bound on the best achievable performance of the model. It is a
benchmark to determine if the model could be improved just by changing the particular parameter. The reduced number of stations (only 17) and sampling of vegetation types imposed limitations in
performing a proper out-of-sample calibration to provide estimates of rsmin and RDMAX for each of the
vegetation types. 3.1. Comparison of LAI and Albedo In this section, we compare the LAI and high-resolution albedo (station) with the original CHTESSEL
input data. The high-resolution MODIS data for each tower are compared with the original CHTESSEL
input data in terms of the mean and root mean squared differences (RMSD), which are computed
for the mean annual cycle. The MODIS albedo is very close to CTR in all stations (see Table 4) with
most of the stations with RMSD around 0.03. There are two exceptions, Fort Peck and Loobos with an
RMSD of 0.07 mostly due to lower values in ALB when compared with CTR during winter, which are 8 of 19 Atmosphere 2020, 11, 1362 associated with snow contamination. Despite the importance of albedo in the surface energy balance,
these results suggest that for these 17 stations, we should expect a small impact on the CHTESSEL
simulation due to the update of the CTR albedo by the high-resolution MODIS albedo. Table 4. Comparison between the CTR and MODIS ALB (MALB) for each tower. The root mean
squared difference is computed for the mean annual cycle. Station
Mean
CTR
Mean
MALB
RMSD
MALB vs. CTR
Amplero
0.16
0.15
0.03
Blodgett
0.13
0.1
0.02
Bugac
0.18
0.16
0.03
Espirra
0.16
0.14
0.02
Fort Peck
0.25
0.18
0.07
Harvard
0.11
0.13
0.03
Hesse
0.13
0.15
0.03
Howard
0.15
0.13
0.02
Howlandm
0.11
0.12
0.01
Hyytiala
0.12
0.11
0.02
Kruger
0.17
0.16
0.03
Loobos
0.17
0.11
0.07
Mopane
0.16
0.17
0.01
Palang
0.14
0.13
0.01
Sylvania
0.11
0.13
0.04
Tumbarumba
0.16
0.12
0.04
University of Michigan
0.11
0.13
0.03
Median
0.15
0.13
0.03 The comparison of CTR and MLAI in Table 5 depicts comparatively large differences in several
stations with the RMSD above one in Bugac, Hesse, Howland and Palang. The time series of the
climatological LAI of CTR, MODIS and CGLS are shown for all stations in the Supplementary
Material (Figures S1–S17). The large differences in the climatology are mostly in the seasonal cycle
amplitude, with MLAI presenting a more pronounced seasonal phenology. These results show some
uncertainty in mapping LAI between the different earth observation products. Despite the differences
between MODIS and CGLS, our results show a larger discrepancy between the products and the CTR
climatology used in CHTESSEL with a median RMSD of 0.86 between CTR and MLAI when compared
with a median RMDS of 0.43 between MODIS and CGLS LAI. 3.1. Comparison of LAI and Albedo While the CTR LAI is also based on
MODIS data, it was derived from a shorter time period (2000–2008) and was rescaled to match a
previous LAI field used at ECMWF [19]. These LAI differences, both in the mean and in the annual
cycle, are expected to impact the model simulations, which are investigated in the following section. 3.2. Fluxes Evaluation CGLS
Amplero
2.43
1.7
1.51
0.9
0.29
Blodgett
3.08
2.28
2.98
0.87
0.47
Bugac
1.93
0.92
1.04
1.04
0.17
Espirra
2.38
1.42
1.13
0.97
0.20
Fort Peck
0.89
0.35
0.34
0.54
0.08
Harvard
3.14
2.45
2.78
0.75
0.58
Hesse
2.35
2.66
2.27
1.65
0.52
Howard
1.76
1.55
1.58
0.28
0.47
Howlandm
3.08
2.40
2.83
1.04
0.82
Hyytiala
2.12
1.69
1.73
0.50
0.51
Kruger
1.76
0.98
0.93
0.80
0.16
Loobos
2.21
1.87
1.87
0.53
0.43
Mopane
1.59
0.87
0.65
0.73
0.25
Palang
5.59
4.42
4.01
1.20
0.40
Sylvania
2.63
1.73
2.24
0.91
0.62
Tumbarumba
3.60
4.31
3.09
0.83
0.72
University of Michigan
2.42
2.11
1.76
0.86
0.25
Median
2.38
1.73
1.76
0.86
0.43 It was not possible to identify a systematic improvement in model performance associated with
the use of the high-resolution LAI dataset. Each station reacts differently, depending on the time period
considered. Some stations in some years benefit from the MODIS product, while other stations or time
periods show a deterioration of model performance. Moreover, the impacts of the high-resolution
LAI dataset on model performance could not be generalized or classified by plant functional type
(PFT) as stations within the same PFT do not react similarly to LAI changes. Stations with a good
performance in CTR (Fort Peck, Harvard, Howard, Howlandm, Hyytiala, Loobos, University of
Michigan) are not impacted by the LAI changes. Additionally, the large differences between the CTR
and LAI climatologies are not reflected in the changes in model performance in some stations like
Hesse or Howlandm. However, some stations suffering from poorer performances in CTR benefit
from MLAI as for example Bugac, Espirra, Palang, Sylvania and Tumba. Considering the four metrics
used to characterise model performance, the temporal correlation benefits the most from the use of the
high-resolution LAI since this product has a larger inter-seasonal variability than CTR. LAI enters the canopy resistance formulation normalizing the minimum stomatal resistance
(rsmin in Equation (2)). It is therefore expected that changes in the LAI climatology would benefit
from an adjustment of the rsmin parameter. Such an adjustment could be achieved by constraining
rsmin so that the ratio rsmin/LAI would stay unchanged for each PFT. However, due to the small
sample of stations for each PFT, such an approach is not feasible. 3.2. Fluxes Evaluation The use of the high-resolution MODIS albedo had a negligible impact on model performance
(not shown). This was expected due to the similarity between the albedo climatologies. Therefore,
the MALB simulations are not shown nor further explored in this study. Figure 1 shows the distribution
of the different metrics for Qh and Qle comparing the CTR and MLAI simulations (among other model
configurations to be discussed in the next section). In the MLAI simulation, the Qle MBE is reduced
from a median value of –3.02 W/m2 in CTR to –2.19. Similarly, the Qh MBE is reduced from 12.08 W/m2
in CTR to 8.48 W/m2 in MLAI. The standard deviation ratio (SD) is also improved with a reduction
from 0.26 and 0.17 for Qle and Qh, respectively, in CTR to 0.19 and 0.14 in MLAI. This improved
variability is also seen in the temporal correlation with an increase from 0.79 to 0.83 in the Qle from
CTR to MLAI and from 0.77 to 0.80 in Qh. Finally, the normalized mean error (NME) has a slight
reduction in Qh from 0.78 in CTR to 0.75 in MLAI and slightly increases for Qle (0.62 in CTR and 0.64
in MLAI). We note that the statistical significance of the changes in the model performance were not 9 of 19 Atmosphere 2020, 11, 1362 assessed because of the small sample size. Despite the small sample, only 17 stations, these results
suggest a modest, but positive impact of replacing CTR LAI by the high-resolution MODIS data for
the station. Table 5. Comparison between CTR and MODIS LAI (MLAI) for each tower. The root mean squared
difference is computed for the mean annual cycle. CGLS, Copernicus Global Land Service. Station
Mean
CTR
Mean
MLAI
Mean
CGLS
RMSD MLAI
vs. CTR
RMSD MODIS
vs. 3.2. Fluxes Evaluation 0.0
0.2
0.4
0.6
0.8
0.26
0.19
0.02
0.17
b) Qle SD
CTR
MLAI
MLAI_RSMIN
MLAI_ROOT 0.4
0.6
0.8
1.0
1.2
1.4
0.62
0.64
0.60
0.56
c) Qle NME
CTR
MLAI
MLAI_RSMIN
MLAI_ROOT 0.4
0.5
0.6
0.7
0.8
0.9
0.79
0.83
0.83
0.87
d) Qle pcorr
CTR
MLAI
MLAI_RSMIN
MLAI_ROOT 0.4 0.15
0.5
1.0
1.5
2.0
2.5
3.0
0.78
0.75
0.73
0.74
g) Qh NME
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0.77
0.80
0.80
0.79
h) Qh pcorr
M
M
M
M
T
CTR
MLAI
MLAI_RSMIN
MLAI_ROOT
CTR
MLAI
LAI_RSMI
MLAI_ROOT
CTR
MLAI
MLAI_RSMIN
MLAI_ROOT
CTR
MLAI
LAI_RSMI
MLAI_ROOT 0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0.17
0.14
0.14
0.15
f) Qh SD
M
CTR
MLAI
MLAI_RSMIN
MLAI_ROOT
CTR
MLAI
LAI_RSMI
MLAI_ROOT )
Figure 1. Latent (a–d, top panels) and sensible (e–h, bottom panels) heat flux evaluation in terms
of mean bias error (MBE), SD, normalized mean error (NME) and r for CTR, LAI, LAI_RSMIN and
LAI_ROOT. The boxplots represent each metric’s distribution for the 17 towers showing the percentiles
of 25, 50 and 75. Symbols denote outliers for values greater than 1.5 times the interquartile range from
the nearest 25th or 75th percentile. )
Figure 1. Latent (a–d, top panels) and sensible (e–h, bottom panels) heat flux evaluation in terms
of mean bias error (MBE), SD, normalized mean error (NME) and r for CTR, LAI, LAI_RSMIN and
LAI_ROOT. The boxplots represent each metric’s distribution for the 17 towers showing the percentiles
of 25, 50 and 75. Symbols denote outliers for values greater than 1.5 times the interquartile range from
the nearest 25th or 75th percentile. There are three sites with a large increase of rsmin from CTR to MLAI_RSMIN: Kruger (100 to 300),
Mopane (175 to 300) and Sylvania (175 to 400). In all three cases, the mean LAI decreases from CTR to
MLAI (see Table 2). This suggests that an adjustment that would keep the ratio rsmin/LAI unchanged
with changes in LAI would not be optimal or an indication that the default rsmin parameters are not
optimal. At these three stations, the increased rsmin results in a reduction of evaporation, as expected,
which is clearly seen in Sylvania (see Figure S3). 3.2. Fluxes Evaluation A set of simulations with varying
rsmin was performed to select an optimal rsmin with the best model performance for each station,
as described in Section 2.4. The optimal rsmin for each site is presented in Table 2. The simulations
with the optimal rsmin (MLAI_RSMIN) have a negligible impact on model performance (see Figure 1,
comparing MLAI and MLAI_RSMIN). The only noteworthy impact is a clear reduction of the SD for
Qle (see Figures 1 and S2), with the remaining performance metrics unchanged when compared with
MLAI. These results show that rsmin acts primarily on the seasonal amplitude of Qle, explaining the
large impact on SD, but neutral in the remaining performance metrics for Qle and Qh. In particular, 10 of 19 10 of 19 Atmosphere 2020, 11, 1362 the neutral impact on Qh SD highlights that the improved seasonal variability in Qle does not
necessarily lead to an improved Qh seasonality. CTR
MLAI
MLAI_RSMIN
MLAI_ROOT
20
10
0
10
20
30
W/m²
-3.02
-2.19
-3.64
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)
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Figure 1. Latent (a–d, top panels) and sensible (e–h, bottom panels) heat flux evaluation in terms
of mean bias error (MBE), SD, normalized mean error (NME) and r for CTR, LAI, LAI_RSMIN and
LAI_ROOT. The boxplots represent each metric’s distribution for the 17 towers showing the percentiles
of 25, 50 and 75. Symbols denote outliers for values greater than 1.5 times the interquartile range from
the nearest 25th or 75th percentile. 3.2. Fluxes Evaluation However, the Qle reduction in Sylvania is associated
with an unusually large rsmin of 400 sm−1, suggesting that this might be compensating missing
processes in the model and/or errors in the driving/observations data (Ukkola et al. [21] reported
that Sylvania was excluded from their study due to precipitation problems). There are three sites
with a large decrease of rsmin: Fort Peck (100 to 25), Blodgett (250 to 150) and Espirra (240 to 75). At these stations, there is also a reduction of the LAI from CTR to MLAI. From these three stations,
Espirra stands out with the largest reduction of rsmin, resulting in an increase of Qle and a decrease
of Qh, particularly during spring (see Figure S4). We note that Qle in Espirra was underestimated in
CTR and was further reduced in MLAI due to the reduction of the LAI (mean LAI changed from 2.38
in CTR to 1.42 in MLAI). The optimal rsmin change from 240 to 75 acts to reduce the rsmin/LAI ratio
from 100 sm−1 to 52 sm−1. We also note that the other two evergreen broadleaf tree stations, Palang
and Tumba, have a mean LAI of about 4.4 m2/m2 with an rsmin/LAI ratio of 54 sm−1. These results
suggest that the high-resolution MODIS LAI extracted for Espirra station underestimates the station
vegetation LAI [49]. 11 of 19 Atmosphere 2020, 11, 1362 The metric distributions in Figure 1 show some dispersion, associated with the occurrence of
a few outliers. The detailed results for each station and score are presented in the Supplementary
Material, in Figures S18–S25. It is possible to identify five stations with recurrent problematic results
for Qh/Qle in different metrics: Blodgett, Espirra, Mopane, Palang and Kruger. In Kruger, CHTESSEL
overestimates substantially the Qle after rainfall events, followed by an underestimation of the Qle
and an overestimation of the Qh (see Figure S5). Kruger is a very dry station with soil moisture
below the wilting point, and the peak evaporation following rainfall events is mainly driven by bare
soil evaporation. The remaining four stations, Blodget, Espirra, Mopane and Palang, share a similar
problem of an early evaporative reduction associated with dry soil moisture conditions resulting in the
overestimation of Qh. Although not seen clearly in the evaluation metrics, Amplero and Tumba also
show similar Qle temporal error patterns. This early evaporation reduction, or evaporative droughts,
as proposed by Ukkola et al. 3.3. Soil Moisture Stress Ukkola et al. [21] identified a systematic overestimation of evaporative droughts in several
LSMs, including CHTESSEL, which is consistent with the results in this study mainly in Amplero,
Blodget, Espirra, Howard, Mopane, Palang and Tumba. In this study, evaporative drought refers
to the regular dry season evaporation reduction, as well as anomalous dry periods as defined by
Ukkola et al. [21]. Figure 2 shows the time series of Qle and soil moisture for Amplero, Blodgett
and Espirra (time series for all stations are provided in the Supplementary Material). In these three
examples, it is possible to identify the early reduction of evaporation associated with the drop of soil
moisture (Figure 2: compare Qle reduction in blue with top soil moisture reduction in red). At these
stations, the evaporation starts to diverge from the observations when soil moisture in the top meter
(where most of the roots are present in CTR; Table S1) falls below field capacity and approaches
the wilting point. During this period, the deep layer soil moisture (dashed red line in Figure 2) is
above or close to field capacity. This is further illustrated by comparing the MLAI Qle simulations
with LAI_NOSMS in Figure 2 (blue vs. dashed-blue line). These idealized simulations, without soil
moisture stress, differ from MLAI exactly during the dry-down period, being closer to the observed
Qle. The LAI_NOSMS simulations present some unrealistic Qle variations (see for example 2003 in
Amplero in Figure 2) associated with the drop of soil moisture below the wilting point that stops
evaporation to avoid water conservation problems that could result in numerical instabilities. Despite
this limitation, the LAI_NOSMS simulations suggest that the evaporative drought in CHTESSEL is
tightly associated with the soil moisture stress formulation. The discrepancy between soil moisture
conditions in the top meter versus the bottom layer during the dry-down and associated evaporative
drought (Figure 2) motivated the exploratory revision of the root distribution. The root distribution with the uniform formulation (Equation (5)) for the different maximum
rooting depths (0.5, 1, 2 and 3 m) is presented in Table S1, allowing the comparison with the CTR
root fraction for each layer and vegetation type. The main changes of the uniform rooting depth
are a decrease of the roots fraction in the top layer(s) and an increase of root fraction in the deeper
layers. 3.2. Fluxes Evaluation [21], is further investigated in the following section. 3.3. Soil Moisture Stress This will give more weight to the deeper layers when computing the root zone soil moisture. The optimal maximum rooting depth for each station (see Section 2.4) in MLAI_ROOT is shown
in Table 2. Of the three short grass stations, Bugac and Fort Peck show an optimal RDMAX of 0.5 m with
negligible differences for MLAI, while Amplero shows a RDMAX of 2 m with an increase of the Qle in
late spring and summer. The increased Qle in Amplero in August/September 2005 (Figure 3) partially
reduces the evaporative drought in MLAI, but results is an overestimation of the Qle in June/July. A closer investigation of the full time series of simulated fluxes and the high-resolution MODIS LAI
(Figure S6) identifies an LAI drop during the 2005 summer, which cannot be represented when using
a climatology. A similar behaviour is also seen in the 2004 summer. Despite the reduction of the
evaporative drought in late summer in Amplero with the uniform root distribution, other factors 12 of 19 Atmosphere 2020, 11, 1362 influence the inter-annual and inter-seasonal variability of the Qle and Qh, which are not captured
by CHTESSEL, as well as observational errors and scale mismatches. Inter-annual variability is also
seen in Howard with a negligible impact of the uniform root distribution in 2005, but with a positive
impact in 2002, 2003 and 2004 (see Figure 3 for 2004 and Figure S7 for the full time series). Mopane
also displays evaporative drought events, but with very dry soil moisture conditions (see Figure S8),
the impact of the uniform root distribution is negligible. Figure 2. Latent heat flux (Qle) and soil moisture in Amplero (a), Blodgett (b) and Espirra (c). For the Qle (left axis), the observations are in grey and the simulations from MLAI in blue and
from MLAI_NOSM in orange. The soil moisture (right axis) for MLAI simulation is shown for the top
3 layers (red solid) and deep layers (red dashed). The light dotted red and blue lines represent soil
moisture at field capacity and the wilting point, respectively. Figure 2. Latent heat flux (Qle) and soil moisture in Amplero (a), Blodgett (b) and Espirra (c). For the Qle (left axis), the observations are in grey and the simulations from MLAI in blue and
from MLAI_NOSM in orange. 3.3. Soil Moisture Stress The soil moisture (right axis) for MLAI simulation is shown for the top
3 layers (red solid) and deep layers (red dashed). The light dotted red and blue lines represent soil
moisture at field capacity and the wilting point, respectively. In the forest sites, all RDMAX are between 2 and 3 m, with the exception of Harvard with 1 m and
Howland and Hyytiala with 0.5 m. These three forest sites, plus Loobos and University of Michigan,
are among the best performing in CTR and MLAI. The impact of the uniform root distribution is small
since all five sites have abundant precipitation and soil moisture conditions that are close to field
capacity. Hesse station is the only case of a consistent deterioration of Qle with the uniform rooting
depth and RDMAX of 2 m, mainly due to a considerable overestimation, contrasting with a reduction of
the Qh bias. This mixed effect in the Qh and Qle justifies the use of several metrics and joint Qh and Qle
in the model evaluation. Other processes and/or driving/observation limitations are likely responsible
for the errors. The remaining four forest sites Blodget, Espirra, Palang and Tumba (see Figure 3)
all display excessive evaporative drought associated with soil moisture dry-down. At these sites,
the experimental formulation of uniform rooting depth with the optimal RDMAX partially reduces the
excessive evaporative drought. The largest impact is seen in Palang and Blodgett, which are among
the sites with the worst Qle and Qh correlations. From these sites shown in Figure 3, the uniform root In the forest sites, all RDMAX are between 2 and 3 m, with the exception of Harvard with 1 m and
Howland and Hyytiala with 0.5 m. These three forest sites, plus Loobos and University of Michigan,
are among the best performing in CTR and MLAI. The impact of the uniform root distribution is small
since all five sites have abundant precipitation and soil moisture conditions that are close to field
capacity. Hesse station is the only case of a consistent deterioration of Qle with the uniform rooting
depth and RDMAX of 2 m, mainly due to a considerable overestimation, contrasting with a reduction of
the Qh bias. This mixed effect in the Qh and Qle justifies the use of several metrics and joint Qh and Qle
in the model evaluation. Other processes and/or driving/observation limitations are likely responsible
for the errors. 3.3. Soil Moisture Stress The remaining four forest sites Blodget, Espirra, Palang and Tumba (see Figure 3)
all display excessive evaporative drought associated with soil moisture dry-down. At these sites,
the experimental formulation of uniform rooting depth with the optimal RDMAX partially reduces the
excessive evaporative drought. The largest impact is seen in Palang and Blodgett, which are among
the sites with the worst Qle and Qh correlations. From these sites shown in Figure 3, the uniform root 13 of 19 13 of 19 Atmosphere 2020, 11, 1362 distribution has the largest impact on Qle, while rsmin and MLAI changes only show minor differences
for CTR in Espirra. distribution has the largest impact on Qle, while rsmin and MLAI changes only show minor difference
for CTR in Espirra. Feb Mar Apr May Jun Jul
Aug Sep Oct Nov Dec
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Palang Qle 2002
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Figure 3. Simulated and observed latent heat flux during one example year with a 14 day running
mean smoothing. Observations are in grey, CTR in black, MLAI in blue, MLAI_ROOT in green and
MLAI_RSMIN in red. 4. 3.3. Soil Moisture Stress Discussion Feb Mar Apr May Jun Jul
Aug Sep Oct Nov Dec
0.0
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MLAI_RSMIN Palang Qle 2002 Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Feb Mar Apr May Jun Jul
Aug Sep Oct Nov De Figure 3. Simulated and observed latent heat flux during one example year with a 14 day running
mean smoothing. Observations are in grey, CTR in black, MLAI in blue, MLAI_ROOT in green and
MLAI_RSMIN in red. 4. Discussion The systematic underestimation of the Qle and overestimation of the Qh for CTR (see Figure 1)
is opposite the findings of Martens et al. (2020) [50], who compared ERA5 data against observations 14 of 19 Atmosphere 2020, 11, 1362 from 143 FLUXNET sites. Similar biases in CHTESSEL considering a subset of 51 FLUXNET stations
were also found [51]. The differences of these studies compared to our results are likely associated
with the different sampling of stations. It is also worth noting that the energy budget closure of eddy
covariance sites can induce systematic errors [52,53]. Therefore, the interpretation of the systematic
biases in the simulated Qle and Qh must be cautious. In this study, the use of several evaluation
metrics that account for both mean biases and variability, with equal weights, partially mitigates the
problems that could arise from systematic observation errors. p
y
Despite the availability of long-term EO records of LAI, in this study, we considered only the
high-resolution LAI climatology. This was driven by two reasons: (i) several stations encompass
periods before the EO record were available (before 1999 in the case of CGLS or 2002 in the case
of MODIS), and (ii) we considered that the first step was to evaluate the impact of the high-resolution
station location conditions, neglecting inter-annual variability. However, our results suggest that the
lack of inter-annual variability in the vegetation state might be responsible for some of the model
problems. Amplero station is an example where we observed some relation between the time-varying
high-resolution LAI (see Figure S6, bottom panel, dotted blue line) and the Qle inter-annual variability. There is robust evidence of the added value of assimilating LAI in land-surface models, enhancing
their capabilities to monitor vegetation phenology and fluxes [54–57]. This is particularly relevant in
models that have a prognostic evolution of vegetation phenology. The soil moisture resistance, or water stress factor, in CHTESSEL has two components:
(i) the derivation of the root zone soil moisture content (using an exponential root distribution)
and (ii) a linear transformation between field capacity (unstressed vegetation) and the wilting point. In this
study, we focused only on the derivation of the root zone soil moisture content, testing a new uniform
root distribution and the associated maximum rooting depth. 4. Discussion There are different approaches to relate
root zone soil moisture and the water stress factor, such as curvilinear relationships or the use of soil
matric potential [58,59]. Such approaches, combined with the rooting depth distribution, are likely to
further enhance the model capability to represent root water uptake. Moreover, Liu et al. [12] reported
that a dynamical rooting depth evolution improved the performance of the Noah-MP-Crop model
under drought-like situations. Additionally, vapour pressure deficit and stem xylem conductance are
also known to influence stomatal regulation [60,61], but were not considered in this study. The presence
of a shallow water table could also influence latent heat flux during drought periods [62]. The water
table is not represented in CHTESSEL, and we are not aware of such effects at the 17 sites considered. Finally, the metrics distribution for MLAI_ROOT (in Figure 1) showed more potential to reduce
the Qle and Qh bias and NME and increase the correlation than the calibration of rsmin alone. This is
achieved by partially addressing the excessive evaporative drought at Blodget, Espirra, Palang and
Tumba stations. A joint optimization of both rsmin and maximum rooting depth is likely to further
improve the model simulations. However, it was not the aim of this study to optimize/calibrate
CHTESSEL for these stations, but to investigate in the current model formulation which aspects
require further attention. The use of the subset of FLUXNET stations to evaluate land surface models’
performance is a common practice [20,21,31,63,64]. 5. Conclusions This study focused on the impact of the representation of canopy resistance on the simulations
of latent and sensible heat fluxes by the CHTESSEL model. Observed data from 17 FLUXNET
towers were used to carry out offline simulations with a particular emphasis on the representation of
evaporation and its relationship with water stress conditions. Three constraints of canopy resistance
were evaluated: (i) the role of vegetation characteristics via the use of high spatial resolution LAI
representing local station conditions; (ii) the impact of the minimum canopy resistance; and (iii) the
impact of a uniform root distribution on soil moisture stress. The replacement of the current LAI
climatology used by CHTESSEL, based on an older MODIS climatology with a coarse resolution,
by a new high-resolution climatology representative of the stations locations did not significantly affect 15 of 19 15 of 19 Atmosphere 2020, 11, 1362 the simulated surface fluxes. The close relationship between LAI and the minimum canopy resistance
was investigated showing some potential to improve the latent heat flux variability via an adjustment
of this parameter. However, these changes did not impact the excessive evaporative drought at
several stations, as reported by Ukkola et al. [21]. In CHTESSEL, we found that this limitation was
tightly associated with the depletion of the top meter soil moisture, while water was still available
at deeper layers. However, the current model formulation for root distribution was not capable of
reaching this deeper reservoir, resulting in an early reduction of evaporation, when compared with the
observations. The replacement of the current exponential roots’ profile by a uniform root distribution
and associated maximum rooting depth reduced the underestimation of evaporation during water
stress conditions. Despite the limitations associated with a reduced number of stations, our results
highlight the importance of root distribution in controlling soil moisture resistance in water stress
conditions. However, root distributions are weakly constrained observationally on the global scale. Therefore, research is necessary to understand the implications of these changes on the global water
and energy budgets, as well as in the coupling with the atmosphere. The proposed uniform root
distribution with a single associated parameter, the maximum rooting depth, is also appealing for
a parameter optimization. These could be further addressed along with the revision of land cover
and vegetation recently proposed for CHTESSEL [65], which identified the necessity to calibrate
vegetation related parameters [51]. 5. Conclusions Considering the coupled nature of the surface water and energy
cycles and the relevance of land-atmosphere coupling, a calibration methodology considering multiple
observational datasets, covering different water/energy components and temporal time-scales, should
be favoured [66], also to possibly include some indirect information of poorly constrained parameters
through variables coupled with them. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4433/11/12/1362/s1,
Table S1: CHTESSEL vegetation types and associated parameters. R1 to R4 denote the root fraction in Layers 1 to
4. The final 4 lines indicate the root fraction when considering a uniform rooting depth with a maximum rooting
depth RDMAX of 0.5, 1, 2 and 2.89 m. Figure S1: Time series of Sensible heat flux, latent heat flux, soil moisture and
LAI in Palang. The turbulent fluxes’ time series compare the observations (grey) with the simulations: CTR (black),
LAI (blue), LAI_RSMIN (red), LAI_ROOT (green) and LAI_NOSMS (dashed blue). The soil moisture time series
shows the CTR the top 3 layers’ meter soil moisture (top meter, solid back) and the bottom layer soil moisture
(dashed black), as well as the soil moisture at the wilting point (blue) and field capacity (red). The LAI time
series compares CTR (black) with the high-resolution MODIS LAI time series (dotted blue), the high-resolution
MODIS climatology (blue), the climatology of MODIS considering the 0.25◦bounding box (dashed blue) and
the CGLS LAI climatology (grey). Figure S2: Latent heat flux SD at the 17 stations for each simulation: CTR,
MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S3: Same as Figure S1, but for Sylvania station. Figure S4: Same
as Figure S1, but for Espirra station. Figure S5: Same as Figure S1, but for Kruger station. Figure S6: Same as
Figure S1, but for Amplero station. Figure S7: Same as Figure S1, but for Howard station. Figure S8: Same as
Figure S1, but for Mopane station. Figure S9: Same as Figure S1, but for Blodgett station. Figure S10: Same as
Figure S1, but for Bugac station. Figure s11: Same as Figure S1, but for Fort Peck station. Figure S12: Same as
Figure S1, but for Harvard station. Figure S13: Same as Figure S1, but for Hesse station. Figure S14: Same as
Figure S1, but for Howlandm station. Figure S15: Same as Figure S1, but for Hyytiala station. Figure S16: Same as
Figure S1, but for Loobos station. Figure S17: Same as Figure S1, but for Tumbarumba station. 5. Conclusions Figure S18: Same as
Figure S1, but for University of Michigan station. Figure S19: Sensible heat flux MBE at the 17 stations for each
simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S20: Latent heat flux MBE at the 17 stations
for each simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S21: Sensible heat flux NME at the
17 stations for each simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S22: Latent heat flux NME
at the 17 stations for each simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S23: Sensible heat flux
SD at the 17 stations for each simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S24: Sensible heat
flux correlation at the 17 stations for each simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S25:
Latent heat flux correlation at the 17 stations for each simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Author Contributions: Conceptualization, D.S., E.D. and P.M.A.M.; formal analysis, D.S.; funding acquisition,
E.D.; writing, original draft preparation, all authors; writing, review and editing, all authors. All authors read and
agreed to the published version of the manuscript Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4433/11/12/1362/s1,
Table S1: CHTESSEL vegetation types and associated parameters. R1 to R4 denote the root fraction in Layers 1 to
4. The final 4 lines indicate the root fraction when considering a uniform rooting depth with a maximum rooting
depth RDMAX of 0.5, 1, 2 and 2.89 m. Figure S1: Time series of Sensible heat flux, latent heat flux, soil moisture and
LAI in Palang. The turbulent fluxes’ time series compare the observations (grey) with the simulations: CTR (black),
LAI (blue), LAI_RSMIN (red), LAI_ROOT (green) and LAI_NOSMS (dashed blue). The soil moisture time series
shows the CTR the top 3 layers’ meter soil moisture (top meter, solid back) and the bottom layer soil moisture
(dashed black), as well as the soil moisture at the wilting point (blue) and field capacity (red). The LAI time
series compares CTR (black) with the high-resolution MODIS LAI time series (dotted blue), the high-resolution
MODIS climatology (blue), the climatology of MODIS considering the 0.25◦bounding box (dashed blue) and
the CGLS LAI climatology (grey). Figure S2: Latent heat flux SD at the 17 stations for each simulation: CTR,
MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S3: Same as Figure S1, but for Sylvania station. Figure S4: Same
as Figure S1, but for Espirra station. Figure S5: Same as Figure S1, but for Kruger station. 5. Conclusions Figure S6: Same as
Figure S1, but for Amplero station. Figure S7: Same as Figure S1, but for Howard station. Figure S8: Same as
Figure S1 but for Mopane station Figure S9: Same as Figure S1 but for Blodgett station Figure S10: Same as g
,
g
g
,
g
Figure S1, but for Howlandm station. Figure S15: Same as Figure S1, but for Hyytiala station. Figure S16: Same as
Figure S1, but for Loobos station. Figure S17: Same as Figure S1, but for Tumbarumba station. Figure S18: Same as
Figure S1, but for University of Michigan station. Figure S19: Sensible heat flux MBE at the 17 stations for each
simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S20: Latent heat flux MBE at the 17 stations
for each simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S21: Sensible heat flux NME at the
17 stations for each simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S22: Latent heat flux NME
at the 17 stations for each simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S23: Sensible heat flux
SD at the 17 stations for each simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S24: Sensible heat
flux correlation at the 17 stations for each simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Figure S25:
Latent heat flux correlation at the 17 stations for each simulation: CTR, MLAI, MLAI_RSMIN and MLAI_ROOT. Author Contributions: Conceptualization, D.S., E.D. and P.M.A.M.; formal analysis, D.S.; funding acquisition,
E.D.; writing, original draft preparation, all authors; writing, review and editing, all authors. All authors read and
agreed to the published version of the manuscript. Funding:
This research was funded by Fundação para a Ciência e a Tecnologia (FCT) Grant Number
PTDC/CTA-MET/28946/2017 (CONTROL). David Stevens was funded by the FCT Grant PD/BD/135250/2017. The authors would like to acknowledge the financial support of the FCT through project UIDB/50019/2020—IDL. René Orth acknowledges the funding from the German Research Foundation (Emmy Noether Grant 391059971). 16 of 19 16 of 19 Atmosphere 2020, 11, 1362 Acknowledgments: This work used eddy covariance data acquired by the FLUXNET community for the La
Thuile FLUXNET release, supported by the following networks: AmeriFlux (US Department of Energy, Biological
and Environmental Research, Terrestrial Carbon Program (DE-FG02-04ER63917 and DE-FG02-04ER63911)),
AfriFlux, AsiaFlux, CarboAfrica, CarboEuropeIP, CarboItaly, CarboMont, ChinaFlux, Fluxnet-Canada (supported
by CFCAS, NSERC, BIOCAP, Environment Canada, and NRCan), GreenGrass, KoFlux, LBA, NECC, OzFlux,
TCOS-Siberia, USCCC. References 1. Carvalhais, N.; Forkel, M.; Khomik, M.; Bellarby, J.; Jung, M.; Migliavacca, M.; Mu, M.; Saatchi, S.; Santoro, M.;
Thurner, M.; et al. Global covariation of carbon turnover times with climate in terrestrial ecosystems. Nature
2014, 514, 213–217. [CrossRef] [PubMed] 1. Carvalhais, N.; Forkel, M.; Khomik, M.; Bellarby, J.; Jung, M.; Migliavacca, M.; Mu, M.; Saatchi, S.; Santoro, M.;
Thurner, M.; et al. Global covariation of carbon turnover times with climate in terrestrial ecosystems. Nature
2014, 514, 213–217. [CrossRef] [PubMed] 1. Carvalhais, N.; Forkel, M.; Khomik, M.; Bellarby, J.; Jung, M.; Migliavacca, M.; Mu, M.; Saatchi, S.; Santoro, M.;
Thurner, M.; et al. Global covariation of carbon turnover times with climate in terrestrial ecosystems. Nature
2014, 514, 213–217. [CrossRef] [PubMed] 2. Orth, R.; Seneviratne, S.I. Analysis of soil moisture memory from observations in Europe. J. Geophys. Res. Atmos. 2012, 117, 1–19. [CrossRef] Orth, R.; Seneviratne, S.I. Analysis of soil moisture memory from observations in Europe. J. Geophys
Res. Atmos. 2012, 117, 1–19. [CrossRef] 3. Koster, R.D.; Mahanama, S.P.; Yamada, T.J.; Balsamo, G.; Berg, A.A.; Boisserie, M.; Dirmeyer, P.A.;
Doblas-Reyes, F.J.; Drewitt, G.; Gordon, C.T.; et al. Contribution of land surface initialization to subseasonal
forecast skill: First results from a multi-model experiment. Geophys. Res. Lett. 2010, 37, 1–6. [CrossRef] 3. Koster, R.D.; Mahanama, S.P.; Yamada, T.J.; Balsamo, G.; Berg, A.A.; Boisserie, M.; Dirmeyer, P.A.;
Doblas-Reyes, F.J.; Drewitt, G.; Gordon, C.T.; et al. Contribution of land surface initialization to subseasonal
forecast skill: First results from a multi-model experiment. Geophys. Res. Lett. 2010, 37, 1–6. [CrossRef] 4. Koster, R.D.; Mahanama, S.P.; Yamada, T.J.; Balsamo, G.; Berg, A.A.; Boisserie, M.; Dirmeyer, P.A.;
Doblas-Reyes, F.J.; Drewitt, G.; Gordon, C.T.; et al. The second phase of the global land-atmosphere
coupling experiment: Soil moisture contributions to subseasonal forecast skill. J. Hydrometeorol. 2011,
12, 805–822. [CrossRef] 4. Koster, R.D.; Mahanama, S.P.; Yamada, T.J.; Balsamo, G.; Berg, A.A.; Boisserie, M.; Dirmeyer, P.A.;
Doblas-Reyes, F.J.; Drewitt, G.; Gordon, C.T.; et al. The second phase of the global land-atmosphere
coupling experiment: Soil moisture contributions to subseasonal forecast skill. J. Hydrometeorol. 2011,
12, 805–822. [CrossRef] 5. Koster, R.D.; Walker, G.K. Interactive vegetation phenology, soil moisture, and monthly temperature
forecasts. J. Hydrometeorol. 2015, 16, 1456–1465. [CrossRef] 5. Koster, R.D.; Walker, G.K. Interactive vegetation phenology, soil moisture, and monthly temperature
forecasts. J. Hydrometeorol. 2015, 16, 1456–1465. [CrossRef] 6. Seneviratne, S.I.; Corti, T.; Davin, E.L.; Hirschi, M.; Jaeger, E.B.; Lehner, I.; Orlowsky, B.; Teuling, A.J. Appendix A The statistical metrics used in this study follow [20]: the mean bias error (MBE), normalized mean
error (NME), standard deviation difference (SD) and correlation coefficient (r) were computed using
daily values with M, O and n representing the model data, observed flux tower data and the number
of days, respectively. n
(
) MBE = ∑n
i=1(Mi −Oi)
n
(A1)
NME = ∑|Mi −Oi|
∑| ¯O −Oi|
(A2)
SD = |1 −
q
∑Mi−¯
M2
n−1
q
∑Oi−¯O2
n−1
|
(A3)
r =
∑n
i=1(Mi −¯M)(Oi −¯O)
q
∑n
i=1(Mi −¯M)2
q
∑n
i=1(Oi −¯O)2
(A4) (A1) (A2) (A3) r =
∑n
i=1(Mi −¯M)(Oi −¯O)
q
∑n
i=1(Mi −¯M)2
q
∑n
i=1(Oi −¯O)2
(A4) (A4) 5. Conclusions We acknowledge the financial support to the eddy covariance data harmonisation
provided by CarboEuropeIP, FAO-GTOS-TCO, iLEAPS, Max Planck Institute for Biogeochemistry, National Science
Foundation, University of Tuscia, Université Laval and Environment Canada and US Department of Energy
and the database development and technical support from Berkeley Water Center, Lawrence Berkeley National
Laboratory, Microsoft Research eScience, Oak Ridge National Laboratory, University of California—Berkeley,
University of Virginia. 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; nor in the decision to
publish the results. enissen, J.M.; Teuling, A.J.; Reichstein, M.; Orth, R.
Critical Soil Moisture Derived From Satellite
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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/). References Nogueira, M.; Albergel, C.; Boussetta, S.; Johannsen, F.; Trigo, I.F.; Ermida, S.L.; Martins, J.P.A.; Dutra, E. Role of vegetation in representing land surface temperature in the CHTESSEL (CY45R1) and SURFEX-ISBA
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Earth observations. Hydrol. Earth Syst. Sci. 2017, 21, 2483–2495. [CrossRef] Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional
affiliations. c⃝2020 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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A Horizontal Magnetic Tweezers and Its Use for Studying Single DNA Molecules
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Micromachines
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Received: 29 January 2018; Accepted: 12 April 2018; Published: 17 April 2018 Abstract: We report the development of a magnetic tweezers that can be used to micromanipulate
single DNA molecules by applying picoNewton (pN)-scale forces in the horizontal plane. The resulting force–extension data from our experiments show high-resolution detection of changes
in the DNA tether’s extension: ~0.5 pN in the force and <10 nm change in extension. We calibrate
our instrument using multiple orthogonal techniques including the well-characterized DNA
overstretching transition. We also quantify the repeatability of force and extension measurements,
and present data on the behavior of the overstretching transition under varying salt conditions. The design and experimental protocols are described in detail, which should enable straightforward
reproduction of the tweezers. Keywords: single molecule micromanipulation; magnetic tweezers; DNA overstretching transition A Horizontal Magnetic Tweezers and Its Use for
Studying Single DNA Molecules Roberto Fabian Jr. 1,*, Christopher Tyson 2, Pamela L. Tuma 3, Ian Pegg 1 and Abhijit Sarkar 1 1
Department of Physics and Vitreous State Laboratory, The Catholic University of America, Washington,
DC 20064, USA; pegg@cua.edu (I.P.); SARKAR@cua.edu (A.S.)
2
Biomedical Engineering Department and Vitreous State Laboratory, The Catholic University of America,
Washington, DC 20064, USA; 88tyson@cua.edu
3
Department of Biology, The Catholic University of America, Washington, DC 20064, USA; tuma@cua.edu 2
Biomedical Engineering Department and Vitreous State Laboratory, The Catholic University of Americ
Washington, DC 20064, USA; 88tyson@cua.edu 3
Department of Biology, The Catholic University of America, Washington, DC 20064, USA; tuma@cua.edu
*
Correspondence: 76fabian@cua.edu; Tel.: +1-202-319-6740 Department of Biology, The Catholic University of America, Washington, DC 20064, USA; tuma@cua.edu
Correspondence: 76fabian@cua.edu; Tel.: +1-202-319-6740
micromachines micromachines micromachines micromachines Micromachines 2018, 9, 188; doi:10.3390/mi9040188 1. Introduction In the past 25 years, many new techniques for the micromanipulation of single DNA molecules
and DNA-protein complexes have been developed. These allow molecular-level measurements of the
elasticity of single DNA molecules through direct mechanical stretching and twisting studies as well
as the exquisite step-by-step dissection of DNA-protein interactions. Such experiments have resulted
in numerous insights into the role of DNA in the genetic processes it participates in, for example
DNA replication [1], DNA transcription [2], and DNA recombination [3], all at the single molecule
level. These data are difficult to obtain using standard ensemble-based biochemical and biophysical
techniques [4]. Magnetic tweezers have emerged as a powerful technique for studying DNA and DNA-protein
interactions at a single molecule level. Magnetic tweezers are uniquely suited for studying the
mechanical response of biopolymers under controlled forces—such experiments are said to be
performed in the constant force ensemble in which the DNA’s thermally-averaged extension is
measured as a function of externally-generated forces. Technical advances now allow applied forces in
the 0.1–100 pN range and extensions down to angstroms to be reliably measured (although typical
setups are limited to detecting extension changes in the order of 1–10 nm) [5–12]. In addition to a
pulling force, the DNA’s twist (or, more generally, linking number) [13] can also be modulated or, in a
fixed-torque assay, direct, simultaneous and independent control of forces and torques on the DNA is
also possible [14–16]. Other progress includes magnetic micro-manipulation of confined DNA [17],
use of proteins as tethers (instead of DNA) [18], and a portable tweezers setup [19]. Micromachines 2018, 9, 188; doi:10.3390/mi9040188 www.mdpi.com/journal/micromachines www.mdpi.com/journal/micromachines 2 of 12 Micromachines 2018, 9, 188 In typical magnetic tweezers, one end of the DNA is directly attached to the surface of the
sample cell while the other end is attached to a micron sized superparamagnetic bead [20]. Above the
sample cell are permanent magnets, typically 2 bar magnets, that generate a force that pulls the
DNA in the vertical plane. The strength of the pulling force can be adjusted by moving the magnets
away or closer to the sample cell. This design is commonly known as a vertical magnetic tweezers. The advantages of this configuration include versatility and simplicity, the ease with which DNA
topology can be modulated (by rotating the magnet to control twist or introduce plectonemes), and
the relative ease with which tethered DNA can be located. 1. Introduction Various schemes have been developed to
measure the extension of the DNA molecule with ever-increasing precision, including techniques based
on counting the diffraction rings from the tethered bead [11,21,22]. Instrument designs that compensate
for sample cell drift—mechanical drift can affect extension measurements in long-duration (~1 h)
experiments—have also been developed [23,24]. However, these advances have added complexity to
vertical magnetic tweezers and required sophisticated calibration techniques to determine the relation
between diffraction rings and DNA extension. Another option is to generate the forces on DNA tethers along the focal plane [24–28]. For example,
Yan et al. [25] developed a magnetic tweezers configuration in which one end of the DNA is tethered
on a non-magnetic bead while the other end of the DNA is tethered to a superparamagnetic bead. The non-magnetic bead is held by a micropipette aspiration while the magnetic bead is suspended near
a permanent bar magnet which applies magnetic forces on the magnetic bead. However, they used a
sample cell which increases buffer evaporation and other external noise. Extension measurements in
horizontal tweezers are, in principle, straightforwardly obtained by subtracting the position of the
two beads. These, in turn, are computed using image processing to determine the intensity-weighted
centroid coordinates of the two beads. An added benefit is that differential extension measurements
allow passive drift cancellation, a feature which is utilized in optical tweezers and some magnetic
tweezers through the use of fiducial markers [29]. Here, we describe an improved version of a horizontal magnetic tweezers design previously
reported in ref. [30]. As reported here, the tweezers is relatively simple to implement and can apply
forces in the range of 0.5 pN to 100 pN (and higher) on single linear DNA molecules connected
to 2 superparamagnetic beads, one to each end. One bead is immobilized by biotin–streptavidin
interactions to the surface of a rigid glass pipette; this bead is connected by the DNA tether to another
superparamagnetic bead suspended in buffer near a bar magnet. The force on the suspended bead
can be modulated by adjusting the bead–magnet distance. 1. Introduction Key features of our design include: (i)
replacing pipette aspiration by a rigid functionalized glass pipette coated with biotin; (ii) incorporating
a sample cell with a single narrow open slit that minimizes buffer evaporation and pressure fluctuation
at the open interfaces; (iii) integrating an inlet and outlet into the sample cell for buffer exchange; (iv)
using a second micromanipulator to manipulate the functionalized rigid glass pipette independently;
(v) attaching the pipette micromanipulator on a rail system firmly fixed to the microscope in order to
increase stability; (vi) floating the sample cell stage on a motorized micromanipulator that allows easy
adjustment of the DNA-bead construct and the magnet distance. As a result, we achieve high precision
measurements—for extensions, <10 nm, and for forces, 0.5 pN. We calibrate our force–extension
measurements using the DNA overstretching transition. 2.1. DNA End-Functionalization The first step is to end-functionalize the DNA tether. In the experiments reported here,
we use linear bacteriophage λ-DNA (N3011S, New England Biolabs, Ipswich, MA, USA) with
sequence-specific 12 base polynucleotide overhangs:
3′-end overhang is gggcggcgaccg and
5′-end overhang is aggtcgccgccc. Following standard protocols [30,31], the 3′-end overhang
of the λ-DNA is ligated to an oligomer with a sequence aggtcgccgcccBattggBattccBttccaBgtttaB 3 of 12 Micromachines 2018, 9, 188 (Integrated DNA Technologies, Coralville, IA, USA), where B represents the biotin attached to
the oligomer. The 5′-end overhang of the λ-DNA is ligated to an oligomer with a sequence
gggcggcgacctBattggBattccBttccaBgtttaB (Integrated DNA Technologies) where B again represents
the biotin attached to the oligomer. Both ends of λ-DNA are thus able to bind to the superparamagnetic
beads coated with streptavidin. 2.2. Surface-Functionalization of the Glass Pipette The second step is to surface-functionalize the glass pipette by coating the pipette tip with biotins. The pipette is formed by drawing 1 mm, thick-walled, glass capillaries (1B100-6, World Precision
Instruments, Sarasota, FL, USA) in a horizontal micropipette puller (Sutter Instruments P-97, Novato,
CA, USA). The following settings are used in the P-97: heat = 701, pull = 125, velocity = 110, and time
= 140, and result in rigid pipettes. The pipette tips are next inserted into a solution that contains 10 mg
silane polyethylene glycol (PEG) biotin (PG2-BNSL-600-2K, NANOCS, New York, NY, USA) dissolved
in 200 µL solution of 90% ethanol (E7023, Sigma Aldrich, St. Louis, MO, USA) in water and left for 30
min at room temperature. 2.3. Sample Cell Preparation The third step is to prepare the sample cell. The sample cell is constructed using two number
0 coverslips of differing lengths which we call coverslip 1 (72198-20, Electron Microscopy Science,
Hatfield, PA, USA) and coverslip 2 (72198-10, Electron Microscopy Science). In addition, a custom
3D-printed spacer (Watershed XC 11122, Proto Labs Inc., Maple Plain, MN, USA) and a 3 mm × 2 mm
× 1 mm neodymium bar magnet (M0301, SUPERMAGNETMAN, Pelham, AL, USA) are also used. The coverslip 1 and coverslip 2 are glued on top and bottom of the spacer forming the ceiling and floor
of the sample cell. The sides of the spacer form three of the four sides of the sample cell; one side is left
open to allow insertion of the pipette. The spacer has an inlet and outlet for buffer exchange. The bar
magnet is glued at the bottom of the coverslip 2. A clear room-temperature vulcanizing (RTV) silicone
sealant (All Purpose 100% Adhesive Sealant, DAP, Baltimore, MD, USA) is used for gluing the two
coverslips and the bar magnet. The overall dimension of the sample cell is 60 mm × 40 mm × 1 mm;
however, the enclosed region is 6 mm × 4 mm × 1 mm, yielding a sample cell volume ~100 µL. 2.4. DNA-Bead Construct The fourth step is to incubate in 1× Tris-EDTA (TE), 150 mM NaCl buffer (henceforth referred
to as the “buffer”) at room temperature for about 10 min 5 µL end-functionalized DNA with 30 µL
pre-washed 2.8 µm diameter superparamagnetic beads (11205D, Thermo Fisher Scientific, Waltham,
MA, USA). After incubation, 5 µL of DNA-bead construct is introduced through the inlet of the sample
cell—see Figure 1b. Once the DNA-bead construct settles to the floor of the sample cell, a Tygon tube
(ACF00001, Saint-Gobain, Courbevoie, France) is connected to the inlet of the sample cell. The other
end of this tube is connected to a syringe. The syringe is connected to a syringe pump (NE-1000,
New Era Pump System, Farmingdale, NY, USA). Another Tygon tube (ACF00002, Saint-Gobain) is
connected to the outlet of the sample cell, while the other end of this tube is connected to another
syringe connected to a syringe pump (NE-1000, New Era Pump System). Prior to connecting the two
tubes into the sample cell, all the tubes and syringes are filled with buffer. The pre-wash procedure for
the superparamagnetic beads is described in detail in the supplementary materials. 4 of 12 Micromachines 2018, 9, 188 Figure 1. Basic principle of the horizontal magnetic tweezers:
(a) the tweezers uses
functionalized glass pipette to manipulate a bead attached to one end of a DNA tether and immo
by biotin-streptavidin interactions to the surface of a rigid glass pipette. The other end of th
is attached to a superparamagnetic bead suspended in buffer and placed at varying distanc
a 3 mm × 2 mm × 1 mm bar magnet. Forces ranging from 0.5 pN to 100 pN (and higher)
produced by moving the DNA-magnet distance from 2000 µm to 100 µm; (b) the design of the
cell is shown. The sample cell has an inlet and an outlet for buffer exchange. The open sid
sample cell allows the insertion of the functionalized glass pipette used to capture and imm
DNA-bead pairs; (c) a block diagram showing the layout of the horizontal magnetic tweezers. Figure 1. Basic principle of the horizontal magnetic tweezers:
(a) the tweezers uses a rigid
functionalized glass pipette to manipulate a bead attached to one end of a DNA tether and immobilized
by biotin-streptavidin interactions to the surface of a rigid glass pipette. 2.5. Horizontal Magnetic Tweezers The fifth step is to integrate the DNA-loaded sample cell and surface-functionalized glass pipette
into the horizontal magnetic tweezers. The sample cell is mounted onto a 3D-printed sample cell
holder made from a carbon-fiber reinforced plastic (CFRP). The sample cell holder is attached to
a linear motorized stage system (X-MCB1, AB103B, XJOY3, Zaber, Vancouver, BC, Canada) which
allows the sample cell to be moved in three axes. The motorized micromanipulator is controlled by
a custom LabVIEW program (National Instruments, Austin, TX, USA) running on a Windows PC. The functionalized glass pipette is clamped into a custom-designed, two-piece, aluminum glass pipette
holder; the holder is attached to a hydraulic, three-axis micromanipulator (MX630L S3432, Siskiyou
Corporation, Grants Pass, OR, USA). Next, after the clamped pipette is brought into focus, we carry out the following procedure
to measure the distance between the magnet and the glass pipette:
(i) using the motorized
micromanipulator we bring the magnet to the pipette so that the face of the magnet touches the
glass pipette; (ii) the magnet is moved parallel to the pipette so that the edge of the pipette is at the
edge of the magnet; (iii) the controller for the motorized micromanipulator is zeroed, thus defining
this point as the origin; (iv) the magnet is moved upward with respect to the pipette until the pipette
rests on the floor of the sample cell and at bottom of the edge of the magnet; (v) repeat step (iii) above;
(vi) the magnet is moved downward with respect to the pipette roughly 500 µm; (vii) repeat step
(iii) above; (viii) the magnet is moved parallel to the pipette to position the edge of the pipette at the
geometrical center of the rectangular magnet face, roughly 1000 mm from the magnet edge; (ix) repeat
step (iii) above. At this point, the pipette reference point is at the geometrical center of the rectangular
magnet face. Both syringe pumps connected to the inlet and the outlet, respectively, are activated to
allow exchange of the buffer with fresh buffer. The motorized stage system and the hydraulic micromanipulator are built around a Nikon
Diaphot TMD inverted light microscope (Nikon, Tokyo, Japan) with a 40×, 0.65 NA, bright-field
objective (Leica, Wetzlar, Germany). Imaging is performed using a Point Grey Grasshopper3 camera
(GS3-U3-23S6M-C) with a frame rate of 120 Hz. 2.4. DNA-Bead Construct The other end of the tether
is attached to a superparamagnetic bead suspended in buffer and placed at varying distances from
a 3 mm × 2 mm × 1 mm bar magnet. Forces ranging from 0.5 pN to 100 pN (and higher) can be
produced by moving the DNA-magnet distance from 2000 µm to 100 µm; (b) the design of the sample
cell is shown. The sample cell has an inlet and an outlet for buffer exchange. The open side of the
sample cell allows the insertion of the functionalized glass pipette used to capture and immobilize
DNA-bead pairs; (c) a block diagram showing the layout of the horizontal magnetic tweezers. 5 of 12 Micromachines 2018, 9, 188 2.5. Horizontal Magnetic Tweezers A zoom lens (Edmund Optics, Barrington, NJ, USA)
is also placed between the camera and the microscope objective. See the Supplementary Materials
section for additional details about the design of the tweezers. 2.6. DNA Pulling Experiments After completing the preceding steps, we are in a position to perform experiments. First, we must
locate a DNA-tethered bead. Once a suitable candidate has been found, we move the magnet closer to
the bead at a speed of 10 µm/s until we observe the DNA overstretching transition. This transition is
very distinctive as the contour length of the DNA increases by 70%. This confirms that we have at least
one DNA tethered to the suspended bead. Next, we move the magnet ~2 mm away from the tethered
bead. Then, we start recording images using a custom Labview program. The custom MatLab code [30] for data analysis uses a particle-tracking algorithm to detect
the centroids of the two tethered beads in each frame and extract their coordinates. This allows
computation of the tether’s extension, ⟨z⟩, as well as the transverse fluctuations of the suspended bead,
⟨∆y2⟩. The applied force on the DNA is determined using the fluctuation–dissipation relation: F = kBT⟨z⟩
⟨∆y2⟩
(1) (1) Here, kB is the Boltzman’s constant, T is the absolute temperature of ~297 K, ⟨z⟩is the DNA tether,
and ⟨∆y2⟩is the variance in the transverse direction of the tethered bead. To improve the precision of the force measurements, we average the force computed using
Equation (1) at each positon/time interval from the magnet. This increases the force resolution as it
smooths over fluctuations from mechanical and other a-thermal noise sources. We likewise average Micromachines 2018, 9, 188 6 of 12 the extensions,⟨z⟩; however, it should be noted that even before this averaging step, each measurement
of ⟨z⟩has been spontaneously thermally averaged because the suspended, tethered bead is embedded
in a thermal bath and the imaging system’s integration time is sufficient to allow proper sampling of
the bead’s position from the relevant Boltzmann distribution; the additional averaging step corrects
for a-thermally-generated noise. We report three types of experiments. In the first set, we calibrate our magnetic tweezers by using
it to determine the tether’s force–extension profile, including the force and extension associated with
the overstretching transition, and then comparing it with the known results. Since we can reliably
generate the overstretching transition in our system, we have an especially powerful way to establish
the accuracy of our measurements. 2.6. DNA Pulling Experiments Force–extension measurements are taken by adjusting the distance
between the supermagnetic bead suspended near the magnet and the magnet using the following
protocol: all distances are measured from the magnet: 2 min each at 2 mm, 1.8 mm, 1.6 mm, 1.4 mm,
1.2 mm, 1.0 mm, 0.9 mm, and 0.8 mm; 1 min time each at 0.7 mm and 0.59 mm, 30 s each at 0.57 mm,
0.56 mm, 0.55 mm and 0.53 mm; 5 s each at 0.49 mm, 0.48 mm, 0.47 mm, 0.45 mm, 0.43 mm and
0.42 mm from the magnet. The position of the magnet is always adjusted at 10 µm/s. The next set of measurements revolves around quantifying the resolution, i.e., precision or
reproducibility, of the force and extension data. This is done by directly estimating the standard
deviation, i.e., standard error (SE), in forces and extensions for experimental replications. We also used
different copies of the magnets to analyze how much variability in the data is introduced by the choice
of magnets. The third set of experiments studied the extent of hysteresis in our force–extension data
and especially the degree to which the overstretching transition is reversible. This can be done
straightforwardly by moving the magnet closer to the tethered bead until the DNA’s contour length
increases to ~70%, i.e., until we find the overstretching transition, then pause briefly, and then move
the magnet away until its contour length decreases by 70%. Bead–magnet distances are adjusted at
a speed of 10 µm/s. We also studied hysteric effects as a function of salt concentration by repeating
these experiments with two different buffers. Figure 1a shows the basic principles of the horizontal magnetic tweezers, Figure 1b shows the
design of the sample cell, and Figure 1c shows the block diagram of the experimental setup. 3. Results and Discussion Figure 2 presents the results of a typical force versus extension measurement for a single DNA
molecule plotted against the modified worm-like chain model (WLC) of DNA [32]: fzb
kBT = 1
4
1 −⟨z⟩
Lo
+ fz
Ko
−2
−1
4 + ⟨z⟩
Lo
−fz
Ko
(2) (2) In Equation (2), fz is the applied force, kB is Boltzmann’s constant, T is the absolute temperature
of ~297 K, b is the persistence length of 50 nm, Lo is the contour length of 16.4 µm, Ko is the elastic
modulus of DNA which is ~1000 pN, and ⟨z⟩is the DNA’s thermally-averaged end-to-end extension,
which is the distance between the intensity-weighted centroids of the two beads—see Figure 2 caption. The solid black circles represent the experimental data while Equation (2) is plotted in red. The standard
errors (SE) in the force are shown as blue lines; we did not include the S.E. of the DNA extension in
Figure 2 because their magnitudes are too small compared to the magnitude of the S.E. of the force. Our force–extension data matches well with the WLC model for forces up to ~40 pN, which covers
the low-force non-linear entropic and high-force, linear, Hookean elastic response regimes. (Entropic
elasticity refers to a range of forces in which the bending fluctuations in the DNA are suppressed
but there is no change to its zero-force contour length; in the Hookean regime, however, the DNA’s
backbone is stretched, thus altering the molecule’s contour length). Between ~40 pN to ~60 pN,
we find an offset in our data with respect to the model’s predicted response. This is not unusual in
this force range since the value of DNA’s elastic modulus, Ko, may change at these forces; additionally, Micromachines 2018, 9, 188 7 of 12 the presence of DNA strand breaks (nicks) and whether single or both strands are tethered can also
influence the elastic response. At a force of ~65 pN, the tethered DNA undergoes the overstretching transition [24,33,34] with its
contour length increasing by ~70%. The inset to Figure 2 shows two snapshots from an experiment
with the left panel showing the tether at full extension, ⟨z⟩~16.4 µm with an applied force of 44.2 pN,
and the right panel showing the tether in the canonical overstretched regime, with ⟨z⟩≈1.7Lo at a
force of 66.8 pN. 3. Results and Discussion Experimental results of force vs DNA extension experiments on λ-DNA using the horizontal
magnetic tweezers. Our data are represented by the solid black circles; corresponding standard errors
in the forces are shown by blue lines. The solid red line represents the worm-like chain model for a
single DNA molecule’s response to force, which agrees well with our data in the 1.0 pN to 40 pN force
range. This corresponds to DNA extension from 13.7 µm to 16.2 µm. Between 40 pN to 60 pN our data
begins to deviate to the right from the worm-like chain model. This is the region where the DNA is
at is full contour length (around 25 pN) and then begins to stretch. At force range of 52.8 pN up to
60.1 pN with corresponding DNA extension of 16.5 µm up to 16.8 µm, our data further shifted to the
right from the worm-like chain model. At force range of 64.7 pN up to 73.7 pN with corresponding
DNA extension of 18.2 µm up to 29.3 µm, the DNA tether undergoes the overstretching transition with
its zero-force contour length Lo increasing by 70% of Lo. The inset shows two snapshots from our DNA
extension experiments. From left to right, the first snapshot shows a single DNA with contour length
of 16.4 µm with the tethered bead 550 µm from the magnet and corresponding to a force of 44.2 pN. The second snapshot shows a single DNA molecule in the overstretching transition with an extension
of 28.5 µm, ~1.7Lo, at 430 µm from the magnet for a force of ~66.8 pN. Figure 2. Experimental results of force vs DNA extension experiments on λ-DNA using the horizontal
magnetic tweezers. Our data are represented by the solid black circles; corresponding standard errors
in the forces are shown by blue lines. The solid red line represents the worm-like chain model for a
single DNA molecule’s response to force, which agrees well with our data in the 1.0 pN to 40 pN force
range. This corresponds to DNA extension from 13.7 µm to 16.2 µm. Between 40 pN to 60 pN our data
begins to deviate to the right from the worm-like chain model. This is the region where the DNA is
at is full contour length (around 25 pN) and then begins to stretch. 3. Results and Discussion Both the measured extension and the force at which the overstretching transition
is initiated agree very well with established results. Moreover, the overall force–extension profile in
this highly non-linear elastic regime is in very good agreement with known results. This provides
an excellent calibration for our force and extension measurements as the overstretching transition
has been observed using multiple, complementary experimental techniques and shows remarkable
consistency between them [24,34,35]. Note that, by construction, the WLC model cannot reproduce the
overstretching transition. The numerical values for magnet distances, corresponding forces, extensions,
and standard errors displayed in Figure 2 are presented in Table 1. We point out that at forces starting
at 50 pN, the superparamagnetic bead fluctuates at a frequency close to the Nyquist frequency [8,29],
which may lead to sampling biases. Figure 2. Experimental results of force vs DNA extension experiments on λ-DNA using the horizontal
magnetic tweezers. Our data are represented by the solid black circles; corresponding standard errors
in the forces are shown by blue lines. The solid red line represents the worm-like chain model for a
single DNA molecule’s response to force, which agrees well with our data in the 1.0 pN to 40 pN force
range. This corresponds to DNA extension from 13.7 µm to 16.2 µm. Between 40 pN to 60 pN our data
begins to deviate to the right from the worm-like chain model. This is the region where the DNA is
at is full contour length (around 25 pN) and then begins to stretch. At force range of 52.8 pN up to
60.1 pN with corresponding DNA extension of 16.5 µm up to 16.8 µm, our data further shifted to the
right from the worm-like chain model. At force range of 64.7 pN up to 73.7 pN with corresponding
DNA extension of 18.2 µm up to 29.3 µm, the DNA tether undergoes the overstretching transition with
its zero-force contour length Lo increasing by 70% of Lo. The inset shows two snapshots from our DNA
extension experiments. From left to right, the first snapshot shows a single DNA with contour length
of 16.4 µm with the tethered bead 550 µm from the magnet and corresponding to a force of 44.2 pN. The second snapshot shows a single DNA molecule in the overstretching transition with an extension
of 28.5 µm, ~1.7Lo, at 430 µm from the magnet for a force of ~66.8 pN. Figure 2. 3. Results and Discussion At force range of 52.8 pN up to
60.1 pN with corresponding DNA extension of 16.5 µm up to 16.8 µm, our data further shifted to the
right from the worm-like chain model. At force range of 64.7 pN up to 73.7 pN with corresponding
DNA extension of 18.2 µm up to 29.3 µm, the DNA tether undergoes the overstretching transition with
its zero-force contour length Lo increasing by 70% of Lo. The inset shows two snapshots from our DNA
extension experiments. From left to right, the first snapshot shows a single DNA with contour length
of 16.4 µm with the tethered bead 550 µm from the magnet and corresponding to a force of 44.2 pN. The second snapshot shows a single DNA molecule in the overstretching transition with an extension
of 28.5 µm, ~1.7Lo, at 430 µm from the magnet for a force of ~66.8 pN. 8 of 12 Micromachines 2018, 9, 188 Table 1. The values of the force, DNA extension and their corresponding standard error (S.E.) of the
tethered DNA at certain distance from the magnet. Magnet Distance (µm)
Force (pN)
S.E. of the Force (pN)
DNA Extension (µm)
S.E. of the DNA Extension (µm)
2000
1.4
0.56
13.7
0.056
1800
1.8
0.59
14.2
0.043
1600
2.4
0.73
14.6
0.032
1400
3.6
0.75
15.0
0.023
1200
5.8
1.15
15.4
0.016
1000
11.5
2.15
15.5
0.014
900
15.2
1.69
15.7
0.003
800
20.9
2.07
15.8
0.002
700
28.8
3.13
16.0
0.002
590
39.7
1.21
16.2
0.001
570
43.5
2.00
16.3
0.001
560
44.0
2.23
16.4
0.002
550
44.2
3.76
16.4
0.002
530
52.8
2.37
16.5
0.002
490
52.1
2.33
16.7
0.002
480
57.3
2.67
16.7
0.004
470
60.1
2.44
16.8
0.005
450
64.7
1.08
18.2
0.007
430
66.8
2.18
28.5
0.010
420
73.7
2.00
29.3
0.008 We studied the reproducibility of our data by computing force and extension standard errors—see
Table 1 columns 3 and 5—and also by assaying the impact on the data of using different copies of
the magnet. As can be seen from columns 3 and 5 of Table 1, the precision of the force and extension
measurements is consistent with other magnetic tweezers designs with forces of ~0.5 pN and extension
changes of ~10 nm measured reproducibly using our instrument. Columns 1 and 2 of Table 1 have
also been plotted in Figure 3. Figure 3. 3. Results and Discussion Magnetic force acting on a 2.8 µm superparamagnetic bead as a function of distance from
the magnet. The solid black circles represent the force at each position from the magnet—see Table 1
columns 1 and 2. The blue lines represent the standard error in the forces—see Table 1 column 3. Figure 3. Magnetic force acting on a 2.8 µm superparamagnetic bead as a function of distance from
the magnet. The solid black circles represent the force at each position from the magnet—see Table 1
columns 1 and 2. The blue lines represent the standard error in the forces—see Table 1 column 3. We also repeated force–extension experiments using five different copies of the magnet to check
if we can reproduce our experimental results—see Figure 4. In addition to comparing our data with
the predictions of the WLC model (solid red line), we also compared our results to the experimental
data presented in Strick et al. [11] (solid black circles). Data from five experiments are displayed
using five non-black or red solid colored circles. The results shown in Figure 4 provide evidence that
force–extension data are not affected by using different copies of the magnet. We note that in spite of the
inhomogeneity in magnets and superparamagnetic beads, over a wide range of magnet–bead distances,
from 2 mm to 0.7 mm, we found a consistent relation between the magnet–bead distance and the
applied force (and thus the tether’s extension). However, below distances of 0.7 mm, the magnet–bead 9 of 12 Micromachines 2018, 9, 188 distance at which a given force was produced varied with the magnet. Although the overstretching
transition occurs at these distances, we found that the measured overstretching forces were highly
consistent and independent of the magnet used. Figure 4. Replications of force–extension measurements using five different magnets and comparison to
calibration data. The WLC is plotted as red solid line. The black solid circles represent the experimental
results on λ-DNA using optical tweezers as presented in Strick et al. [11]. The remaining solid colored
circles represent data from our experimental replications. Figure 4. Replications of force–extension measurements using five different magnets and comparison to
calibration data. The WLC is plotted as red solid line. The black solid circles represent the experimental
results on λ-DNA using optical tweezers as presented in Strick et al. [11]. The remaining solid colored
circles represent data from our experimental replications. 4. Future Direction We
are
currently
carrying
out
single
molecule
micromanipulation
experiments
on
DNA-compacting proteins. Looking ahead, we intend to modify the tweezers to incorporate
fluorescence measurements, and improve our data analysis and validation approaches by using
techniques described in refs. [38–41]. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-666X/9/4/188/s1,
Horizontal Magnetic Tweezers Design, The prewash procedure for the superparamagnetic beads. Acknowledgments: We gratefully acknowledge the assistance of the following individuals: Keir Neuman,
Yeonee Seol, Jie Yan, Anneliese Striz, Alfonso Lopez Coral, Jennifer Groebner and John Green. A. Sarkar would
like to acknowledge funding from the Catholic University of America and Vitreous State Laboratory. Author Contributions: Roberto Fabian carried out the experiments and data analysis using MATLAB code
written by Christopher Tyson. Roberto Fabian, Christopher Tyson and Abhijit Sarkar designed the instrument. Pamela Tuma, Ian Pegg and Abhijit Sarkar supervised the experiments and assisted with the data analysis. Roberto Fabian, Christopher Tyson, Pamela Tuma, Ian Pegg and Abhijit Sarkar wrote the manuscript. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results. 3. Results and Discussion 10 of 12 Micromachines 2018, 9, 188 10 of 12 For comparison, Figure 5b shows hysteretic effects in the DNA overstretching transition when the
experiment is repeated under otherwise identical conditions except 1× TE buffer with 0 mM NaCl is
used. The asymmetry between the extensional and the contractile phases is marked and consistent with
similar data from other groups [36,37]. This result may be interpreted as the presence of a large section
of melted DNA beyond the overstretching force which prevents the two strands for re-annealing once
the force is relaxed—see [36] for a detailed discussion. 3. Results and Discussion Also evident in Figure 4 is the excellent agreement between the data from reference [11] obtained
using optical tweezers to stretch λ-DNA, and our data across the entire force range. Also evident in Figure 4 is the excellent agreement between the data from reference [11] obtained
using optical tweezers to stretch λ-DNA, and our data across the entire force range. We wanted to establish if our overstretching data were reversible and to study how the
DNA’s elastic response was affected by the buffer’s salt concentration. The results are shown in
Figure 5. Figure 5a shows that the DNA overstretching transition is reversible showing very little
hysteresis, i.e., looking left to right, the extension profile is symmetrical with the contraction profile. This experiment was done by moving the magnet closer to the tethered DNA until it underwent the
overstretching transition (length abruptly increased from Lo (=16.4 µm) to 1.7Lo (=28 µm)) and then
after a pause, moved the magnet away from the tethered DNA until its length decreased back to
Lo—please see the Methods section for more details on the experimental protocol. We used 1× TE
buffer with 150 mM NaCl with experiments performed at room temperature. Figure 5. Hysteresis in DNA overstretching transition: (a) the extension-time graph of the DNA during
a reversible overstretching transition. This experiment is done on 1× Tris-EDTA (TE) buffer with
150 mM NaCl at room temperature. Looking left to right, the extensional and contractile phases are
symmetrical; (b) the extension-time graph of the DNA during a hysteretic overstretching transition. This experiment is done on 1× TE buffer in the absence of salt at room temperature. Looking left to
right, the asymmetry between the extensional and contractile responses is clear visible. Bead-magnet
distance are adjusted at a speed of 10 µm/s in (a,b). Figure 5. Hysteresis in DNA overstretching transition: (a) the extension-time graph of the DNA during
a reversible overstretching transition. This experiment is done on 1× Tris-EDTA (TE) buffer with
150 mM NaCl at room temperature. Looking left to right, the extensional and contractile phases are
symmetrical; (b) the extension-time graph of the DNA during a hysteretic overstretching transition. This experiment is done on 1× TE buffer in the absence of salt at room temperature. Looking left to
right, the asymmetry between the extensional and contractile responses is clear visible. Bead-magnet
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Evaluation of cytological diagnostic accuracy for canine splenic neoplasms: An investigation in 78 cases using STARD guidelines
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OPEN ACCESS Citation: Tecilla M, Gambini M, Forlani A, Caniatti
M, Ghisleni G, Roccabianca P (2019) Evaluation of
cytological diagnostic accuracy for canine splenic
neoplasms: An investigation in 78 cases using
STARD guidelines. PLoS ONE 14(11): e0224945. https://doi.org/10.1371/journal.pone.0224945 Editor: Douglas H. Thamm, Colorado State
University, UNITED STATES
Received: July 21, 2019
Accepted: October 24, 2019
Published: November 7, 2019 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.pone.0224945 Copyright: © 2019 Tecilla et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. RESEARCH ARTICLE Marco Tecilla☯, Matteo GambiniID☯*, Annalisa Forlani¤, Mario Caniatti, Gabriele Ghisleni,
Paola Roccabianca Diagnostic Pathology Service, Dipartimento di Medicina Veterinaria (DIMEVET), Università degli Studi di
Milano, Lodi, Italy ☯These authors contributed equally to this work. ¤ Current address: IDEXX Laboratories, Wetherby, West Yorkshire, United Kingdom
* matteo.gambini@unimi.it a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 Abstract Cytology represents a useful diagnostic tool in the preliminary clinical approach to canine
splenic lesions, and may prevent unnecessary splenectomy. However, few studies have
evaluated diagnostic accuracy of cytology in the diagnosis of canine splenic neoplasms. The aim of this study was to determine overall accuracy, sensitivity, specificity, positive and
negative predictive values (i.e. diagnostic accuracy indexes) of cytology for canine splenic
neoplasms following Standards for the Reporting of Diagnostic Accuracy Studies (STARD)
guidelines. A consecutive series of canine splenic cytological samples was retrospectively
retrieved from the database of the Diagnostic Pathology Service of the Department of Veter-
inary Medicine (DIMEVET—University of Milan). Histopathology was set as the diagnostic
reference standard. Cytological cases were enrolled when slides were available for review
and when the same lesion was submitted for histopathology. Seventy-eight (78) lesions
were included in the study. By histopathology, 56 were neoplastic and 22 were non-neoplas-
tic. Cytology had an overall accuracy of 73.08% (95% C.I. 61.84%-82.50%), sensitivity of
64.29% (95% C.I. 50.36%-76.64%), specificity of 95.45% (95% C.I. 77.16%-99.88%), and
positive and negative predictive values of 97.3% (95% C.I. 84.01%-99.60%) and 51.22%
(95% C.I. 42.21%-60.15%), respectively. Low sensitivity and negative predictive value were
balanced by very high specificity and positive predictive value. When positive for neoplasia,
cytology represents a useful diagnostic tool to rule in splenic neoplasia, prompting surgery
independently from other diagnostic tests. Conversely, a negative cytological result requires
additional investigations to confirm the dog to be disease free. Cytological diagnostic accuracy and canine splenic neoplasms splenic malignant tumor of dogs [2]. Still, HES represents fewer than 25% of overall splenic
lesions [3] with up to 74% of dogs being diagnosed with benign lesions [4] such as hematoma
and hyperplasia [2,5]. However, due to the poor prognosis associated with HES, splenectomy
is still the routine approach to most canine splenic masses for both diagnostic and therapeutic
purposes [6–8]. Funding: The authors received no specific funding
for this work. All the authors were employed by the
Università degli Studi di Milano at the time by
which the study was conducted and concluded. One of the authors [A.F.] is currently employed by
IDEXX Laboratories (Wetherby, West Yorkshire,
UK). This Company did not provide support in the
form of salary for any of the authors at the time by
which the study was conducted, neither had any
additional role in the study design, research
material supply, data collection and analysis,
decision to publish, or preparation of the
manuscript. While the two-month post-splenectomy survival rate is lower in dogs with HES (32%) com-
pared to dogs with hematomas (83%), the general survival rate after splenectomy is 52%,
regardless of underlying splenic pathology [3]. While this can be interpreted as fair survival
data, a proportion of dogs (7.6%) will develop complications secondary to splenectomy
because of thrombotic or coagulopathic syndromes [9]. Additional adverse effects following
splenectomy in dogs have included peri- and post-operative ventricular arrhythmias [9–11],
reduced blood filtration and renewal [12,13], impairment of humoral immune response [14],
reduced immune-surveillance against bacteria and parasites [15–19], and higher incidence of
gastric dilatation-volvulus [6,20–22]. For these reasons, any preoperative diagnostic approach
to splenic lesions, including cytology, may be beneficial in preventing unnecessary splenec-
tomy. Notwithstanding the common belief that splenic aspiration can be dangerous especially
when investigating cavitated masses [6,7,23], complications from splenic aspiration proce-
dures are rarely elicited even in thrombocytopenic animals [8,24–26]. For comparison, in
human medicine splenic fine needle aspiration (FNA) cytology is seldom associated with com-
plications [27,28], resulting in 5.2% secondary complications with fewer than 1% considered
severe and consisting mostly of controllable hemorrhage [29]. Competing interests: One of the authors [A.F.] is
currently employed by IDEXX Laboratories
(Wetherby, West Yorkshire, UK) which did not
provide support in the form of salary for any of the
authors at the time by which the study was
conducted, neither had any additional role in the
study design, research material supply, data
collection and analysis, decision to publish, or
preparation of the manuscript. This does not alter
our adherence to PLOS ONE policies on sharing
data and materials. Thus, attempts to minimize unnecessary splenectomy should prompt an increased use of
additional diagnostic techniques as preoperative screening tests to characterize splenic disease. Fine needle aspiration cytology can provide diagnostic information useful to distinguish
inflammatory, benign and malignant nodular lesions and to assess generalized splenomegaly
[7,24,30]. Despite the relatively high frequency of splenic diseases in dogs, data regarding usefulness
and validity of diagnostic cytology are fragmentary. In veterinary medicine, no studies have
comprehensively assessed overall accuracy, sensitivity, specificity, positive and negative predic-
tive values of canine splenic cytology against histopathology utilizing the Standards for the
Reporting of Diagnostic Accuracy Studies (STARD) guidelines [8,23–25,31–33]. STARD
guidelines have been created to avoid incomplete reporting in diagnostic accuracy studies and
to improve the general quality of the latter, reducing problems related to study identification,
critical appraisal, and replication [34]. Overall agreement between cytology and histology of
canine splenic lesions is the most frequent index reported, ranging from 38 to 100% [8,23–
25,31–33]. Specifically, this index has been evaluated on a limited number of splenic cytologi-
cal specimens (range 5–40) [8,23–25,31–33]. In most reports, sensitivity, specificity, positive
and negative predictive values of splenic cytology are not calculated and cannot be properly
estimated [8,23–25,31–33] because caseloads simultaneously include multiple species
[24,25,31–33], multiple tissues and organs [31–33], or “equivocal” or “provisional” cytological
and histological diagnoses [23,24,33]. Application of STARD guidelines in the current study
allowed cross-tabulation of cytological results (i.e. the index test) against those of histopathol-
ogy (i.e. the reference standard) to generate sensitivity and specificity data [34]. These data
have not been included in previous studies and will be useful for future researchers comparing
diagnostic methods for canine splenic neoplasms. Introduction Ultrasonographic examination of nodular splenic lesions in dogs is not reliable to differentiate
with certainty benign and malignant processes, necessitating the use of additional, ideally min-
imally invasive, diagnostic tests [1]. Hemangiosarcoma (HES) is the most common primary Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. 1 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 Criteria of selection of cases In this retrospective study, the electronic cytological database of the Diagnostic Pathology
Service of the Department of Veterinary Medicine (DIMEVET) of the University of Milan
was searched for splenic samples collected between January 1st 1998 to January 31st 2018. The
database was searched for specific key words in the following combinations: 1) “dog” and
“spleen”, 2) “dog” and “splenic”. A consecutive series of canine splenic cytologies was
obtained. Cytological samples came from external referring private practices or from the Vet-
erinary Teaching Hospital (VTH) of the DIMEVET, or were prepared from fresh surgical
biopsies and necropsies. Samples were submitted or collected to evaluate splenomegaly or
nodular lesions. The histopathology database was then searched for the histopathology corresponding to the
same lesion examined by cytology. Histopathological samples were obtained from splenic
biopsies (nodular lesions) or whole spleens (from splenectomies or necropsies) submitted to
the Diagnostic Pathology Service of the DIMEVET, or were represented by slides submitted by
external pathologists as second opinion cases. A time interval >45 days between cytological
and histological sampling was an exclusion criterion. Histopathological samples collected via
needle core biopsies were excluded from the study as they may bear reduced diagnostic reli-
ability compared to incisional and excisional histological samples [8,37]. Cases were included
in the study only when cytological and histological slides of the same lesion were available for
review. Additional information collected from the archives for cases included in the study were:
sex, age, breed, cytological sampling technique (e.g. fine needle aspiration–FNA, touch
imprint, scraping) and gross appearance of the lesion (i.e. diffuse versus nodular lesion). To improve data completeness, transparency, and reproducibility, the study was con-
ducted following the STARD guidelines [34] to the best of authors’ ability. All canine splenic
samples included in the current retrospective work, regardless of the sampling technique
applied, were submitted to the Diagnostic Pathology Service of the DIMEVET for diagnostic
purposes of spontaneous developing diseases. No animals were sampled or euthanized for
research use. The use of animal tissue in the current study was approved by the Ethics Com-
mittee in charge for animal welfare of the University of Milan (Organismo Preposto al Bene-
ssere degli Animali, OPBA) with protocol number OPBA_86_2019. Sensitive information
regarding owners and animals were stored, managed and preserved according to European
and Italian laws. To avoid unnecessary splenectomy, a diagnostic test with a high sensitivity and negative
predictive value is desirable because these indexes measure the percentage of diseased dogs
correctly diagnosed with splenic neoplasia and the probability that dogs with a negative cytol-
ogy truly do not have a neoplasm, respectively. In this context, the aim of this study was to PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 2 / 15 Cytological diagnostic accuracy and canine splenic neoplasms determine the diagnostic accuracy of cytology in the diagnosis of canine splenic neoplasms uti-
lizing the corresponding histopathology as the diagnostic reference standard [24,31,33,35,36],
following STARD guidelines [34]. Additionally, sensitivity of cytology in the diagnosis of spe-
cific tumor types and in the identification of nodular versus diffuse neoplasms was evaluated. Case review All cytological and histopathological samples were independently reviewed in a blinded fash-
ion by three cytologists (M.C.—ECVP, G.G.—ECVCP, M.G.—resident) and by three anatomi-
cal pathologists (A.F.–ECVP, P.R.—ECVP, M.T.—resident), respectively. Both cytologists and
anatomical pathologists were blinded to signalment information related to each case. All cytological and histopathological samples were independently reviewed in a blinded fash-
ion by three cytologists (M.C.—ECVP, G.G.—ECVCP, M.G.—resident) and by three anatomi-
cal pathologists (A.F.–ECVP, P.R.—ECVP, M.T.—resident), respectively. Both cytologists and
anatomical pathologists were blinded to signalment information related to each case. For each cytological case, one slide for each sampling technique was reviewed. First, slides
were examined at low-power magnification (i.e. 10x objective lens) to assess the adequacy of
the specimen. Poorly cellular samples were those characterized by marked hemodilution in the
absence of both stromal elements and a mixed population of leukocytes [30]. Poorly cellular
samples, poorly smeared samples (i.e. too thick or where most cells were ruptured), and sam-
ples where stain quality impaired adequate definition of the cell type (e.g. formalin-contami-
nated smears), were considered inconclusive [30]. Inconclusive cases were excluded from the
statistical analysis as previously reported [8,24,32,33,35–37]. Cytological diagnoses were expressed according to those reported in the literature
[4,30,38,39]. To facilitate comparison of the agreement between cytological and histological
results, each cytological sample was further classified as neoplastic or non-neoplastic according
to the main pathologic process. Non-neoplastic samples were those characterized by degenera-
tive, reactive (including extramedullary hematopoiesis) [23,30], and inflammatory changes, as
well as normal specimens consisting of stromal elements with mixed leukocyte population
[30,38]. Reviewing cytologists were not allowed to use diagnostic modifiers such as “probably”,
“most likely”, “suggestive of”, as previously reported [37], nor to provide equivocal diagnoses
(i.e. reporting more than one differential diagnosis). When a univocal diagnosis was not
reached, cytologists reviewed the case collaboratively to find an agreement. Only the definitive
diagnosis was included in the consecutive statistical analysis. Neoplastic cytological samples were further subdivided by tumor type into the following
subcategories: benign soft tissue tumor (BSTT) including angioma, angiosarcoma (HES), soft
tissue sarcoma (STS) excluding angiosarcoma, lymphoma (LYM), mast cell tumor (MCT), his-
tiocytic tumors including hemophagocytic sarcoma (HS), other round cell tumors including
plasma cell tumor, myeloid leukemia and undifferentiated round cell tumor (ORCT), carci-
noma (CARC), malignant neoplasm not otherwise specified (MNNOS). Case review Cases were included only when the three anatomical pathologists were in agreement
because histopathology served as the diagnostic reference standard to evaluate cytological
diagnostic accuracy. Neoplasms were classified applying the World Health Organization’s his-
tologic classification of tumors in domestic animals [40–48]. To further standardize histopath-
ological diagnoses, anatomic pathologists were invited to classify some specific pathological
entities (i.e. lymphomas, histiocytic proliferative disorders, nodular lesions previously classi-
fied as “fibrohistiocytic nodules”) according to criteria reported in recent literature [49–52]. For nodular lesions, histological samples were considered conclusive and therefore only
included in the statistical analysis if at least one slide containing at least one margin between
the nodule and the adjacent splenic parenchyma was available for review [2,3,5]. Histological samples were then classified as neoplastic and non-neoplastic. Neoplastic cases
were further subdivided utilizing the same subcategories applied to cytological samples. Sample processing Cytological samples were air dried and stained with May-Gru¨nwald-Giemsa (Merck KGaA,
Frankfurt, Germany). Tissue samples for histopathology were fixed in 10% neutral buffered
formalin, processed routinely, and embedded in paraffin wax. Sections of 5 μm were stained
with Hematoxylin and Eosin. Second opinion cases were provided as Hematoxylin and Eosin
stained slides by the referring pathologists. 3 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 Cytological diagnostic accuracy and canine splenic neoplasms Data analysis For all cases, the cytological diagnosis was compared with its paired histopathological diagno-
sis. Since histological samples maintain tissue architecture and are not biased by cellularity
[8,32,37,53], histopathology was set as the reference standard as previously reported
[24,31,33,35,36]. 4 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 Cytological diagnostic accuracy and canine splenic neoplasms To determine diagnostic accuracy indexes, cytological specimens were classified according
to four correlation categories (true positive, true negative, false positive, false negative). Specifi-
cally, the True Positive (TP) category included all cytological samples diagnosed as neoplastic
with a corresponding neoplastic histopathology. The True Negative (TN) category comprised
all cytological samples diagnosed as non-neoplastic with a corresponding non-neoplastic his-
topathology. The False Positive (FP) category included all cytological samples diagnosed as
neoplastic with a corresponding non-neoplastic histopathology. The False Negative (FN) cate-
gory comprised all non-neoplastic cytological diagnoses with a corresponding neoplastic
histopathology. To evaluate the sensitivity of cytology in the diagnosis of specific tumor types, only those
cases histologically confirmed as neoplastic were taken into account. The subcategories
assigned to each cytological and corresponding histological sample were then compared. When cytological and histological diagnoses matched for both neoplastic categorization and
tumor type subcategorization, the case was defined as “true positive with complete agreement”. When cytological and histological diagnoses matched for the neoplastic categorization but did
not match for the tumor type subcategorization, the case was defined as “true positive with
partial agreement”. When a histopathological diagnosis categorized as neoplastic corre-
sponded to a cytological diagnosis categorized as non-neoplastic, the case was considered in
disagreement and defined as “false negative case”. PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 Statistical methods Cytological-histological correlation categories (TP, FP, TN, FN) were included in a 2x2 table
and used to calculate point estimates of overall accuracy, sensitivity, specificity, positive and
negative predictive values [36,54]. Overall accuracy was defined as the ability of cytology to
correctly identify neoplastic and non-neoplastic lesions, and was calculated as the sum of cases
in which cytology and histology agreed in diagnosing a lesion as neoplastic (i.e. TP) or non-
neoplastic (i.e. TN), divided by the total number of cases included in the study [32,55]. Given
that pre-determined acceptability criteria for diagnostic performance of splenic cytology to
distinguish between neoplastic and non-neoplastic lesions have not been previously estab-
lished, overall accuracy, sensitivity, specificity, positive and negative predictive values were
considered low if <70%, moderate if 70% and <80%, high if 80% and <90%, and very
high if 90% [54]. To increase data comparability with other studies, positive and negative
likelihood ratios were calculated [36]. Ninety-five percent (95%) confidence interval was calcu-
lated for each of the above mentioned indices of diagnostic test accuracy using a web-based
application (MEDCALC—https://www.medcalc.org/calc/diagnostic_test.php). The level of agreement between cytology and histopathology in the diagnosis of splenic neo-
plastic conditions was further investigated calculating the Cohen’s kappa coefficient (κ), which
was then corrected by the standard error. The value of k can be indicative of no agreement (if
k <0), slight agreement (k = 0–0.20), fair agreement (k = 0.21–0.40), moderate agreement
(k = 0.41–0.60), substantial agreement (k = 0.61–0.80), almost perfect agreement (k = 0.81–
0.99), or perfect agreement (k = 1) [36,54]. Cohen’s kappa and standard error were calculated
utilizing GraphPad QuickCalcs Web site (GraphPad Inc.—https://www.graphpad.com/
quickcalcs/kappa2/). Sensitivity of cytology in differentiating splenic tumor types was defined as the ability of
cytology to correctly identify as neoplastic a sample belonging to a specific neoplastic subcate-
gory. Therefore, sensitivity for each tumor type was calculated as the sum of cases in complete
and partial agreement (i.e. true positive cases) divided by the total number of cases with that
specific neoplasm [31]. 5 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 Cytological diagnostic accuracy and canine splenic neoplasms Similarly, the sensitivity of cytology in the diagnosis of neoplastic lesions according to
their distribution pattern (i.e. diffuse or nodular) was evaluated. For each distribution pat-
tern, sensitivity was calculated as the sum of cases in complete and partial agreement (i.e. Statistical methods true positive) divided by the total number of cases with a specific distribution pattern. Sensitivity of cytology according to distribution pattern was calculated for neoplastic
lesions in general (i.e. the general sensitivity value obtained in our study), as well as for those
specific neoplastic subcategories including cases with either diffuse or nodular distribution
pattern. Chi-square analysis applied to pairwise comparison was performed to evaluate whether sta-
tistically significant differences existed in the sensitivity of cytology for different tumor types,
as well as in the diagnosis of neoplastic lesions with diffuse or nodular distribution pattern
[36,37]. Specifically, the sensitivity of cytology for each neoplastic subcategory was compared
with the sensitivity for splenic neoplasms in general, the sensitivity for all other neoplastic sub-
categories, and the sensitivity for any other neoplastic subcategory. Similarly, the difference
between sensitivity for nodular or diffuse lesions among neoplasm in general and for each
neoplastic subcategory was statistically investigated. Chi-square analysis was performed only
on sensitivity values different from 0% and 100%, using MEDCALC (https://www.medcalc. org/calc/comparison_of_proportions.php). A p-value <.05 was considered statistically
significant. PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 Cytological diagnostic accuracy and canine splenic neoplasms Table 1. Prevalence, agreement levels and sensitivity of cytology in the diagnosis of each neoplastic subcategory. Prevalence
TP cases with complete agreement
TP cases with partial agreement
FN cases
Sensitivity
Confidence Interval (95%)
TOTAL
71.79% (56/78)
42.86% (24/56)
21.43% (12/56)
35.71% (20/56)
64.29%
50.36%–76.64%
HES
28.57% (16/56)
68.75% (11/16)
6.25% (1/16)
25% (4/16)
75%
47.62%–92.73%
LYM
28.57% (16/56)
37.50% (2/16)
12.50% (6/16)
50% (8/16)
50%
24.65%–75.35%
STS
12.50% (7/56)
42.86% (3/7)
28.57% (2/7)
28.57% (2/7)
71.43%
29.04%–96.33%
BSTT
8.93% (5/56)
0% (0/5)
0% (0/5)
100% (5/5)
0%
0.00%–52.18%
HS
7.14% (4/56)
25% (1/4)
50% (1/4)
25% (1/4)
75%
19.41%–99.37%
MCT
7.14% (4/56)
100% (4/4)
0% (0/4)
0% (0/4)
100%
39.76%–100%
CARC
5.36%(3/56)
66.67% (2/3)
33.33% (1/3)
0% (0/3)
100%
29.24%–100%
ORCT
1.79% (1/56)
0% (0/1)
100% (1/1)
0% (0/1)
100%
2.50%–100%
BSTT, benign soft tissue tumor including angioma; CARC, carcinoma; FN, false negative; HES, angiosarcoma; HS, histiocytic neoplasm (including hemophagocytic
syndrome); LYM, lymphoma; MCT, mast cell tumor (MCT); ORCT, other round cell tumor; STS, soft tissue sarcoma other than angiosarcoma; TP, true positive. https://doi.org/10.1371/journal.pone.0224945.t001 , agreement levels and sensitivity of cytology in the diagnosis of each neoplastic subcategory. BSTT, benign soft tissue tumor including angioma; CARC, carcinoma; FN, false negative; HES, angiosarcoma; HS, histiocytic neoplasm (including hemophagocytic
syndrome); LYM, lymphoma; MCT, mast cell tumor (MCT); ORCT, other round cell tumor; STS, soft tissue sarcoma other than angiosarcoma; TP, true positive. https://doi.org/10.1371/journal.pone.0224945.t001 Animals and samples From a total of 950 splenic cytological samples retrieved between 1998–2018, 92 cytological
samples from 91 dogs were included in the study; one dog was sampled for two distinct splenic
lesions. A total of 858 splenic cytological cases were excluded for the following reasons: lack of
a corresponding histopathological sample (832 cases), unavailable cytological and/or histologi-
cal samples (16 cases), and needle core biopsies (10 cases). Among the selected cytological
cases, 14 were considered inconclusive, and therefore excluded from the consecutive statistical
analysis. Detailed evaluation of diagnostic accuracy was performed on 78/92 reviewed cytolog-
ical samples (retrieval rate: 84.78%) obtained from 77 dogs [37]. Sex was available for 76/77 dogs: 19 were spayed females, 6 neutered males, 21 intact
females, and 30 intact males. Mean age was 9.05 years (age range 2 months-16 years; age was
not available for 2 cases). Twenty-six (26) breeds other than mongrels were represented; in one
case breed was not provided. The time interval between cytological sampling and corresponding histopathology collec-
tion ranged from 0 to 44 days for all cases. Of the 78 cases included in the study, 81 cytological slides were evaluated (3 cases were sam-
pled with two different techniques, i.e. touch imprinting and scraping). Cytological samples
consisted of 43/81 touch imprints (53.09%) collected from both surgical biopsies and necrop-
sies, 28 FNAs (34.57%), of these 21 were ultrasound guided, 1 was CT-scan guided, 1 was
obtained during surgery, while in 5 FNA biopsies no additional sampling information was
available. In 6 cases scrapings were obtained from surgical and necropsy specimens (7.41%). In 4 cases (4.94%) the sampling technique was not specified. Complete agreement among anatomical pathologists was reached for all the 78 correspond-
ing histopathological samples. Histopathological specimens were distributed as follows: 51 sur-
gical samples from partial or complete splenectomies (51/78 cases, 65.38%), 24 spleens from
necropsies (24/78, 30.77%), and 3 cases submitted as a second opinion (3/78, 3.85%). 6 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 confidence interval; FN, false negative; HES, angiosarcoma; LYM, lymphoma; MCT, mast cell tumor (MCT); TP, true positiv CI, confidence interval; FN, false negative; HES, angiosarcoma; LYM, lymphoma; MCT, mast cell tumor (MCT); TP, true positive. s://doi.org/10.1371/journal.pone.0224945.t002 https://doi.org/10.1371/journal.pone.0224945.t002 Cytological and histological diagnoses All cytological and corresponding histopathological diagnoses (78 cases) are listed in S1 Table. The diagnoses for the cytological-histological pairs excluded due to inconclusive cytology are
listed in S2 Table. No diagnostic differences were found for samples collected using two different sampling
techniques, and therefore they were considered as one case in the consecutive statistical analy-
sis. Cytologically, 37/78 cases were diagnosed as neoplastic (47.44%) and 41/78 as non-neo-
plastic (52.56%). All cases diagnosed as neoplastic were classified as malignant, and indeed, no
benign neoplasms were cytologically observed. Histologically, 56/78 cases (71.79%) were neoplastic (S1 Table) and 22/78 cases (28.21%)
were non-neoplastic. Malignant tumors were 51 (51/56 tumors, 91.07%) and 5 were benign. The prevalence of each tumor type is reported in Table 1. No malignant neoplasm not other-
wise specified was included in the study. Of the 78 splenic lesions, 60 were nodular (76.92%), and 17 were diffuse (21.79%), while no
information regarding the distribution pattern was available for 1 case (1.28%). Of the 56 neo-
plastic lesions, 43/56 cases (76.79%) were nodular and 12 cases (21.43%) were diffuse. The case
for which distribution pattern was not provided was a liposarcoma (1.79%). This case was
excluded from the evaluation of cytology sensitivity according to neoplasm distribution pat-
tern. The proportion of cases with nodular or diffuse pattern for each tumor type are reported
in Table 2. Table 2. Prevalence, agreement levels and sensitivity of cytology in the diagnosis of each neoplastic subcategory on the basis of distribution pattern. Nodular
Diffuse
# of cases
TP
FN
Sensitivity
(95% CI)
# of cases
TP
FN
Sensitivity
(95% CI)
TOTAL
43/56 (76.79%)
26/43
17/43
60.47%
(44.41%–75.02%)
12/56 (21.43%)
9/12
3/12
75%
(42.81%–94.51%)
HES
14/16 (87.50%)
10/14
4/14
71.43%
(41.90%–91.61%)
2/16 (12.50%)
2/2
0/2
100%
(15.81%–100%)
LYM
10/16 (62.50%)
5/10
5/10
50%
(18.71%–81.29%)
6/16 (37.50%)
3/6
3/6
50%
(11.81%–88.19%)
MCT
1/4 (25%)
1/1
0/1
100%
(2.50%–100%)
3/4 (75%)
3/3
0/3
100%
(29.24%–100%)
CI, confidence interval; FN, false negative; HES, angiosarcoma; LYM, lymphoma; MCT, mast cell tumor (MCT); TP, true positive. ment levels and sensitivity of cytology in the diagnosis of each neoplastic subcategory on the basis of distribution pattern. Prevalence, agreement levels and sensitivity of cytology in the diagnosis of each neoplastic subcategory on the basis of d PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 7 / 15 Cytological diagnostic accuracy and canine splenic neoplasms Table 3. Cytological and histological diagnoses Cytological-histological correlation categories. Diagnosis
Histology: neoplastic
Histology: non-neoplastic
Total
Cytology: neoplastic
36 (TP)
1 (FP)
37
Cytology: non-neoplastic
20 (FN)
21 (TN)
41
Total
56
22
78
FN, false negative; FP, false positive; TN, true negative; TP, true positive. https://doi.org/10.1371/journal.pone.0224945.t003 Table 3. Cytological-histological correlation categories. FN, false negative; FP, false positive; TN, true negative; TP, true positive. NLR, negative likelihood ratio; NPV, negative predictive value; PLR, positive likelihood ratio; PPV, positive
predictive value. NLR, negative likelihood ratio; NPV, negative predictive value; PLR, positive likelihood ratio; PPV, positive
predictive value. https://doi.org/10.1371/journal.pone.0224945.t004 PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 Cyto-histological correlation Following the tabulation of cytological and histological diagnoses (S1 Table), 36 cases (46.15%)
were classified as TP, 21 (26.92%) were TN, 20 (25.64%) were FN, and 1 (1.28%) was a FP
(Table 3). The FP case had a cytological diagnosis of lymphoma that corresponded histologically to a
purulent bacterial splenitis (in this case the full spleen was available for analysis and no tumor
was found; however, severe marginal zone hyperplasia was present). Neoplastic and non-neoplastic lesions were correctly identified in 57/78 cases (Table 3),
therefore overall accuracy of cytology was 73.08% (Table 4). Sensitivity of cytology in the diag-
nosis of splenic neoplasms was 64.29%, specificity was 95.45%, positive predictive value was
97.30%, and negative predictive value was 51.22% (Table 4). Positive and negative likelihood
ratios were 14.14 and 0.37, respectively (Table 4). According to Cohen’s test the level of agreement was considered as “moderate”, with a κ
value of 0.473 corresponding to a standard error of 0.086. The distribution of TP and FN cases for each tumor type is reported in Table 1. The sensi-
tivity of cytology in the diagnosis of each neoplastic subcategory was 100% for MCT, CARC
and ORCT, 75% for HES, 75% for HS, 71.43% for STS, 50% for LYM, and 0% for BSTT
included in the study. Further details regarding complete and partial agreement between
cytological and histological diagnoses as well as confidence intervals of sensitivity value for
each tumor type are listed in Table 1. Chi-square analysis of cytological sensitivity was applica-
ble only to HES, HS, STS, and LYM. No statistically significant sensitivity differences were
observed (p-value ranging from 0.1506 to 1.0). Table 4. Prevalence of neoplastic lesions, with point estimate and 95% confidence interval of diagnostic accuracy
indexes, likelihood ratios and Cohen’s k. Diagnostic accuracy index
Value
Confidence Interval (95%)
Prevalence
71.79%
60.47%–81.41%
Overall accuracy
73.08%
61.84%–82.50%
Sensitivity
64.29%
50.36%–76.64%
Specificity
95.45%
77.16%–99.88%
PPV
97.30%
84.01%–99.60%
NPV
51.22%
42.21%–60.15%
PLR
14.14
2.06–96.94
NLR
0.37
0.26–0.54
K value
0.473
0.304–0.643 Table 4. Prevalence of neoplastic lesions, with point estimate and 95% confidence interval of diagnostic accuracy
indexes, likelihood ratios and Cohen’s k. PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 8 / 15 Cytological diagnostic accuracy and canine splenic neoplasms The proportion of TP and FN cases with nodular or diffuse pattern for each tumor type are
reported in Table 2. Discussion In this study we report overall accuracy, sensitivity, specificity, positive and negative predic-
tive values of cytology for the diagnosis of canine splenic neoplasms. Similar studies [8,23–
25,31–33] have limited the evaluation of cytological diagnostic accuracy to overall agreement
with histopathology, hampering comparison with our results. Our study has evidenced a
moderate overall accuracy of cytology. Specifically, although this technique had a high speci-
ficity and positive predictive value for the diagnosis of splenic neoplasia, sensitivity and
negative predictive value were lower, indicating that cytological diagnosis of splenic neopla-
sia is reliable, but a negative result cannot be used to exclude the possibility of splenic
neoplasia. According to overall accuracy and Cohen’s k values, cytology is not a reliable alternative to
histopathology in the definitive diagnosis of splenic tumors in most cases. When compared
with previous studies, our overall accuracy value (73.08%) laid in between the higher range of
83.87–100% [23–25] and the lower 38–69.7% range [8,31–33] reported in other studies. To
allow comparison, the overall accuracy (intended as the sum of complete and partial diagnostic
agreements) was calculated from the raw data of previously published caseloads [8,23–25,31–
33] when not made explicit in the corresponding manuscript. Low sensitivity and negative predictive value of this study indicate that a cytology negative
for neoplasia should prompt further investigations to confirm a dog to be truly free from neo-
plastic disease. This contrasts with our initial hypothesis that cytology may represent a useful
tool to avoid unnecessary splenectomy. Instead, high specificity and positive predictive value
identify cytology as a good and reliable tool to rule in the diagnosis of splenic neoplasia with a
high degree of confidence. In practical terms, a cytology positive for neoplasia may lead to a
faster surgical treatment, avoiding lag times and higher costs associated with application of
diagnostic imaging techniques such as contrast-enhanced ultrasound and computed tomogra-
phy (CT) [1,56]. Our results are in line with studies evaluating diagnostic accuracy of cytology
applied to various organs in dogs [31,32,35,36,54], with sensitivity and negative predictive
value generally lower than specificity and positive predictive value, respectively. Regarding the reliability of cytology in the diagnosis of specific tumor types, the lack of sta-
tistically significant differences between subcategories may be related to an imbalance in the
number of cases for each tumor type. Cyto-histological correlation Sensitivity of cytology in the diagnosis of neoplastic lesions in general
according to their distribution pattern was 60.47% for nodular and 75% for diffuse neoplasms,
with no statistically significant difference between the two values (p = 0.3593). For some tumor
types the sensitivity of cytology on the basis of the distribution pattern was not calculated,
given that only nodular (BSTT, STS, HS, CARC) or diffuse (ORCT) neoplastic lesions were
represented in these categories. Sensitivity in the diagnosis of nodular and diffuse lymphomas
was for both 50%, with no statistically significant difference between the two values (p = 1.0). Sensitivity for nodular angiosarcomas was 71.43% and 100% for diffuse angiosarcomas, while
sensitivity for both nodular and diffuse mast cell tumors was 100%. Considering these results,
Chi-square analysis of sensitivity on the basis of the distribution pattern was not performed for
angiosarcomas and mast cell tumors. PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 Discussion Also, our results may be influenced by the tumor cell
type evaluated, since exfoliation rate varies substantially between round cell, epithelial and
mesenchymal tumors [30,31,35,53]. Specifically, mesenchymal tumors have the lowest ten-
dency to exfoliate [30,31,35,53] explaining the low sensitivity in diagnosing benign mesenchy-
mal tumors. Moreover, identification of vascular tumors (i.e. angiomas and HES) among false PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 9 / 15 Cytological diagnostic accuracy and canine splenic neoplasms negative cases is not surprising since the architecture of these tumors often leads to significant
peripheral blood contamination in aspirates [4,30,32,57]. The low sensitivity of cytology in the
diagnosis of splenic lymphomas relates to the specific distribution of tumor types in the spleen,
where indolent nodular lymphomas (i.e. mantle cell lymphoma and marginal zone lymphoma)
are frequent as was in this caseload. These are nodular lymphomas composed of small to
medium sized cells with minimal atypia and a low mitotic rate [49,58]. Thus, mantle cell lym-
phoma and marginal zone lymphoma can be easily misinterpreted as reactive lymphoid hyper-
plasia on cytology, and histopathology is often necessary for a definitive diagnosis that relies
on the evaluation of tumor architecture [49]. Although not statistically significant, our results paralleled those of previous reports identi-
fying higher cytological accuracy in the diagnosis of diffuse compared to focal lesions
[23,32,57]. One false positive diagnosis of neoplasia (i.e. lymphoma) was included in this study. Splenic
marginal zone hyperplasia is a common finding in dogs [49,59], and cytological sampling
from these areas may result in a monomorphic specimen mimicking marginal zone lym-
phoma. This is a risk that pathologists have to bear in mind; thus the diagnosis of nodular low-
grade lymphoma should be supported by histological evaluation of architectural changes, espe-
cially in dogs. A recent report [60] has demonstrated a high overall concordance between his-
topathology, immunohistochemistry and PCR for antigen receptor rearrangement (PARR) in
the diagnosis of marginal zone lymphoma, mantle cell lymphoma and lymphoid or complex
nodular hyperplasia. Therefore, further development of combined methods also applicable to
cytological specimens may provide a less invasive and more valuable diagnostic approach to
the diagnosis of splenic nodular lymphoid lesions. Although histopathology is generally considered the diagnostic reference standard
[24,31,33,35,36], several limitations should also be considered for this technique in the diagno-
sis of splenic tumors. PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 Discussion Specifically, the diagnosis of splenic hematomas and hemangiosarcomas
is considered difficult, especially if spleens are not submitted entirely and if adequate samples
from the margin of the lesion are not collected [2,3,5]. Noteworthy, splenic hematomas and
HES may not be grossly distinguishable [2,3,5,23,61], and the first may represent a component
of the latter [61]. The current study is characterized by several limitations, mainly due to its retrospective
nature. One major limit was the inclusion of specimens obtained by different sampling tech-
niques, with a high number of impression smears collected from both surgical biopsies and
necropsies. Additionally, the inclusion of splenic cytological samples from necropsies and the
university setting of this work may have further biased the study toward cases with a more
aggressive behavior and with features of malignancy easier to diagnose. This may not reflect
daily clinical practice in which FNA is the most common sampling technique to pre-opera-
tively assess splenic lesions. Also, as previously observed [31,53], different sampling methods
may have resulted in an improvement of sensitivity of cytology in this study, especially for
those neoplasms characterized by low exfoliation rate. On the other hand, this observation can
be viewed also in positive terms. Indeed, in a practical setting the preliminary evaluation of
surgical biopsies or entire spleens by cytology prior to fixation could be implemented to facili-
tate the diagnosis and to reduce turnaround time. Additionally, this approach can provide
pathologists with material useful not only for a preliminary diagnosis, but also for immunocy-
tochemistry and for PARR on fresh specimens. Despite this caseload being larger than previously reported ones, the small number of cases
evaluated may explain the relatively wide confidence intervals observed around point estimates
of our diagnostic accuracy indexes. Results may have been further biased by the inclusion PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 10 / 15 Cytological diagnostic accuracy and canine splenic neoplasms criteria applied in the current study, leading to the exclusion of more than 90% cytological
samples of canine spleen in our archives. Unfortunately, full agreement with STARD guidelines could not be obtained in this study
since the type of treatment administered between cytological and histological sampling, and
the incidence of adverse events following splenic sampling, could not be retrieved from our
electronic archives. Acknowledgments We thank Professor Valerio Bronzo for theoretical assistance with the setting of Chi-square
analysis. Discussion In conclusion, to the best of our knowledge, this is the first study conjunctively reporting
overall accuracy, sensitivity, specificity, positive and negative predictive values of cytology in
the diagnosis of canine splenic neoplasms compared to histopathology. Diagnostic accuracy
indexes identified limitations of negative cytological results in excluding a dog to be truly free
from neoplasia; however, high specificity and positive predictive value highlighted cytology as
a valuable tool in the diagnostic approach to splenic neoplasms. Supporting information S1 Table. Cytological and corresponding histological diagnosis for each case included in
the statistical analysis, with correlation category and lesion distribution pattern. Neoplastic
subcategory and level of agreement are provided when applicable. Dogs from which more
than one sample was obtained are marked with (). The age of dogs is reported in years, if not
otherwise specified. Abbreviations: B, biopsy; BSTT, benign soft tissue tumor including angi-
oma; CA, complete agreement; CARC, carcinoma; DA, disagreement; EMH, extramedullary
hematopoiesis; F, female; FN, false negative; FNA, fine needle aspirate; FP, false positive; HES,
angiosarcoma; HS, histiocytic neoplasm (including hemophagocytic syndrome); LYM, lym-
phoma; M, male; MCT, mast cell tumor (MCT); mm, months; MNNOS, malignant neoplasm
not otherwise specified; N, necropsy; NF, neutered female; NM, neutered male; NPL, neoplas-
tic; NON-NPL, non-neoplastic; NOS, not otherwise specified; n/a, not applicable; n/d, not
determined; ORCT, other round cell tumor; PA, partial agreement; RLH, reactive lymphoid
hyperplasia; SC, scraping; STS, soft tissue sarcoma other than angiosarcoma; TN, true negative;
TP, true positive; 2OP, second opion case. (XLSX) S2 Table. Cytological and corresponding histological diagnosis for each cytologically
inconclusive case, with lesion distribution pattern. Neoplastic subcategory is provided when
applicable. Abbreviations: B, biopsy; BSTT, benign soft tissue tumor including angioma;
CARC, carcinoma; EMH, extramedullary hematopoiesis; F, female; FNA, fine needle aspirate;
HES, angiosarcoma; LYM, lymphoma; M, male; N, necropsy; NF, neutered female; NM, neu-
tered male; NPL, neoplastic; NON-NPL, non-neoplastic; n/a, not applicable; STS, soft tissue
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Ausg K Kleintiere Heimtiere. 2012; 40: 250–260. PMID: 22911256 6. Fossum TW, Caplan ER. Chapter 24—Surgery of the Hemolymphatic System. In: Fossum TW, editor. Small animal surgery. Fourth Ed. St. Louis, Missouri: Elsevier Mosby; 2013. pp. 685–704. 7. Cleveland MJ, Casale S. Incidence of malignancy and outcomes for dogs undergoing splenectomy for
incidentally detected nonruptured splenic nodules or masses: 105 cases (2009–2013). J Am Vet Med
Assoc. 2016; 248: 1267–1273. https://doi.org/10.2460/javma.248.11.1267 PMID: 27172343 8. Watson AT, Penninck D, Knoll JS, Keating JH, Sutherland-Smith J. Safety and correlation of test results
of combined ultrasound-guided fine-needle aspiration and needle core biopsy of the canine spleen. Vet
Radiol Ultrasound. 2011; 52: 317–322. https://doi.org/10.1111/j.1740-8261.2010.01778.x PMID:
21554481 9. Wendelburg KM, O’Toole TE, McCobb E, Price LL, Lyons JA, Berg J. Risk factors for perioperative
death in dogs undergoing splenectomy for splenic masses: 539 cases (2001–2012). J Am Vet Med
Assoc. 2014; 245: 1382–1390. https://doi.org/10.2460/javma.245.12.1382 PMID: 25459483 10. Marino DJ, Matthiesen DT, Fox PR, Lesser MB, Stamoulis ME. Ventricular Arrhythmias in Dogs Under-
going Splenectomy : Vet Surg. 1994; 23: 101–106. https://doi.org/10.1111/j.1532-950x.1994.tb00453.x
PMID: 8191668 11. Pastarapatee N, Kijtawornrat A, Buranakarl C. Imbalance of autonomic nervous systems involved in
ventricular arrhythmia after splenectomy in dogs. J Vet Med Sci. 2017; 79: 2002–2010. https://doi.org/
10.1292/jvms.17-0482 PMID: 29070771 12. Miko I, Nemeth N, Sogor V, Kiss F, Toth E, Peto K, et al. Author Contributions Conceptualization: Annalisa Forlani, Mario Caniatti, Paola Roccabianca. Conceptualization: Annalisa Forlani, Mario Caniatti, Paola Roccabianca. Data curation: Marco Tecilla, Matteo Gambini, Annalisa Forlani. Data curation: Marco Tecilla, Matteo Gambini, Annalisa Forlani. Formal analysis: Marco Tecilla, Matteo Gambini. Formal analysis: Marco Tecilla, Matteo Gambini. Formal analysis: Marco Tecilla, Matteo Gambini. 11 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 Cytological diagnostic accuracy and canine splenic neoplasms Investigation: Marco Tecilla, Matteo Gambini, Annalisa Forlani, Mario Caniatti, Gabriele
Ghisleni, Paola Roccabianca. Methodology: Matteo Gambini, Annalisa Forlani, Paola Roccabianca. Project administration: Paola Roccabianca. Resources: Annalisa Forlani, Mario Caniatti, Paola Roccabianca. Supervision: Mario Caniatti, Paola Roccabianca. Validation: Marco Tecilla, Matteo Gambini. Validation: Marco Tecilla, Matteo Gambini. Visualization: Marco Tecilla, Matteo Gambini, Annalisa Forlani. Writing – original draft: Marco Tecilla, Annalisa Forlani, Paola Roccabianca. Writing – review & editing: Marco Tecilla, Matteo Gambini, Annalisa Forlani, Mario
Caniatti, Gabriele Ghisleni, Paola Roccabianca. References Comparative erythrocyte deformability investi-
gations by filtrometry, slit-flow and rotational ektacytometry in a long-term follow-up animal study on
splenectomy and different spleen preserving operative techniques: Partial or subtotal spleen resection
and spleen autotransplantation. Clin Hemorheol Microcirc. 2017; 66: 83–96. https://doi.org/10.3233/
CH-160231 PMID: 28128749 13. Miko I, Nemeth N, Peto K, Furka A, Toth L, Furka I. Changes of red blood cell aggregation parameters
in a long-term follow-up of splenectomy, spleen-autotransplantation and partial or subtotal spleen PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 12 / 15 Cytological diagnostic accuracy and canine splenic neoplasms resections in a canine model. Clin Hemorheol Microcirc. 2017; 67: 91–100. https://doi.org/10.3233/CH-
170264 PMID: 28598833 14. Kaltreider HB, Barth E, Pellegrini C. The effect of splenectomy on the appearance of specific antibody-
forming cells in lungs of dogs after intravenous immunization with sheep erythrocytes. Exp Lung Res. 1981; 2: 231–238. https://doi.org/10.3109/01902148109052318 PMID: 6975209 15. Richardson EF, Brown NO. Hematological and biochemical changes and results of aerobic bacteriologi-
cal culturing in dogs undergoing splenectomy. J Am Anim Hosp Assoc. 1996; 32: 199–210. https://doi. org/10.5326/15473317-32-3-199 PMID: 8731133 16. Tisˇkina V, Capligina V, Must K, Berzina I, Ranka R, Jokelainen P. Fatal Babesia canis canis infection in
a splenectomized Estonian dog. Acta Vet Scand. 2016; 58: 1–6. 17. Kawamura M, Maede Y, Namioka S. Mitogenic responsibilities of lymphocytes in canine babesiosis and
the effects of splenectomy on it. Jpn J Vet Res. 1987; 35: 1–10. PMID: 3599600 18. Kemming G, Messick JB, Mueller W, Enders G, Meisner F, Muenzing S, et al. Can we continue
research in splenectomized dogs? Mycoplasma haemocanis: Old problem—New insight. Eur Surg
Res. 2004; 36: 198–205. https://doi.org/10.1159/000078853 PMID: 15263824 19. Lester SJ, Hume JB, Phipps B. Haemobartonella canis infection following splenectomy and transfusion. Can Vet J. 1995; 36: 444–445. PMID: 7585424 20. Sartor AJ, Bentley AM, Brown DC. Association between previous splenectomy and gastric dilatation-
volvulus in dogs: 453 cases (2004–2009). J Am Vet Med Assoc. 2013; 242: 1381–1384. https://doi.org/
10.2460/javma.242.10.1381 PMID: 23634682 21. Allen P, Paul A. Gastropexy for Prevention of Gastric Dilatation-Volvulus in Dogs: History and Tech-
niques. Top Companion Anim Med. 2014; 29: 77–80. https://doi.org/10.1053/j.tcam.2014.09.001 PMID:
25496925 22. Maki LC, Males KN, Byrnes MJ, El-Saad AA, Coronado GS. Incidence of gastric dilatation-volvulus fol-
lowing a splenectomy in 238 dogs. Can Vet J. 2017; 58: 1275–1280. PMID: 29203936 23. Christensen NI, Canfield PJ, Martin PA, Krockenberger MB, Spielman DS, Bosward KL. PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 References Caniatti M, da Cunha NP, Avallone G, Romussi S, Mortellaro CM, Tranquillo V, et al. Diagnostic accu-
racy of brush cytology in canine chronic intranasal disease. Vet Clin Pathol. 2012; 41: 133–140. https://
doi.org/10.1111/j.1939-165X.2011.00388.x PMID: 22250805 36. Fournier Q, Cazzini P, Bavcar S, Pecceu E, Ballber C, Elders R. Investigation of the utility of lymph
node fine-needle aspiration cytology for the staging of malignant solid tumors in dogs. Vet Clin Pathol. 2018; 47: 489–500. https://doi.org/10.1111/vcp.12636 PMID: 30011068 37. Berzina I, Sharkey LC, Matise I, Kramek B. Correlation between cytologic and histopathologic diagno-
ses of bone lesions in dogs: a study of the diagnostic accuracy of bone cytology. Vet Clin Pathol. 2008;
37: 332–338. https://doi.org/10.1111/j.1939-165X.2008.00050.x PMID: 18761529 38. Raskin RE. Chapter 4—Hemolymphatic System. In: Raskin RE, Meyer DJBT, editors. Canine and
Feline Cytology—A Color Atlas and Interpretation Guide. Third Ed. St. Louis, Missouri: W.B. Saun-
ders; 2016. pp. 91–137. https://doi.org/10.1016/B978-1-4557-4083-3.00004-8 39. Me´nard M, Fontaine M, Morin M. Fine needle aspiration biopsy of malignant tumors in dogs and cats: a
report of 102 cases. Can Vet J = La Rev Vet Can. 1986; 27: 504–10. Available: http://www.ncbi.nlm.nih. gov/pubmed/17422728 40. Valli VE. Chapter 3—B-Cell Neoplasms. In: Valli VE, editor. Veterinary Comparative Hematology. First
Ed. Ames, Iowa: Blackwell Publishing; 2007. pp. 119–274. 41. Valli VE. Chapter 4—T-Cell and NK-Cell Neoplasms. In: Valli VE, editor. Veterinary Comparative Hema-
tology. First Ed. Ames, Iowa: Blackwell Publishing; 2007. pp. 275–366. 42. Valli VE. Chapter 5—Acute Myeloid Leukemias. In: Valli VE, editor. Veterinary Comparative Hematol-
ogy. First Ed. Ames, Iowa: Blackwell Publishing; 2007. pp. 367–424. 43. Valli VE. Chapter 6—Chronic Myeloproliferative Diseases. In: Valli VE, editor. Veterinary Comparative
Hematology. First Ed. Ames, Iowa: Blackwell Publishing; 2007. pp. 425–460. 44. Valli VE. Chapter 7—Myeloproliferative/Myelodysplastic Diseases. In: Valli VE, editor. Veterinary Com-
parative Hematology. First Ed. Ames, Iowa: Blackwell Publishing; 2007. pp. 461–467. 45. Valli VE. Chapter 9—Hodgkin’s Lymphoma. In: Valli VE, editor. Veterinary Comparative Hematology. First Ed. Ames, Iowa: Blackwell Publishing; 2007. pp. 491–504. 46. Hendrick MJ. Chapter 5—Mesenchymal Tumors of the Skin and Soft Tissues. In: Meuten DJ, editor. Tumors in domestic animals. Fifth Ed. Ames, Iowa: Wiley/Blackwell; 2017. pp. 142–175. http://ez. statsbiblioteket.dk:2048/login?url=https://ebookcentral.proquest.com/lib/asb/detail.action?docID=
4737344 47. Valli VE, Bienzle D, Meuten DJ, Linder KE. Chapter 7—Tumors of the Hemolymphatic System. In: Meu-
ten DJ, editor. Tumors in domestic animals. Fifth Ed. Ames, Iowa: Wiley/Blackwell; 2017. pp. 203–
321. http://ez.statsbiblioteket.dk:2048/login?url=https://ebookcentral.proquest.com/lib/asb/detail. action?docID=4737344 48. Moore PF. Chapter 8—Canine and Feline Histiocytic Diseases. References Cytopathologi-
cal and histopathological diagnosis of canine splenic disorders. Aust Vet J. 2009; 87: 175–181. https://
doi.org/10.1111/j.1751-0813.2009.00421.x PMID: 19382924 24. Ballegeer EA, Forrest LJ, Dickinson RM, Schutten MM, Delaney FA, Young KM. Correlation of ultraso-
nographic appearance of lesions and cytologic and histologic diagnoses in splenic aspirates from dogs
and cats: 32 cases (2002–2005). J Am Vet Med Assoc. 2007; 230: 690–696. https://doi.org/10.2460/
javma.230.5.690 PMID: 17331053 25. O’Keefe DA, Couto CG. FNA of the spleen as an aid in the diagnosis of splenomegaly. J Vet Intern
Med. 1987; 1: 102–109. 26. Spangler WL, Kass PH. Pathologic factors affecting postsplenectomy survival in dogs. J Vet Intern
Med. 1997; 11: 166–171. https://doi.org/10.1111/j.1939-1676.1997.tb00085.x PMID: 9183768 27. Gochhait D, Dey P, Rajwanshi A, Nijhawan R, Gupta N, Radhika S, et al. Role of Fine needle aspiration
cytology of spleen. APMIS. 2015; 123: 190–193. https://doi.org/10.1111/apm.12339 PMID: 25469450 28. Go´mez-Rubio M, Lo´pez-Cano A, Rendo´n P, Muñoz-Benvenuty A, Macı´as M, Garre C, et al. Safety and
diagnostic accuracy of percutaneous ultrasound-guided biopsy of the spleen: A multicenter study. J Clin
Ultrasound. 2009; 37: 445–450. https://doi.org/10.1002/jcu.20608 PMID: 19582827 29. Barone S, Baer MR, Sait SNJ, Lawrence D, Block AW, Wetzler M. Ultrasound-guided fine needle biopsy
of the spleen: High clinical efficacy and low risk in a multicenter Italian study. Am J Hematol. 2001; 67:
93–99. https://doi.org/10.1002/ajh.1085 PMID: 11343380 30. Christopher MM. Cytology of the spleen. Veterinary Clinics of North America—Small Animal Practice. 2003. pp. 135–152. https://doi.org/10.1016/S0195-5616(02)00082-7 31. Cohen M, Bohling MW, Wright JC, Welles EA, Spano JS. Evaluation of sensitivity and specificity of cyto-
logic examination: 269 cases (1999–2000). J Am Vet Med Assoc. 2003; 222: 964–7. Available: http://
www.ncbi.nlm.nih.gov/pubmed/12685787 PMID: 12685787 32. Eich C, Whitehair J, Moroff S, Heeb L. The accuracy of intraoperative cytopathological diagnosis com-
pared with conventional histopathological diagnosis. J Am Anim Hosp Assoc. 2014; 36: 16–18. https://
doi.org/10.5326/15473317-36-1-16 PMID: 10667401 33. Braun AO, Hauser B. Korrelation zwischen zytologischen und histologischen Haut-, Lymphknoten- Und
Milzbefunden bei 500 Hunden und Katzen. Schweiz Arch Tierheilkd. 2007; 149: 249–257. 34. Bossuyt PM, Reitsma JB, Bruns DE, Gatsonis CA, Glasziou PP, Irwig L, et al. STARD 2015: An
updated list of essential items for reporting diagnostic accuracy studies [Internet]. Clinical Chemistry. 2015. pp. 1446–1452. https://doi.org/10.1373/clinchem.2015.246280 PMID: 26510957 13 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 November 7, 2019 Cytological diagnostic accuracy and canine splenic neoplasms 35. References In: Meuten DJ, editor. Tumors in
domestic animals. Fifth Ed. Ames, Iowa: Wiley/Blackwell; 2017. pp. 322–336. http://ez. statsbiblioteket.dk:2048/login?url=https://ebookcentral.proquest.com/lib/asb/detail.action?docID=
4737344 49. Valli VE, Vernau W, De Lorimier LP, Graham PS, Moore PF. Canine indolent nodular lymphoma. Vet
Pathol. 2006; 43: 241–256. https://doi.org/10.1354/vp.43-3-241 PMID: 16672571 50. Valli VE, Myint M, Barthel A, Bienzle D, Caswell J, Colbatzky F, et al. Classification of canine malignant
lymphomas according to the world health organization criteria. Vet Pathol. 2011; 48: 198–211. https://
doi.org/10.1177/0300985810379428 PMID: 20861499 51. Moore PF. A Review of Histiocytic Diseases of Dogs and Cats. Vet Pathol. 2014; 51: 167–184. https://
doi.org/10.1177/0300985813510413 PMID: 24395976 52. Moore AS, Frimberger AE, Sullivan N, Moore PF. Histologic and Immunohistochemical Review of
Splenic Fibrohistiocytic Nodules in Dogs. J Vet Intern Med. 2012; 26: 1164–1168. https://doi.org/10. 1111/j.1939-1676.2012.00986.x PMID: 22882592 53. Vos JH, van den Ingh TS, van Mil FN. Non-exfoliative canine cytology: the value of fine needle aspira-
tion and scraping cytology. Vet Q. 1989; 11: 222–231. https://doi.org/10.1080/01652176.1989.9694228
PMID: 2690456 54. Bonfanti U, Bertazzolo W, Gracis M, Roccabianca P, Romanelli G, Palermo G, et al. Diagnostic value of
cytological analysis of tumours and tumour-like lesions of the oral cavity in dogs and cats: A prospective
study on 114 cases. Vet J. 2015; 205: 322–327. https://doi.org/10.1016/j.tvjl.2014.10.022 PMID:
25466576 55. Ku CK, Kass PH, Christopher MM. Cytologic–histologic concordance in the diagnosis of neoplasia in
canine and feline lymph nodes: a retrospective study of 367 cases. Vet Comp Oncol. 2017; 15: 1206–
1217. https://doi.org/10.1111/vco.12256 PMID: 27523399 PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 14 / 15 Cytological diagnostic accuracy and canine splenic neoplasms 56. Clifford CA, Pretorius ES, Weisse C, Sorenmo KU, Drobatz KJ, Siegelman ES, et al. Magnetic reso-
nance imaging of focal splenic and hepatic lesions in the dog. J Vet Intern Med. 2004; 18: 330–338. https://doi.org/10.1892/0891-6640(2004)18<330:mriofs>2.0.co;2 PMID: 15188820 57. Sharkey LC, Dial SM, Matz ME. Maximizing the Diagnostic Value of Cytology in Small Animal Practice. Vet Clin North Am—Small Anim Pract. 2007; 37: 351–372. https://doi.org/10.1016/j.cvsm.2006.11.004
PMID: 17336679 58. Valli VE, Kass PH, Myint MS, Scott F. Canine Lymphomas: Association of Classification Type, Disease
Stage, Tumor Subtype, Mitotic Rate, and Treatment With Survival. Vet Pathol. 2013; 50: 738–748. https://doi.org/10.1177/0300985813478210 PMID: 23444036 59. Valli VE. Chapter 2—Normal and Benign Reactive Hematopoietic Tissues—Spleen. Veterinary Com-
parative Hematology. First Edit. Blackwell Publishing; 2007. pp. 47–76. 60. Sabattini S, Lopparelli RM, Rigillo A, Giantin M, Renzi A, Matteo C, et al. PLOS ONE | https://doi.org/10.1371/journal.pone.0224945
November 7, 2019 References Canine Splenic Nodular Lym-
phoid Lesions: Immunophenotyping, Proliferative Activity, and Clonality Assessment. Vet Pathol. 2018;
55: 645–653. https://doi.org/10.1177/0300985818777035 PMID: 29807508 61. Patten SG, Boston SE, Monteith GJ. Outcome and prognostic factors for dogs with a histological diag-
nosis of splenic hematoma following splenectomy: 35 cases (2001–2013). Can Vet J. 2016; 57: 842–
846. PMID: 27493283 15 / 15
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https://pure.knaw.nl/ws/files/37314494/7037_Pilotto.pdf
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Meta-analysis of multidecadal biodiversity trends in Europe
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Nature communications
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Meta-analysis of multidecadal biodiversity trends in Europe Pilotto, Francesca; Kühn, Ingolf; Adrian, Rita; Alber, Renate; Alignier, Audrey; Andrews,
Christopher; Bäck, Jaana; Barbaro, Luc; Beaumont, Deborah; Beenaerts, Natalie;
Benham, Sue; Boukal, David S.; Bretagnolle, Vincent; Camatti, Elisa; Canullo, Roberto;
Cardoso, Patricia G.; Ens, Bruno J.; Everaert, Gert; Evtimova, Vesela; Feuchtmayr,
Heidrun; García-González, Ricardo; Gómez García, Daniel; Grandin, Ulf; Gutowski, Jerz
M.; Hadar, Liat; Halada, Lubos; Halassy, Melinda; Hummel, Herman; Huttunen, Kaisa-
Leena; Jaroszewicz, Bogdan; Jensen, Thomas C.; Kalivoda, Henrik; Schmidt, Inger
Kappel; Kröncke, Ingrid; Leinonen, Reima; Martinho, Filipe; Meesenburg, Henning;
Meyer, Julia; Minerbi, Stefano; Monteith, Don; Nikolov, Boris P.; Oro, Daniel; Ozoliņš,
Dāvis; Padedda, Bachisio M.; Pallett, Denise; Pansera, Marco; Pardal, Miguel Ângelo;
Petriccione, Bruno; Pipan, Tanja; Pöyry, Juha; Schäfer, Stefanie M.; Schaub, Marcus;
Schneider, Susanne C.; Skuja, Agnija; Soetaert, Karline; Spriņģe, Gunta; Stanchev,
Radoslav; Stockan, Jenni A.; Stoll, Stefan; Sundqvist, Lisa; Thimonier, Anne; Van Hoey
Gert; Van Ryckegem, Gunther; Visser, Marcel E.; Vorhauser, Samuel; Haase, Peter
published in
Nature Communications
2020
DOI (link to publisher)
10.1038/s41467-020-17171-y
document version
Publisher's PDF, also known as Version of record
document license
CC BY
Link to publication in KNAW Research Portal Pilotto, Francesca; Kühn, Ingolf; Adrian, Rita; Alber, Renate; Alignier, Audrey; Andrews,
Christopher; Bäck, Jaana; Barbaro, Luc; Beaumont, Deborah; Beenaerts, Natalie;
Benham, Sue; Boukal, David S.; Bretagnolle, Vincent; Camatti, Elisa; Canullo, Roberto;
Cardoso, Patricia G.; Ens, Bruno J.; Everaert, Gert; Evtimova, Vesela; Feuchtmayr,
Heidrun; García-González, Ricardo; Gómez García, Daniel; Grandin, Ulf; Gutowski, Jerzy
M.; Hadar, Liat; Halada, Lubos; Halassy, Melinda; Hummel, Herman; Huttunen, Kaisa-
Leena; Jaroszewicz, Bogdan; Jensen, Thomas C.; Kalivoda, Henrik; Schmidt, Inger
Kappel; Kröncke, Ingrid; Leinonen, Reima; Martinho, Filipe; Meesenburg, Henning;
Meyer, Julia; Minerbi, Stefano; Monteith, Don; Nikolov, Boris P.; Oro, Daniel; Ozoliņš,
Dāvis; Padedda, Bachisio M.; Pallett, Denise; Pansera, Marco; Pardal, Miguel Ângelo;
Petriccione, Bruno; Pipan, Tanja; Pöyry, Juha; Schäfer, Stefanie M.; Schaub, Marcus;
Schneider, Susanne C.; Skuja, Agnija; Soetaert, Karline; Spriņģe, Gunta; Stanchev,
Radoslav; Stockan, Jenni A.; Stoll, Stefan; Sundqvist, Lisa; Thimonier, Anne; Van Hoey,
Gert; Van Ryckegem, Gunther; Visser, Marcel E.; Vorhauser, Samuel; Haase, Peter
published in document version
Publisher's PDF, also known as Version of record
document license
CC BY Link to publication in KNAW Research Portal Link to publication in KNAW Research Portal citation for published version (APA)
Pilotto, F., Kühn, I., Adrian, R., Alber, R., Alignier, A., Andrews, C., Bäck, J., Barbaro, L., Beaumont, D.,
Beenaerts, N., Benham, S., Boukal, D. S., Bretagnolle, V., Camatti, E., Canullo, R., Cardoso, P. G., Ens, B. Royal Netherlands Academy of Arts and Sciences
(KNAW) Meta-analysis of multidecadal biodiversity trends in Europe
Pilotto, Francesca; Kühn, Ingolf; Adrian, Rita; Alber, Renate; Alignier, Audrey; Andrews,
Christopher; Bäck, Jaana; Barbaro, Luc; Beaumont, Deborah; Beenaerts, Natalie;
Benham, Sue; Boukal, David S.; Bretagnolle, Vincent; Camatti, Elisa; Canullo, Roberto;
Cardoso, Patricia G.; Ens, Bruno J.; Everaert, Gert; Evtimova, Vesela; Feuchtmayr,
Heidrun; García-González, Ricardo; Gómez García, Daniel; Grandin, Ulf; Gutowski, Jerzy
M.; Hadar, Liat; Halada, Lubos; Halassy, Melinda; Hummel, Herman; Huttunen, Kaisa-
Leena; Jaroszewicz, Bogdan; Jensen, Thomas C.; Kalivoda, Henrik; Schmidt, Inger
Kappel; Kröncke, Ingrid; Leinonen, Reima; Martinho, Filipe; Meesenburg, Henning;
Meyer, Julia; Minerbi, Stefano; Monteith, Don; Nikolov, Boris P.; Oro, Daniel; Ozoliņš,
Dāvis; Padedda, Bachisio M.; Pallett, Denise; Pansera, Marco; Pardal, Miguel Ângelo;
Petriccione, Bruno; Pipan, Tanja; Pöyry, Juha; Schäfer, Stefanie M.; Schaub, Marcus;
Schneider, Susanne C.; Skuja, Agnija; Soetaert, Karline; Spriņģe, Gunta; Stanchev,
Radoslav; Stockan, Jenni A.; Stoll, Stefan; Sundqvist, Lisa; Thimonier, Anne; Van Hoey,
Gert; Van Ryckegem, Gunther; Visser, Marcel E.; Vorhauser, Samuel; Haase, Peter
published in
Nature Communications
2020
DOI (link to publisher)
10.1038/s41467-020-17171-y
document version
Publisher's PDF, also known as Version of record
document license
CC BY
Link to publication in KNAW Research Portal
citation for published version (APA)
Pilotto, F., Kühn, I., Adrian, R., Alber, R., Alignier, A., Andrews, C., Bäck, J., Barbaro, L., Beaumont, D.,
Beenaerts, N., Benham, S., Boukal, D. S., Bretagnolle, V., Camatti, E., Canullo, R., Cardoso, P. G., Ens, B. J.,
Everaert, G., Evtimova, V., ... Haase, P. (2020). Meta-analysis of multidecadal biodiversity trends in Europe. Nature Communications, 11(1). https://doi.org/10.1038/s41467-020-17171-y General rights
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research search. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the KNAW public portal. Meta-analysis of multidecadal biodiversity trends in Europe J.,
Everaert, G., Evtimova, V., ... Haase, P. (2020). Meta-analysis of multidecadal biodiversity trends in Europe. Nature Communications, 11(1). https://doi.org/10.1038/s41467-020-17171-y General rights
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and investigate your claim. ARTICLE Meta-analysis of multidecadal biodiversity trends
in Europe
https://doi.org/10.1038/s41467-020-17171-y
OPEN Francesca Pilotto
et al.# Local biodiversity trends over time are likely to be decoupled from global trends, as local
processes may compensate or counteract global change. We analyze 161 long-term biological
time series (15–91 years) collected across Europe, using a comprehensive dataset comprising
~6,200 marine, freshwater and terrestrial taxa. We test whether (i) local long-term biodi-
versity trends are consistent among biogeoregions, realms and taxonomic groups, and (ii)
changes in biodiversity correlate with regional climate and local conditions. Our results reveal
that local trends of abundance, richness and diversity differ among biogeoregions, realms and
taxonomic groups, demonstrating that biodiversity changes at local scale are often complex
and cannot be easily generalized. However, we find increases in richness and abundance with
increasing temperature and naturalness as well as a clear spatial pattern in changes in
community composition (i.e. temporal taxonomic turnover) in most biogeoregions of
Northern and Eastern Europe. #A list of authors and their affiliations appears at the end of the paper. #A list of authors and their affiliations appears at the end of the paper. NATURE COMMUNICATIONS | (2020) 11:3486 | https://doi.org/10.1038/s41467-020-17171-y | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y T and temporal resolution, and mostly short time series. Other
studies focused on a smaller number of sites or more restricted
biotic scope. A study of 22 sites in different realms from Central
Europe detected stronger effects of temperature on population
trends in terrestrial than aquatic habitats (i.e. populations of
terrestrial
“species
with
warmer
temperature
preferences
increased more than [terrestrial] species with colder temperature
preferences” (page 3), but found no such relationship for aquatic
taxa)23. Finally,
Gibson-Reinemer
et
al.24
found
that
the
increasing species turnover in mountain communities was
stronger in ectotherm communities and in tropical compared to
temperate regions. These results point towards region, biotic, and
realm specific patterns in local biodiversity trends, but a com-
prehensive overview directly comparing trends among com-
plementary time series has been lacking so far. T
he current biodiversity crisis, manifested in a global decline
of species, affects many taxonomic groups and biotic
realms1–4. These changes may be less evident at specific
locations, since local factors, such as small-scale colonization and
species turnover may compensate or even counteract trends
occurring at larger spatial scales. Heterogeneity in patterns of
change in biodiversity observed at a local scale5,6 has been
described in several studies. Meta-analysis of multidecadal biodiversity trends
in Europe
https://doi.org/10.1038/s41467-020-17171-y
OPEN For example, strong declines in local
biomass and distribution have been reported for terrestrial
insects7–10 and birds11–13, but reports of local increases in bio-
diversity are also widespread and span multiple taxonomic
groups5 including freshwater invertebrates14,15, fishes16, birds12,13
and plants17–19. Ecosystem functions and their benefits to people at local to
global scales ultimately depend on the taxonomic and functional
diversity of local communities5,20. The same relationship applies
to conservation measures: while they need to be harmonized at
larger scales, most measures need to be tailored to local condi-
tions. Therefore, it is crucial to understand the variation in bio-
diversity trends between localities and how and why these may
vary across biota, regions and local conditions21. The present study analyzes trends in various taxonomic groups
measured at specific locations in nine different biogeoregions
across the European continent. We ask: (1) are long-term trends
in biodiversity detectable at individual localities across Europe? (2) If so, to what extent are such trends attributable to changes in
climate at a regional scale and/or changes in local conditions? (3)
Do observed trends in biodiversity vary predictably among bio-
georegions, realms and taxonomic groups and, thus, inform our
understanding and prediction of larger scale patterns? Due to the
ongoing global change we expect to observe: (1) long-term
reductions in biodiversity indicated by declining species richness
and abundances (2) increasing variability in community com-
position, indicated by higher temporal species turnover and (3)
differential responses across taxonomic groups and biogeor-
egions, reflecting differences in the extent of and vulnerability to
climate change (i.e. Southern Europe should be more negatively
affected than Northern Europe25). Analyses of the trends in local biodiversity over large spatial
scales and multiple taxonomic groups are needed to fully
understand the patterns of local biodiversity change and the
discrepancies between local and global biodiversity trends. Unfortunately such syntheses are rare. Current evidence from
multi-taxa biodiversity trend comparisons is limited and equi-
vocal, and direct comparison across studies is hampered by
substantial differences in the temporal and spatial coverage and
resolution of the data. Dornelas et al.6 studied 100 time series of
terrestrial, freshwater and marine taxonomic groups around the
world and found no systematic temporal changes in α-diversity of
local communities. However, the authors detected significant
increases in β-diversity and increasing trends in species richness
for terrestrial plants in the temperate region6. Meta-analysis of multidecadal biodiversity trends
in Europe
https://doi.org/10.1038/s41467-020-17171-y
OPEN The most com-
prehensive study to date to our knowledge22 showed that species
turnover, i.e., a measure of temporal community variability, is
stronger in marine than freshwater and terrestrial assemblages
and that this is often decoupled from changes in species richness. However, these results were based on a relatively coarse spatial We compile 161 long-term (minimum 15 years) biomonitoring
time series from 115 sites, mostly belonging to the International
Long-Term Ecological Research network (ILTER26), in 21 European
countries (Fig. 1), covering nine biogeoregions, three realms and
eight taxonomic groups (Fig. 1, Supplementary Table 1, Supple-
mentary Data). For each time series, we compute total abundance of
the community, taxonomic richness, diversity and temporal turn-
over and estimated their monotonic trends over the study period. Biogeoregions
b
a
c
Realms
TE
TE
FW
FW
MA
MA
Taxonomic groups
Alpine
Atlantic
Black Sea
Boreal
Continental
Mediterranean
Pannonian
North Sea
Adriatic Sea
Outside
Site
Alpine
Atlantic
Black Sea
Boreal
Continental
Mediterranean
Pannonian
North Sea
Alg
Alg
Bir
Bir
Fish
Fish
InvA
InvA
InvT
InvT
Mam
Mam
Pl
Pl
Pla
Pla
Alg
Bir
Fish
InvA
InvT
Mam
Pl
Pla
Adriatic
Alpine
Atlantic
Black Sea
Boreal
Continental
Mediterranean
Pannonian
North Sea
Adriatic
Fig. 1 Distribution of the time series across biogeoregions, realms and taxonomic groups. a Relative distribution of studied taxonomic groups across
biogeoregions (magenta dots: study sites). Note that the most south-eastern site (in Israel) belongs to the Mediterranean region. b Relative distribution of
studied taxonomic groups and biogeoregions across realms. c Relative distribution of studied biogeoregions across taxonomic groups. FW freshwater, MA
marine and transitional zone, TE terrestrial, Alg benthic algae, Bir birds, InvA aquatic invertebrates, InvT terrestrial invertebrates, Mam mammals, Pl
plankton, Pla terrestrial plants. The pie charts show the proportion of taxonomic groups for each biogeoregion and realm, and the proportion of
biogeoregions for each realm and taxonomic group. The shapefiles of the biogeographical regions and marine subregions were obtained from EEA74. Drawings of taxonomic groups are from phylopic.org. Source data are provided as a Source Data file. Meta-analysis of multidecadal biodiversity trends
in Europe
https://doi.org/10.1038/s41467-020-17171-y
OPEN b b
Realms
TE
FW
MA
Alg
Bir
Fish
InvA
InvT
Mam
Pl
Pla c Realms Taxonomic groups Biogeoregions
a
Alpine
Atlantic
Black Sea
Boreal
Continental
Mediterranean
Pannonian
North Sea
Adriatic Sea
Outside
Site
Alg
Bir
Fish
InvA
InvT
Mam
Pl
Pla Alg
Bir
Fish
InvA
InvT
Mam
Pl
Pla
Alpine
Atlantic
Black Sea
Boreal
Continental
Mediterranean
Pannonian
North Sea
Adriatic Pla TE
FW
MA
Alpine
Atlantic
Black Sea
Boreal
Continental
Mediterranean
Pannonian
North Sea
Adriatic Fig. 1 Distribution of the time series across biogeoregions, realms and taxonomic groups. a Relative distribution of studied taxonomic groups across
biogeoregions (magenta dots: study sites). Note that the most south-eastern site (in Israel) belongs to the Mediterranean region. b Relative distribution of
studied taxonomic groups and biogeoregions across realms. c Relative distribution of studied biogeoregions across taxonomic groups. FW freshwater, MA
marine and transitional zone, TE terrestrial, Alg benthic algae, Bir birds, InvA aquatic invertebrates, InvT terrestrial invertebrates, Mam mammals, Pl
plankton, Pla terrestrial plants. The pie charts show the proportion of taxonomic groups for each biogeoregion and realm, and the proportion of
biogeoregions for each realm and taxonomic group. The shapefiles of the biogeographical regions and marine subregions were obtained from EEA74. Drawings of taxonomic groups are from phylopic.org. Source data are provided as a Source Data file. Fig. 1 Distribution of the time series across biogeoregions, realms and taxonomic groups. a Relative distribution of studied taxonomic groups across
biogeoregions (magenta dots: study sites). Note that the most south-eastern site (in Israel) belongs to the Mediterranean region. b Relative distribution of
studied taxonomic groups and biogeoregions across realms. c Relative distribution of studied biogeoregions across taxonomic groups. FW freshwater, MA
marine and transitional zone, TE terrestrial, Alg benthic algae, Bir birds, InvA aquatic invertebrates, InvT terrestrial invertebrates, Mam mammals, Pl
plankton, Pla terrestrial plants. The pie charts show the proportion of taxonomic groups for each biogeoregion and realm, and the proportion of
biogeoregions for each realm and taxonomic group. The shapefiles of the biogeographical regions and marine subregions were obtained from EEA74. Drawings of taxonomic groups are from phylopic.org. Source data are provided as a Source Data file. Fig. 1 Distribution of the time series across biogeoregions, realms and taxonomic groups. a Relative distribution o NATURE COMMUNICATIONS | (2020) 11:3486 | https://doi.org/10.1038/s41467-020-17171-y | www.nature.com/naturecommunicatio 2 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y ARTICLE NATURE COMMUNICA Table 1 Biodiversity trends. Results Overall trends. We observed overall increasing trends in taxo-
nomic richness, diversity and turnover, across all time series,
while there was no significant trend in abundance (Table 1). Biodiversity trends. The trends of all biodiversity metrics dif-
fered among biogeoregions (Table 1). Abundances declined in the
Atlantic region and increased in the North Sea over time. Species
richness and diversity increased in the Black Sea, Boreal region
and North Sea. Species turnover increased over time in time series
from the Alpine, Boreal and Continental regions, and decreased
in time series from the Black Sea and North Sea (Table 1, Fig. 2). Biodiversity trends. The trends of all biodiversity metrics dif-
fered among biogeoregions (Table 1). Abundances declined in the
Atlantic region and increased in the North Sea over time. Species
richness and diversity increased in the Black Sea, Boreal region
and North Sea. Species turnover increased over time in time series
from the Alpine, Boreal and Continental regions, and decreased
in time series from the Black Sea and North Sea (Table 1, Fig. 2). We did not detect any clear trends in abundance in any realm,
while trends in taxon richness, diversity and turnover varied
across realms. We recorded increasing taxon richness in the
freshwater realm, increasing taxon richness and diversity in the
marine realm, and increasing turnover in the terrestrial realm
(Table 1; Fig. 3). We did not detect any clear trends in abundance in any realm,
while trends in taxon richness, diversity and turnover varied
across realms. We recorded increasing taxon richness in the
freshwater realm, increasing taxon richness and diversity in the
marine realm, and increasing turnover in the terrestrial realm
(Table 1; Fig. 3). g
We found a significant decline in the abundance of terrestrial
invertebrates. Species richness, diversity and turnover trends
differed among taxonomic groups (Table 1). Species richness and
diversity increased in birds and aquatic invertebrates. This
contrasted with the decreasing diversity in benthic algae (note
that algae data were only available for sites within a single river
catchment). Turnover trends significantly increased for plants. Other taxa did not show a trend in the respective biodiversity
metrics (Table 1, Fig. 4). Influence of climatic trends and site characteristics. Results The most
important predictors (i.e., highest absolute values of z-scores) of
abundance and richness trends were site naturalness (i.e., a
measure of local anthropogenic pressure), temperature trend (i.e.,
S-statistics of air or water temperature trend, according to the
realm) and their interaction (Table 2). Increases in temperature
and site naturalness were correlated with increased abundance
and richness. The negative interaction between site naturalness
and temperature trend indicates stronger increases in abundance
and richness with increasing temperature at sites with lower
naturalness than at sites with high naturalness (Supplementary
Fig. 1). The most important predictor of diversity trends was
longitude (positive correlation). Species turnover trends were
mostly affected by elevation (positive correlation) and by the
positive interaction between site naturalness and temperature
trend, indicating stronger increases in turnover with increasing
temperature at sites with higher naturalness than at sites with low
naturalness (Table 2, Supplementary Fig. 1). Study length had
much less influence on the trends, indicating that the differences
in study intervals among time series did not bias our results
(Table 2). Biodiversity trends for the whole dataset (overall trends) and within the different biogeoregions,
realms and taxonomic groups, as resulting from meta-analysis mixed models. Note that only
significant results (p ≤0.05) are reported for the biogeoregion, realm and taxonomic group-
specific analysis. Biodiversity trends for the whole dataset (overall trends) and within the different biogeoregions,
realms and taxonomic groups, as resulting from meta-analysis mixed models. Note that only
significant results (p ≤0.05) are reported for the biogeoregion, realm and taxonomic group-
specific analysis. Meta-analysis of multidecadal biodiversity trends
in Europe
https://doi.org/10.1038/s41467-020-17171-y
OPEN Abundance
Richness
Diversity
Turnover
Overall trend
z
−1.4
2.95
2
3.59
d.f. 152
160
160
160
p
0.162
0.003
0.045
0.003
Biogeoregion
Wald-type
test of model
coefficients
QM
24.397
65.83
21.23
38.6
d.f. 8
9
9
9
p
0.002
<0.001
0.012
<0.001
Atlantic
z
−2.73
p
0.006
North Sea
z
2.73
5.77
2.55
−2.42
p
0.006
<0.001
0.011
0.015
Black Sea
z
3.41
2.67
−2.73
p
<0.001
0.008
0.006
Boreal
z
4.35
2.09
2.05
p
<0.001
0.037
0.04
Alpine
z
3.79
p
<0.001
Continental
z
2.49
p
0.01
Realm
Wald-type
test of model
coefficients
QM
4.17
22.75
12.58
13.15
d.f. 3
3
3
3
p
0.243
<0.001
0.006
0.004
Freshwater
z
2.73
p
0.006
Marine
z
3.9
3.37
p
<0.001
<0.001
Terrestrial
z
3.25
p
0.001
Taxonomic group
Wald-type
test of model
coefficients
QM
24.67
57.09
20.06
d.f. 8
8
8
p
0.002
<0.001
0.01
Terrestrial
invertebrates
z
−2.95
p
0.003
Birds
z
4.11
4.78
p
<0.001
<0.001
Aquatic
invertebrates
z
2.42
2.26
p
0.015
0.024
Benthic algae
z
−5.33
p
<0.001
Plants
z
3.7
p
<0.001
Biodiversity trends for the whole dataset (overall trends) and within the different biogeoregions,
realms and taxonomic groups, as resulting from meta-analysis mixed models. Note that only Europe and increases in richness and abundance with increasing
temperature and naturalness. Table 1 Biodiversity trends. Abundance
Richness
Diversity
Turnover NATURE COMMUNICATIONS | (2020) 11:3486 | https://doi.org/10.1038/s41467-020-17171-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y Abundance
Richness
Diversity
e
Turnover
Decrease
Increase
No changes
Outside
150
100
Trend (S-statistic)
50
0
–50
–100
Adr
Alp
Atl
BIS
Bor
Con
Abundance
Richness
Diversity
Turnover
Med
NoS
Pan
a
c
d
b
Biodiversity trends in the different biogeoregions. The results of meta-analysis mixed models are shown for the four studied biodiversity metric
ance (a), richness (b), diversity (c) and turnover (d). Green: significant increasing trends (p ≤0.05); orange: significantly declining trends (p ≤0.05
dark grey for Adriatic Sea): no significant trends (p > 0.05). For biogeoregion identity see Fig. 1. e Values of S-statistics (model estimated mean, err
/−C.I.). Adr: Adriatic (n = 1 time series), Alp: Alpine (n = 33 time series), Atl Atlantic (n = 56 time series), BlS Black Sea (n = 5 time series), B
(n = 32 time series), Con Continental (n = 17 time series), Med Mediterranean (n = 9 time series), NoS North Sea (n = 7 time series), and Pa
nian (n = 1 time series). Dark blue: abundance, pink: richness, yellow: diversity, light blue: turnover. Solid line and dot: p ≤0.05; dashed line and ope
p > 0 05 Source data are provided as a Source Data file Abundance
Richness
Decrease
Increase
No changes
Outside
a
b Abundance
Decrease
Increase
No changes
Outside
a Richness
b b a Abundance Diversity
c
d Diversity
e
Turnover
c
d Turnover
d d c e
150
100
Trend (S-statistic)
50
0
–50
–100
Adr
Alp
Atl
BIS
Bor
Con
Abundance
Richness
Diversity
Turnover
Med
NoS
Pan e Fig. 2 Biodiversity trends in the different biogeoregions. The results of meta-analysis mixed models are shown for the four studied biodiversity metrics:
abundance (a), richness (b), diversity (c) and turnover (d). Green: significant increasing trends (p ≤0.05); orange: significantly declining trends (p ≤0.05);
black (dark grey for Adriatic Sea): no significant trends (p > 0.05). For biogeoregion identity see Fig. 1. e Values of S-statistics (model estimated mean, error
bar: +/−C.I.). Adr: Adriatic (n = 1 time series), Alp: Alpine (n = 33 time series), Atl Atlantic (n = 56 time series), BlS Black Sea (n = 5 time series), Bor
Boreal (n = 32 time series), Con Continental (n = 17 time series), Med Mediterranean (n = 9 time series), NoS North Sea (n = 7 time series), and Pan
Pannonian (n = 1 time series). Discussion Our results demonstrate considerable heterogeneity in the extent
and direction of change in biodiversity metrics in recent decades
between biogeoregions, realms and taxonomic groups in Europe. We identified increases in taxon richness in Northern and Eastern
Europe. Records in these regions are primarily represented by
terrestrial and aquatic invertebrate datasets. These observed pat-
terns are in line with recent modelled predictions of future
responses to global changes25 and are likely due to climate-
change induced poleward range shifts across taxa27–30. We also
detected declines in species abundances for terrestrial inverte-
brates and in the Atlantic biogeoregion (from which most of the We then apply a meta-analytic approach to identify the patterns
among biogeoregions, realms and taxonomic groups. Our time
series have an unbalanced distribution across biogeoregions, realms
and taxonomic groups, which is a common issue in macroecological
studies6,22,23. We account for the unbalanced design by testing the
robustness of our results using a sensitivity analysis (Supplementary
notes) and when interpreting the results. Our results show that
trends in common biodiversity metrics differ among biogeoregions,
realms, and taxonomic groups. Particularly, we find stronger
changes in community composition in Northern and Eastern 3 ARTICLE NATURE COMMUNICATIONS | (2020) 11:3486 | https://doi.org/10.1038/s41467-020-17171-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y MA
TE
FW
Abundance
Richness
Diversity
Increase
No changes
Turnover
Abundance
Richness
Diversity
Turnover
80
60
40
20
0
–20
–40
FW
MA
TE
Trend (S-statistic)
a
b
c
d
e
ig. 3 Biodiversity trends in the three realms. The results of meta-analysis mixed models are shown for the four studied biodiversity metrics: abun
a), richness (b), diversity (c) and turnover (d). Green: significant increasing trends (p ≤0.05); black: no significant trends (p > 0.05). e Values of
tatistics (model estimated mean, error bar: +/−C.I.). Dark blue: abundance, pink: richness, yellow: diversity, light blue: turnover. Solid line and do
.05; dashed line and open circle: p > 0.05. FW freshwater (n = 51 time series); MA marine and transitional zones (n = 18 time series); TE terrestria
2 time series). Source data are provided as a Source Data file. Richness
b MA
TE
FW
Abundance
Diversity
Increase
No changes
a
c MA
TE
FW
Abundance
Increase
No changes
a
c a Richness Increase
No changes Turnover
d d d c 80
60
40
20
0
–20
–40
FW
Trend (S-statistic)
e TE Fig. 3 Biodiversity trends in the three realms. The results of meta-analysis mixed models are shown for the four studied biodiversity metrics: abundance
(a), richness (b), diversity (c) and turnover (d). Green: significant increasing trends (p ≤0.05); black: no significant trends (p > 0.05). e Values of S-
statistics (model estimated mean, error bar: +/−C.I.). Dark blue: abundance, pink: richness, yellow: diversity, light blue: turnover. Solid line and dot: p ≤
0.05; dashed line and open circle: p > 0.05. FW freshwater (n = 51 time series); MA marine and transitional zones (n = 18 time series); TE terrestrial (n =
92 time series). Source data are provided as a Source Data file. colonization of more tolerant and generalist species36 and/or
invasive taxa, resulting in biotic homogenization despite no
change in overall species richness36,37. Such patterns were, for
example, observed in vascular plants at coarse scales in Europe
due to the extinction of rare native species and spread of alien
species38. Thus, temporal changes in taxonomic composition, i.e.,
turnover, are likely to be more sensitive than simply taxonomic
richness (i.e., alpha-diversity) and abundance in responses to
global change39,40. Accordingly, we show that temporal changes
in taxon turnover are more pertinent across biogeoregions than
the other three studied biodiversity metrics. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y Dark blue: abundance, pink: richness, yellow: diversity, light blue: turnover. Solid line and dot: p ≤0.05; dashed line and open
circle: p > 0.05. Source data are provided as a Source Data file. terrestrial invertebrate time series in our dataset are derived). Our
results, based on data with larger spatial, temporal and taxonomic
coverage, and finer temporal resolution, corroborate recent
reports of worldwide declines of local terrestrial insect commu-
nities31–33. studies on local changes in biodiversity also detected no overall
changes5,22,34 in apparent contradiction to the documented
global-scale biodiversity loss (e.g. IPBES4). However, the extinc-
tion of rare species, by definition, is often restricted to the very
few local places where these species occur and may thus go
undetected in large extent quantitative studies35. Furthermore,
the loss of specialist taxa could be compensated locally by the Several studied biogeoregions, realms and biotic groups
showed no significant trends in biodiversity metrics. Other 4 ARTICLE NATURE COMMUNICATIONS | (2020) 11:3486 | https://doi.org/10.1038/s41467-020-17171-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y Dar
ue: abundance pink: richness yellow: diversity light blue: turnover Solid line and dot: p ≤0 05; dashed line and open circle: p > 0 05 Number of time Diversity
Turnover
Decrease
Increase
No changes
e
c
d
150
100
Trend (S-statistic)
50
0
–50
–100
–150
Abundance
Richness
Diversity
Turnover
Plants
Terrestrial
invertebrates
Mammals
Birds
Benthic
algae
Plankton
Aquatic
invertebrates
Fish
Fig. 4 Biodiversity trends for the studied taxonomic groups. The results of meta-analysis mixed models are shown for the four studied biodiversity
metrics: abundance (a), richness (b), diversity (c) and turnover (d). Green: significant increasing trends (p ≤0.05); orange: significant declining trends
(p ≤0.05); black: no significant trends (p > 0.05). Drawings from phylopic.org. e values of S-statistics (model estimated mean, error bar: +/−C.I.). Dark
blue: abundance, pink: richness, yellow: diversity, light blue: turnover. Solid line and dot: p ≤0.05; dashed line and open circle: p > 0.05. Number of time
series (n): Plants: 34, terrestrial invertebrates: 53, mammals: 1, birds: 16, benthic algae: 7, plankton: 9, aquatic invertebrates: 38, fish: 3. Source data are
provided as a Source Data file. Decrease
Increase
No changes Diversity
Turnover
Decrease
Increase
No changes
c
d e
150
100
Trend (S-statistic)
50
0
–50
–100
–150
Abundance
Richness
Diversity
Turnover
Plants
Terrestrial
invertebrates
Mammals
Birds
Benthic
algae
Plankton
Aquatic
invertebrates
Fish e Fig. 4 Biodiversity trends for the studied taxonomic groups. The results of meta-analysis mixed models are shown for the four studied biodiversity
metrics: abundance (a), richness (b), diversity (c) and turnover (d). Green: significant increasing trends (p ≤0.05); orange: significant declining trends
(p ≤0.05); black: no significant trends (p > 0.05). Drawings from phylopic.org. e values of S-statistics (model estimated mean, error bar: +/−C.I.). Dark
blue: abundance, pink: richness, yellow: diversity, light blue: turnover. Solid line and dot: p ≤0.05; dashed line and open circle: p > 0.05. Number of time
series (n): Plants: 34, terrestrial invertebrates: 53, mammals: 1, birds: 16, benthic algae: 7, plankton: 9, aquatic invertebrates: 38, fish: 3. Source data are
provided as a Source Data file. changes are due to direct human impact, indirect effects (see e.g.,
Didham et al.47, in the context of biological invasions) or recovery
processes48. consistent with other studies, e.g.14,25. However, we could also
identify a combined effect of naturalness and temperature on
abundance, richness and turnover trends. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y The observed
increases in temporal taxon turnover are spatially structured
across Europe, involving mostly biogeoregions of Northern and
Eastern Europe. Such changes in community composition can
reflect non-equilibrium dynamics (including time-lags and tran-
sient
phenomena41)
with,
for
example,
climate
change42, pollution or the introduction and spread of alien species43. Moreover, over the past 30–40 years (i.e. the period covered by
most time series in our dataset) positive impacts of environmental
regulation, e.g., reductions in atmospheric emissions and hence
acid rain, could also be important drivers of biotic change (see
e.g., Monteith et al.44). In a recent analysis of UK vegetation data,
an overall increase in vegetation species richness was linked to
recovery from acidification45. Accordingly, increases in species
turnover could also reflect a process of biological recovery from
past disturbances. Another possible interacting factor is the
change in land use, which follows different temporal trajectories
in different European regions, and thus could concur in
explaining regional differences in biodiversity trends46. Future
research should clarify to what extent the increasing taxon
turnover is led by the spread of generalist and invasive species
compared to declines in rare species, and whether the observed 5 ARTICLE NATURE COMMUNICATIONS | (2020) 11:3486 | https://doi.org/10.1038/s41467-020-17171-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y Abundance
Richness
Diversity
Turnover
Decrease
Increase
No changes
e
a
c
d
b
150
100
Trend (S-statistic)
50
0
–50
–100
–150
Abundance
Richness
Diversity
Turnover
Plants
Terrestrial
invertebrates
Mammals
Birds
Benthic
algae
Plankton
Aquatic
invertebrates
Fish
4 Biodiversity trends for the studied taxonomic groups. The results of meta-analysis mixed models are shown for the four studied biodiversity
cs: abundance (a), richness (b), diversity (c) and turnover (d). Green: significant increasing trends (p ≤0.05); orange: significant declining trends
0.05); black: no significant trends (p > 0.05). Drawings from phylopic.org. e values of S-statistics (model estimated mean, error bar: +/−C.I.). Dark
abundance, pink: richness, yellow: diversity, light blue: turnover. Solid line and dot: p ≤0.05; dashed line and open circle: p > 0.05. Number of time
TICLE
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y Abundance
Richness
Decrease
a
c
d
b Diversity
Turnover
Decrease
Increase
No changes
e
c
d
150
100
Trend (S-statistic)
50
0
–50
–100
–150
Abundance
Richness
Diversity
Turnover
Plants
Terrestrial
invertebrates
Mammals
Birds
Benthic
algae
Plankton
Aquatic
invertebrates
Fish
4 Biodiversity trends for the studied taxonomic groups. The results of meta-analysis mixed models are shown for the four studied biodiver
rics: abundance (a), richness (b), diversity (c) and turnover (d). Green: significant increasing trends (p ≤0.05); orange: significant declining t
≤0.05); black: no significant trends (p > 0.05). Drawings from phylopic.org. e values of S-statistics (model estimated mean, error bar: +/−C.I.)
e: abundance, pink: richness, yellow: diversity, light blue: turnover. Solid line and dot: p ≤0.05; dashed line and open circle: p > 0.05. Number o
es (n): Plants: 34, terrestrial invertebrates: 53, mammals: 1, birds: 16, benthic algae: 7, plankton: 9, aquatic invertebrates: 38, fish: 3. Source da
vided as a Source Data file. Diversity
Turnover
Decrease
Increase
No changes
e
c
d
150
100
Trend (S-statistic)
50
0
–50
–100
–150
Abundance
Richness
Diversity
Turnover
Plants
Terrestrial
invertebrates
Mammals
Birds
Benthic
algae
Plankton
Aquatic
invertebrates
Fish
g. 4 Biodiversity trends for the studied taxonomic groups. The results of meta-analysis mixed models are shown for the four studied biodiversity
etrics: abundance (a), richness (b), diversity (c) and turnover (d). Green: significant increasing trends (p ≤0.05); orange: significant declining trends
≤0.05); black: no significant trends (p > 0.05). Drawings from phylopic.org. e values of S-statistics (model estimated mean, error bar: +/−C.I.). ARTICLE However, our data, as well as
most of the data used in other studies6,10,22,23, is being considered
in the absence of the longer historical perspective required to
capture the overall changes in biodiversity in the Anthropocene,
as we lack baseline data from times when human impacts were
lower (e.g. pre-industrial era). This limitation is a common issue
in studies of long-term biodiversity change (but see ref. 53) that
can have a detrimental effect in restoration ecology54,55. The lack
of baseline data could be overcome by the integration of ecolo-
gical and paleobiological approaches (e.g. Battarbee et al.56),
which could extend the temporal dimension back to, e.g., the end
of the last ice age. Such approaches could have important con-
servation implications57 and allow for comparisons between
impacted and unimpacted sites within the same geographical
area, which could reveal differences between changes driven by
climate and by direct anthropogenic pressures (e.g. changes in
land use or pollution)58. Looking into the future, there is an
urgent need to harmonize biodiversity monitoring schemes59,60
that would also allow improved up- and downscaling of trends as
well as integrating cross-domain feedback loops61. strongest changes in biodiversity metrics with increasing tem-
perature (i.e. steeper increase in abundance and richness and
steep decline in turnover). We speculate that degraded sites are
more prone to invasion of generalist and invasive species, while
natural sites may be more resilient49. A major obstacle in assessing biodiversity trends is that long-
term data are unevenly distributed among taxonomic groups and
are biased towards charismatic taxa, such as birds and mammals,
and towards groups with long study traditions, such as vascular
plants and marine fishes, while invertebrates are relatively
neglected, except butterflies and bees in some countries. Our
study encompasses an unusually large variety of taxonomic
groups, including those largely overlooked in previous large-scale
biodiversity studies6,20. Some of these overlooked groups that we
included in our analysis, such as aquatic invertebrates, showed
unexpected increases in richness and diversity, likely due to the
aforementioned processes (e.g. recovery from stressors, spread of
generalist or invasive taxa and taxa adapted to warmer tem-
peratures). On the other hand, we recorded declines in species
abundances for terrestrial invertebrates in the Atlantic biogeor-
egion, consistent with previous findings31,32,50. Our results
therefore emphasize that patterns of change in the biodiversity of
the most studied ‘iconic’ groups cannot be extrapolated across
other taxa. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y Table 2 Influence of climatic trends and site characteristics
on biodiversity trends. Explanatory variables
z
Abundance
Intercept
−1.48
Temperature trend
1.22
Precipitation trend
0.18
Naturalness
1.73
Latitude
−0.99
Study length
−0.30
Temperature trend: Naturalness
−2.39
Richness
Intercept
3.66
Temperature trend
2.04
Precipitation trend
0.38
Naturalness
1.14
Latitude
0.49
Longitude
2.16
Elevation
−1.20
Study length
0.37
Temperature trend: Naturalness
−2.02
Diversity
Intercept
1.97
Temperature trend
−0.56
Precipitation trend
−2.05
Latitude
−0.49
Longitude
3.49
Elevation
−1.66
Study length
1.27
Turnover
Intercept
3.35
Temperature trend
−0.60
Precipitation trend
1.66
Naturalness
−0.52
Latitude
1.66
Longitude
−1.21
Elevation
4.09
Study length
1.03
Temperature trend: Naturalness
3.39
The table shows the effect sizes of the explanatory variables on the studied biodiversity metrics. of support as they do not fit into the temporal extent of most
funding schemes. In contrast to space-for-time or aggregated
snapshot (e.g. opportunistic data or atlases of species distribu-
tions) approaches, the long-term tracking of communities in
specific locations minimizes the risk of biases related to shifts in
sampling locations, sampling areas and protocols51. Table 2 Influence of climatic trends and site characteristics
on biodiversity trends. p
g
p
g
p
Our dataset is, to the best of our knowledge, the most com-
prehensive in terms of the spatial and temporal extent and
taxonomic and realm representation of high temporal resolution
long-term biodiversity monitoring records in Europe. Most sites
included in this study are part of the global ILTER network and
the vast majority are characterized by low anthropogenic pres-
sure, which may have led to an underestimation of the true scale
of biodiversity changes at continental scale. Perhaps most
importantly, most studied sites are shielded from direct effects of
changes in land-use and loss of habitat, e.g., conversion to
intensive agriculture and urbanization. To overcome such sources
of bias, long-term monitoring programs should include a larger
representation of more intensively used (e.g. agricultural) areas
and incorporate sites vulnerable to significant anthropogenic
perturbations51. On the other hand, our approach reduces the
potential risk of tracking immediate biodiversity responses to
localized disturbance or successional recovery processes, which
can bias the estimates of biodiversity change52. y
g
Although most of our time series do not predate the 1980s
(only one study goes back to the 1920s), we were still able to
detect evidence of substantial reorganization of communities
within relatively short time frames. NATURE COMMUNICATIONS | (2020) 11:3486 | https://doi.org/10.1038/s41467-020-17171-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y More specifically, and
counterintuitive to common expectation, we found that sites
considered to be in a less natural state are those experiencing the p
Our model results showing positive correlations of temperature
and naturalness
with richness and
abundance trends are 6 Methods
D t Data compilation. We circulated a call for biodiversity data within the ILTER
network and additional partners to fill in geographical gaps. The criteria for data
selection were: (1) each time series covers at least 15 years, (2) with preferably at
least ten survey events during that time, (3) sampling occurred at the same site
(no space-for-time substitution), and (4) survey method, seasonal and taxonomic
resolution were consistent throughout the whole study period for each time series. The final dataset included 161 time series from 89 ILTER sites and 26 additional
long-term monitoring sites from other networks. The time span of the studied time
series ranged between 15 and 91 years (median: 20), so that one time series started
in 1921 while the others started between the 1980 and 2003 (median: 1994). The
end years range between 2005 and 2018 (median: 2015; Supplementary Fig. 2). Additional meta-analysis mixed models were used to test the influence of
selected abiotic variables describing site characteristics and climate on the
biodiversity trends. We included the following variables: latitude, longitude,
elevation, site naturalness, S-statistics65 of temperature and precipitation trends,
and the length of each time series. These explanatory variables showed little
collinearity (|r| < 0.6), and were thus all retained as potential predictors. Similar to
Everaert et al.71, we applied an information-theoretic approach to model selection
and multi-model inference72, to determine the relative importance of those
explanatory variables on the trends in biodiversity metrics. For this we created a
candidate set of models with all possible linear combinations of explanatory
variables and we extracted the corrected Akaike's information criterion (AICc) of
each model using the R package glmulti73. We retained only plausible models with
δAICc ≤2 of the best model (i.e. the one(s) with the lowest AICc72) and computed
the relative importance of each predictor variable as the sum of the Akaike's
weights of all the selected models in which that variable was included. We
computed the model-averaged coefficients (±95% C.I.) for each predictor variable
in each selected model, weighted by the Akaike's weights (Supplementary Table 2). To evaluate the effect of the interaction among climatic and local stressors, we
added to the selected models the interactions between site naturalness and
temperature trends, and the interaction between site naturalness and precipitation
trends. We then compared the resulting models (without interaction, with single
interaction, with both interactions) and chose the one with the lowest AIC value. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y drivers of these changes. This demonstrates the urgent need to
complement biodiversity time series with environmental data that
can be used to explain the observed patterns but are often not
collected on a routine basis (with exceptions in limnology and
oceanography). The complexity of biodiversity dynamics that our
results and those of other studies highlight is not to be interpreted
as an obstacle to the development of conservation measures. Rather we argue that a better understanding of the patterns,
trends and changes in biodiversity and environmental conditions
will allow conservation measures to be better tailored to specific
locations and taxonomic groups in order to significantly retard or
even reverse further biodiversity loss62. drivers of these changes. This demonstrates the urgent need to
complement biodiversity time series with environmental data that
can be used to explain the observed patterns but are often not
collected on a routine basis (with exceptions in limnology and
oceanography). The complexity of biodiversity dynamics that our
results and those of other studies highlight is not to be interpreted
as an obstacle to the development of conservation measures. Rather we argue that a better understanding of the patterns,
trends and changes in biodiversity and environmental conditions
will allow conservation measures to be better tailored to specific
locations and taxonomic groups in order to significantly retard or
even reverse further biodiversity loss62. For the first step of the analysis, we used the Mann–Kendall trend test to
identify monotonic trends in each biodiversity and climate time series over the
study period65,66. We detected serially correlated time series using auto- and cross-
covariance and correlation functions67, and we applied the modified
Mann–Kendall with Hamed and Rao68 variance correction approach. We used S-
statistic and its variance as effect size of the trend65 for the next step of the analysis. A similar meta-analytical approach has already been applied in a previous study on
ecological time series69. pp
Mann–Kendall with Hamed and Rao68 variance correction approach. We used S-
statistic and its variance as effect size of the trend65 for the next step of the analysis. A similar meta-analytical approach has already been applied in a previous study on
ecological time series69. At 23 study sites, multiple time series were available for a given taxonomic
group, e.g., when surveys were conducted at multiple transects or plots or with
multiple traps. Methods
D t We have implemented this procedure because, to the best of our knowledge, it is
not possible to include selected interactions into the information-theoretic
approach to model selection and including all interactions would have resulted in
an overly complex model. Biodiversity data. Biodiversity data were expressed as abundance or biomass of
surveyed taxa at each survey occasion, in some instances as percent coverage (e.g. for some time series of benthic algae and plants). Survey methods and season
varied among time series, but were kept constant within each time series
throughout the entire study period. In most cases, surveys were carried out once a
year, but some time series had gaps or more frequent survey intervals (e.g. weekly
resolution for phytoplankton, zooplankton and moths from the Finnish and Czech
sites). In the latter case, we filtered the data in the time series to select only the
months/seasons that were consistently surveyed throughout the whole study per-
iod, and we pooled the data (as sums or averages) within each year. Taxonomic
resolution was kept constant for each time series throughout the entire study
period, generally at the species or genus level, with a few exceptions (i.e. a few
macroinvertebrates groups were identified at family resolution). g
y
For each year within each time series, we computed four biodiversity metrics:
total number of organisms or biomass (hereby referred to as abundance),
taxonomic richness (i.e. number of taxa), Simpson´s diversity and temporal taxon
turnover. The latter was computed as the proportion of taxa gained and lost
between two subsequent years relative to the total number of taxa observed, using
Eq. (1), as implemented in the R package codyn63. p
approach to model selection and including all interactions would have resulted in
an overly complex model. Data availability All datasets analyzed during the current study have been deposited in public repositories,
at the links reported in Supplementary Data. Please note that some of the datasets will be
publicly accessible after a period of embargo, from 1st January 2021. The source data
underlying all figures are provided as a Source Data file. Source data are provided with
this paper. We gathered information on local anthropogenic pressures in a standardized
questionnaire (Supplementary Methods). The questionnaire asked the data
providers to assess the impact (from 1 = no to 4 = strong impact) of a series of
pressures at the site (e.g. urbanization, sources of pollution, agriculture, etc.; for a
full list see Supplementary Methods) and indicate whether impacts were constant
or changed throughout the study period. Data providers were also asked to
estimate the overall environmental quality of the site (i.e. naturalness; from 1 = low
to 5 = high) and to state whether it was constant or changed throughout the study
period. We used this information to define the quality-class of the sites, based on
the overall assessment. The majority of sites (n = 67) scored 5 (i.e. high
naturalness), 42 sites scored 4, 37 sites scored 3 and 15 sites scored 2. Code availability The R code used for all analyses is available at the GitHub repository: https://github.com/
Biodiversity-trends-in-Europe-ILTER/R-code. Source data are provided with this paper. Received: 20 November 2019; Accepted: 16 June 2020; Received: 20 November 2019; Accepted: 16 June 2020; Received: 20 November 2019; Accepted: 16 June 2020; Data analysis. We used a two-step procedure that allowed us to combine very
heterogeneous original datasets (see paragraph Biodiversity data). First, we ana-
lyzed each time series separately to quantify time-series-specific biodiversity trends. Second, we used the effect sizes of the individual time-series-specific biodiversity
trends to synthesize the overall trends and identify common patterns and drivers. The results of this second (meta-)analysis are reported in the paper. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y To avoid pseudoreplication, we combined those time series using
meta-analysis mixed models (using the R package metafor70) and extracted the
cumulative effect sizes and their variances prior to the second step of the analysis. The total of 161 time series reported above refers to the aggregated final set of time
series. The second step of the analysis aimed at synthesizing the trends across the
different time series. For that, we fitted meta-analysis mixed models to account for
random effects. To compute the overall biodiversity trends and to explore how the
trends varied among biogeoregions, realms and taxonomic groups, we included
biogeoregion (nine levels), realm (three levels: freshwater, marine and transitional
zone, and terrestrial) and taxonomic group (eight levels) as explanatory variables in
the models with no intercept. We did so separately because biogeoregion, realm
and taxonomic group were not independent (see Supplementary Table 1). The
results of these models hence show whether or not the overall S-statistics for
individual trends in the groups differ significantly from zero. We classified the time series into nine biogeoregions, three realms and eight
taxonomic groups (Fig. 1). Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this article. Abiotic variables. For each study site we extracted the daily mean temperature
and daily total precipitation data from the gridded observational dataset for pre-
cipitation, temperature and sea level pressure in Europe (spatial resolution: 0.25
degrees64), and computed the mean annual temperature and total annual pre-
cipitation. For aquatic ecosystems we used in situ water temperature measured at
the surface and calculated the mean temperature across the yearly monitoring
periods. ARTICLE g
g
p
The inherent complexity of ecological systems manifested by
diverging long-term responses of local biodiversity still hamper
any upscaling of these trends to a continental or even global scale. This might explain the partly contradictory results not only
within but also among large-scale studies that are based on local
biodiversity data6,22. For example, our study revealed higher
taxon turnover in the terrestrial realm at European scale while, at
a global scale, turnover seems higher in marine assemblages22. However, these studies do agree on the lack of an overall decline
in species richness and on the increase in taxon turnover over
time. We argue that these contradictory results can be driven by
the insufficient number and quality of systemic and harmonized
biodiversity monitoring activities at a local scale and by the
insufficient length of the underlying time series. Regarding the
latter, we should bear in mind that prior to the start of most of
our biodiversity time series (mainly in the 1980s), many species
had already declined in abundance or gone extinct. Moreover, the
vast majority of larger scale studies describing biodiversity
changes have not been able to clearly identify the environmental These findings reiterate the need to not only maintain but to
increase the numbers of long-term monitoring schemes of local
ecosystems. Such schemes can provide unique insights for ecol-
ogists and conservationists, yet they are often threatened by a lack 7 7 ARTICLE Number of taxa gained þ number of taxa lost
total number of taxa ð1Þ To account for biases in biogeoregions, realms and taxonomic groups, we also
performed sensitivity analyses (Supplementary notes and Supplementary Table 3),
which confirmed that the results were robust. We classified the time series into nine biogeoregions, three realms and eight
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from the Mondego estuary (Portugal) were supported by the Centre for Functional
Ecology Strategic Project (UID/BIA/04004/2019) within the PT2020 Partnership
Agreement and COMPETE 2020, and by FEDER through the project ReNATURE
(Centro 2020, Centro-01-765-0145-FEDER-000007). We would like to thank Lim-
burgse Koepel voor Natuurstudie (LiKoNa) for the data related to the National Park Peer review information Nature Communications thanks the anonymous reviewer(s) for
their contribution to the peer review of this work. [Peer reviewer reports are available.] Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://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 institutional affiliations. Protection Agency. Data collection at Esthwaite Water and a subset of UK ECN
sites was supported by Natural Environment Research Council award number NE/
R016429/1 as part of the UK-SCaPE programme delivering National Capability. Sponsorship of other UK ECN sites contributing data was provided by Agri-Food and
Biosciences Institute, Biotechnology and Biological Sciences Research Council,
Department of Environment Food and Rural Affairs, Natural Resources Wales, Defense
Science Technology Laboratory, Environment Agency, Forestry Commission, Forest
Research, the James Hutton Institute (The Rural & Environment Science & Analytical
Services Division of the Scottish Government), Natural England, Rothamsted Research,
Scottish Government, Scottish Natural Heritage and the Welsh Government. Data
from the Mondego estuary (Portugal) were supported by the Centre for Functional
Ecology Strategic Project (UID/BIA/04004/2019) within the PT2020 Partnership
Agreement and COMPETE 2020, and by FEDER through the project ReNATURE
(Centro 2020, Centro-01-765-0145-FEDER-000007). We would like to thank Lim-
burgse Koepel voor Natuurstudie (LiKoNa) for the data related to the National Park Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The images or other third party
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D.G.G., U.G., J.M.G., L. Hadar, L. Halada, M.H., H.H., K.L.H., B.J., T.C.J., H.K., I.K.S., I. K R L F M H M J M S M D M B P N D Oro D Ozoliņš B M P D P M P M F.P. and P.H. conceived the study. F.P. analyzed the data, with inputs by I.K. F.P. wrote
the manuscript, with major contributions by P.H. and I.K. R. Adrian, R. Alber, A.A., C. A., J.B., L.B., D.B., N.B., S.B., D.S.B., V.B., E.C., R.C., P.C., B.J.E., G.E., V.E., H.F., R.G.G.,
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of the manuscript. 71. Everaert, G., Deschutter, Y., De Troch, M., Janssen, C. R. & De
Schamphelaere, K. Multimodel inference to quantify the relative importance
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65. Kendall, M. G. Rank correlation methods. (1948). 9 NATURE COMMUNICATIONS | (2020) 11:3486 | https://doi.org/10.1038/s41467-020-17171-y | www.nature.com/naturecommunications Competing interests 74. EEA. Biogeographical regions and Marine regions and subregions under the
Marine Strategy Framework Directive. https://www.eea.europa.eu/data-and-
maps (2016). Acknowledgements Supplementary information is available for this paper at https://doi.org/10.1038/s41467-
020-17171-y. Supplementary information is available for this paper at https://doi.org/10.1038/s41467-
020-17171-y. We are grateful to the ILTER network and the eLTER PLUS project (Grand Agreement
No. 871128) for financial support. We acknowledge the E-OBS dataset from the EU-
FP6 project ENSEMBLES (http://ensembles-eu.metoffice.com) and the data providers
in the ECA&D project (http://www.ecad.eu). The evaluation of forest plant diversity
was based on data collected by partners of the official UNECE ICP Forests Network We are grateful to the ILTER network and the eLTER PLUS project (Grand Agreement
No. 871128) for financial support. We acknowledge the E-OBS dataset from the EU-
FP6 project ENSEMBLES (http://ensembles-eu.metoffice.com) and the data providers
in the ECA&D project (http://www.ecad.eu). The evaluation of forest plant diversity
was based on data collected by partners of the official UNECE ICP Forests Network Correspondence and requests for materials should be addressed to F.P. or P.H. No. 871128) for financial support. We acknowledge the E-OBS dataset from the EU-
FP6 project ENSEMBLES (http://ensembles-eu.metoffice.com) and the data providers
in the ECA&D project (http://www.ecad.eu). The evaluation of forest plant diversity
was based on data collected by partners of the official UNECE ICP Forests Network
(http://icp-forests.net/contributors); part of the data were co-financed by the European
Commission, project LIFE 07 ENV/D/000218 “Further Development and Imple-
mentation of an EU-level Forest monitoring Systeme (FutMon)”. Data on wintering
water birds in Bulgaria were provided by the national Executive Environment Agency
with the Ministry of Environment and Waters. Data from the Finnish moth monitoring
scheme were supported by the Finnish Ministry of the Environment. Data from the
Swedish ICP Integrated Monitoring sites were financed by the Swedish Environmental
Protection Agency. Data collection at Esthwaite Water and a subset of UK ECN
sites was supported by Natural Environment Research Council award number NE/
R016429/1 as part of the UK-SCaPE programme delivering National Capability. Sponsorship of other UK ECN sites contributing data was provided by Agri-Food and
Biosciences Institute, Biotechnology and Biological Sciences Research Council,
Department of Environment Food and Rural Affairs, Natural Resources Wales, Defense
Science Technology Laboratory, Environment Agency, Forestry Commission, Forest
Research, the James Hutton Institute (The Rural & Environment Science & Analytical
Services Division of the Scottish Government), Natural England, Rothamsted Research,
Scottish Government, Scottish Natural Heritage and the Welsh Government. © The Author(s) 2020 1Senckenberg Research Institute and Natural History Museum Frankfurt, Gelnhausen, Germany. 2Environmental Archaeology Lab, Department of
Historical, Philosophical and Religious Studies, Umeå University, Umeå, Sweden. 3Department of Community Ecology, Helmholtz Centre for
Environmental Research - UFZ, Halle, Germany. 4Martin Luther University Halle-Wittenberg, Geobotany and Botanical Garden, Halle, Germany.
5German Centre for Integrative Biodiversity Research (iDiv) Halle - Jena - Leipzig, Leipzig, Germany. 6Department of Ecosystem Research, Leibniz
Institute of Freshwater Ecology and Inland Fisheries & Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Berlin, Germany.
7Biological Laboratory, Agency for Environment and Climate Protection, Bolzano, Italy. 8UMR 0980 BAGAP, INRAE – Institut Agro – ESA,
Rennes, France. 9LTSER Zone Atelier Armorique, 35042 Rennes, France. 10UK Centre for Ecology & Hydrology, Bush Estate, Penicuik,
Midlothian, UK. 11Institute for Atmospheric and Earth system Research, Department of Forest Sciences, University of Helsinki, Helsinki, Finland.
12Dynafor, INRAE, University of Toulouse, France & CESCO, Muséum National d’Histoire Naturelle, Sorbonne-Univ, Paris, France & LTSER Zone
Atelier Pyrénées Garonne, Auzeville-Tolosane, France. 13Rothamsted Research, North Wyke, Okehampton, Devon, UK. 14Centre for Environmental
Sciences, Hasselt University, Hasselt, Belgium. 15Forest Research, Farnham, UK. 16University of South Bohemia, Faculty of Science, Department of
Ecosystem Biology & Soil and Water Research Infrastructure, Ceske Budejovice, Czech Republic. 17Czech Academy of Sciences, Biology Centre,
Institute of Entomology, Ceske Budejovice, Czech Republic. 18CEBC, UMR7372, CNRS & La Rochelle University, 79360 Villiers en bois, France.
19LTSER Zone Atelier Plaine & Val de Sèvre, 79360 Beauvoir sur Niort, France. 20Institute of Marine Sciences, National Research Council,
Venice, Italy. 21School of Biosciences and Veterinary Medicine, unit Plant Diversity and Ecosystems Management, University of Camerino,
Camerino, Italy. 22CIIMAR, Interdisciplinary Centre of Marine and Environmental Research of the University of Porto, Porto, Portugal. 23Sovon
Dutch Centre for Field Ornithology, Nijmegen, The Netherlands. 24Flanders Marine Institute, Ostend, Belgium. 25Institute of Biodiversity and
Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria. 26UK Centre for Ecology & Hydrology, Lancaster Environment Centre,
Lancaster, UK. 27Instituto Pirenaico de Ecología (CSIC), Jaca, Spain. 28Department of Aquatic Sciences and Assessment, Swedish University of
Agricultural Sciences, Uppsala, Sweden. 29Department of Natural Forests, Forest Research Institute, Białowieża, Poland. 30Ramat Hanadiv, Zikhron
Ya’akov, Israel. 31Institute of Landscape Ecology SAS, Branch Nitra, Slovakia. 32MTA Centre for Ecological Research, Institute of Ecology and
Botany, Vácrátót, Hungary. 33Royal Netherlands Institute for Sea Research, and Utrecht University, Yerseke, The Netherlands. 34Department of
Ecology and Genetics, University of Oulu, Oulu, Finland. 35Oulanka Research Station, University of Oulu Infrastructure Platform, Kuusamo, Finland.
36Białowieża Geobotanical Station, Faculty of Biology, University of Warsaw, Białowieża, Poland. 37Norwegian Institute for Nature Research, NINA-
Oslo, Norway. 38Institute of Landscape Ecology SAS, Bratislava, Slovakia. 39Geosciences and Natural Resource Management, University of
Copenhagen, Copenhagen, Denmark. 40Senckenberg am Meer, Marine Research Department, Wilhelmshaven, Germany. 41Kainuu Centre for
Economic Development, Transport and the Environment, Kajaani, Finland. 42Centre For Functional Ecology (CFE), Department of Life Sciences,
University of Coimbra, Coimbra, Portugal. 43Northwest German Forest Research Institute, Göttingen, Germany. 44Forest Services, Autonomous
Province of Bolzano - South Tyrol, Bolzano, Italy. 45CEAB (CSIC), 17300 Blanes, Spain. 46IMEDEA (CSIC-UIB), 07190 Esporles, Spain. 47Institute of
Biology, University of Latvia, Salaspils, Latvia. 48Dipartimento di Architettura, Design e Urbanistica, Università degli Studi di Sassari, Sassari, Italy.
49UK Centre for Ecology & Hydrology, Wallingford, UK. 50Carabinieri, Biodiversity and Park Protection Department, Castel di Sangro Biodiversity
Unit, L’Aquila, Italy. 51ZRC SAZU Karst Research Institute, Ljubljana & UNESCO Chair on Karst Education University of Nova Gorica,
Vipava, Slovenia. 52Finnish Environment Institute (SYKE), Biodiversity Centre, Helsinki, Finland. 53Swiss Federal Institute for Forest Snow and
Landscape Research WSL, Birmensdorf, Switzerland. 54Norwegian Institute for Water Research, Oslo, Norway. 55Ecological Sciences, James
Hutton Institute, Craigiebuckler, Aberdeen, UK. 56University of Applied Sciences Trier, Environmental Campus Birkenfeld, Birkenfeld, Germany.
57University of Duisburg-Essen, Essen, Germany. 58Swedish Meteorological and Hydrological Institute, Gothenburg, Sweden. 59Flanders Research
Institute for Agriculture, Fishery and Food, Oostende, Belgium. 60Research Institute for Nature and Forest, Brussels, Belgium. 61Department of
Animal Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, The Netherlands. ✉email: francesca.pilotto@umu.se; peter.
haase@senckenberg.de Acknowledgements If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/. © The Author(s) 2020 Francesca Pilotto
1,2✉, Ingolf Kühn
3,4,5, Rita Adrian6, Renate Alber7, Audrey Alignier8,9,
Christopher Andrews
10, Jaana Bäck11, Luc Barbaro
12, Deborah Beaumont13, Natalie Beenaerts
14,
Sue Benham
15, David S. Boukal
16,17, Vincent Bretagnolle18,19, Elisa Camatti
20, Roberto Canullo
21,
Patricia G. Cardoso22, Bruno J. Ens
23, Gert Everaert
24, Vesela Evtimova
25, Heidrun Feuchtmayr
26,
Ricardo García-González27, Daniel Gómez García27, Ulf Grandin
28, Jerzy M. Gutowski
29, Liat Hadar30,
Lubos Halada
31, Melinda Halassy
32, Herman Hummel
33, Kaisa-Leena Huttunen34,35,
Bogdan Jaroszewicz
36, Thomas C. Jensen
37, Henrik Kalivoda38, Inger Kappel Schmidt
39,
Ingrid Kröncke40, Reima Leinonen41, Filipe Martinho
42, Henning Meesenburg
43, Julia Meyer40,
Stefano Minerbi44, Don Monteith
26, Boris P. Nikolov
25, Daniel Oro45,46, Dāvis Ozoliņš47, Francesca Pilotto
1,2✉, Ingolf Kühn
3,4,5, Rita Adrian6, Renate Alber7, Audrey Alignier8,9,
Christopher Andrews
10, Jaana Bäck11, Luc Barbaro
12, Deborah Beaumont13, Natalie Beenaerts
14,
Sue Benham
15, David S. Boukal
16,17, Vincent Bretagnolle18,19, Elisa Camatti
20, Roberto Canullo
21,
Patricia G. Cardoso22, Bruno J. Ens
23, Gert Everaert
24, Vesela Evtimova
25, Heidrun Feuchtmayr
26,
Ricardo García-González27, Daniel Gómez García27, Ulf Grandin
28, Jerzy M. Gutowski
29, Liat Hadar30,
Lubos Halada
31, Melinda Halassy
32, Herman Hummel
33, Kaisa-Leena Huttunen34,35,
Bogdan Jaroszewicz
36, Thomas C. Jensen
37, Henrik Kalivoda38, Inger Kappel Schmidt
39,
Ingrid Kröncke40, Reima Leinonen41, Filipe Martinho
42, Henning Meesenburg
43, Julia Meyer40,
Stefano Minerbi44, Don Monteith
26, Boris P. Nikolov
25, Daniel Oro45,46, Dāvis Ozoliņš47, NATURE COMMUNICATIONS | (2020) 11:3486 | https://doi.org/10.1038/s41467-020-17171-y | www.nature.com/naturecommunicatio 10 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y Vipava, Slovenia. 52Finnish Environment Institute (SYKE), Biodiversity Centre, Helsinki, Finland. 53Swiss Federal Institute for Forest Snow and
Landscape Research WSL, Birmensdorf, Switzerland. 54Norwegian Institute for Water Research, Oslo, Norway. 55Ecological Sciences, James
Hutton Institute, Craigiebuckler, Aberdeen, UK. 56University of Applied Sciences Trier, Environmental Campus Birkenfeld, Birkenfeld, Germany.
57University of Duisburg-Essen, Essen, Germany. 58Swedish Meteorological and Hydrological Institute, Gothenburg, Sweden. 59Flanders Research
Institute for Agriculture, Fishery and Food, Oostende, Belgium. 60Research Institute for Nature and Forest, Brussels, Belgium. 61Department of
Animal Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, The Netherlands. ✉email: francesca.pilotto@umu.se; peter.
haase@senckenberg.de NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17171-y ARTICLE Bachisio M. Padedda
48, Denise Pallett49, Marco Pansera20, Miguel Ângelo Pardal42, Bruno Petriccione
50,
Tanja Pipan
51, Juha Pöyry
52, Stefanie M. Schäfer49, Marcus Schaub
53, Susanne C. Schneider54,
Agnija Skuja47, Karline Soetaert33, Gunta Spriņģe47, Radoslav Stanchev25, Jenni A. Stockan55, Stefan Stoll56,57,
Lisa Sundqvist58, Anne Thimonier
53, Gert Van Hoey
59, Gunther Van Ryckegem
60, Marcel E. Visser
61,
Samuel Vorhauser7 & Peter Haase
1,57✉ 1Senckenberg Research Institute and Natural History Museum Frankfurt, Gelnhausen, Germany. 2Environmental Archaeology Lab, Department of
Historical, Philosophical and Religious Studies, Umeå University, Umeå, Sweden. 3Department of Community Ecology, Helmholtz Centre for
Environmental Research - UFZ, Halle, Germany. 4Martin Luther University Halle-Wittenberg, Geobotany and Botanical Garden, Halle, Germany. 5German Centre for Integrative Biodiversity Research (iDiv) Halle - Jena - Leipzig, Leipzig, Germany. 6Department of Ecosystem Research, Leibniz
Institute of Freshwater Ecology and Inland Fisheries & Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Berlin, Germany. 7Biological Laboratory, Agency for Environment and Climate Protection, Bolzano, Italy. 8UMR 0980 BAGAP, INRAE – Institut Agro – ESA,
R
F
9LTSER Z
At li
A
i
35042 R
F
10UK C
t
f
E
l
& H d
l
B
h E t t
P
i
ik lier Armorique, 35042 Rennes, France. 10UK Centre for Ecology & Hydrology, Bush Estate, Penicuik, 11 NATURE COMMUNICATIONS | (2020) 11:3486 | https://doi.org/10.1038/s41467-020-17171-y | www.nature.com/naturecommunications
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Creche e família ou creche e famílias: o contexto dessa relação na contemporaneidade
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Abstract The postmodern world is marked by a multiplicity of identities, formed and continually transformed by the cultural
systems that surround them. Thus, this paper aims to discuss the relationship between nursery and families in the
contemporary context and highlight the need to understand families in their plurality. Drawing from Lahire (1995,
2002, 2004, 2006), this paper highlights the importance of understanding the families in the plural form,
recognizing their specificities and planning actions that exceed the vision of the modern nuclear family. Hall
(2014) and Lyotard (2013) conceptualize post-modernity and characterize the subject of contemporaneity. Nurseries do not operate exclusively with one type of family, but it is constantly relating with the plural meanings
of families. Keywords: Nursery. Childhood. Families. Contemporaneity. Resumo O mundo pós-moderno é marcado por uma multiplicidade de identidades, formadas e transformadas
continuamente pelos sistemas culturais que as rodeiam. A creche, assim, não atua exclusivamente com um tipo de
família, mas está constantemente relacionando-se com os sentidos plurais das famílias. Dessa forma, este artigo
objetiva discutir a relação entre creche e famílias no contexto da contemporaneidade e, para tanto, utiliza-se da
pesquisa bibliográfica. Autores como Lahire (1995, 2002, 2006), nos ajudam a pensar sobre essa relação e
reconhece as experiências socializadoras heterogêneas das famílias e a perspectiva de que é impossível agir como
se estivéssemos ligados e a um espaço cultural homogêneo. Hall (2014) e Lyotard (2013) conceituam a pós-
modernidade e caracterizam os sujeitos da contemporaneidade. As conclusões desse estudo apontam para a
importância de se entender as famílias no plural, reconhecendo suas especificidades e planejando ações que
ultrapassem a visão da família nuclear moderna. p
Palavras-chave: Creche. Infância. Famílias. Contemporaneidade. * Endereço eletrônico: leticiacasannova@gmail.com 41 41 Creche e família ou creche e famílias: o contexto dessa relação na contemporaneidade Letícia Veiga Casanova* Introdução não basta quando se busca a educação compartilhada,
que prevê a troca, a interação entre pais e professores,
o envolvimento entre instituição educacional e
familiar. E, para tanto, conhecer as famílias torna-se
fundamental. De segunda a sexta-feira, geralmente a partir
das 7 horas, os portões das creches já estão abertos
para receber pais, mães, irmãos, avós que trazem suas
crianças, deixando-as com os profissionais e
seguindo caminho ao trabalho ou aos seus afazeres
diários. É possível perceber que alguns demoram-se
mais nas despedidas feitas na porta da sala e fazem
questão de informar à professora sobre como sua
criança passou a noite ou contam alguma situação
acontecida em casa. Outros se limitam ao bom dia e
entregam seu filho e a mochila por cima do
portãozinho. Cada família possui suas especificidades,
seus modos próprios de relacionar-se com o mundo,
de entender e de estabelecer contato com as pessoas
que fazem parte de seu cotidiano. Como afirma
Szymanski (2010), o importante é conhecer o tipo de
vida que estão oferecendo ao filho/à filha e não
somente identificar o tipo de família. Conhecer o que as famílias são e como se
caracterizam
pode
nos
trazer
informações
importantes. Como afirma Lahire (1995, p. 72), “[...]
o fato de os diferentes membros das famílias
contextualizadas agirem como agem, de seus filhos
serem o que são e comportarem-se como tal nos
espaços escolares não é fruto de causas únicas que
agiriam poderosamente sobre eles”, as famílias estão
envolvidas em um conjunto de fatos que os tornam o
que são. A relação família e escola inicia-se na creche
quando os bebês deixam o cuidado exclusivo da mãe
e passam a vivenciar outras experiências com outras
pessoas, em um outro ambiente que não o lar. São
duas referências, família e professor, que se
relacionam diante de um ponto em comum: a criança. Assim, nas instituições de educação dos pequenos,
conhecer cada criança torna-se imprescindível para o
sucesso do processo. Porém, só conhecer as crianças q
Conjunto de fatos que se estabelecem nas Letícia Veiga Casanova 42 sim um ambiente que favoreça o desenvolvimento
integral de cada criança, um lugar para aprender. configurações das famílias e percorrem o grau de
escolarização, seu trabalho, a renda familiar, sua
relação com os outros membros da família e da
comunidade, suas vivências e suas experiências. Introdução Nesse sentido, é importante destacarmos que há uma
variedade de formas familiares nas sociedades
humanas e as relações que estabelecem com sua vida
cotidiana e com os significados que atribuem ao
mundo são diversos. Saraceno e Naldini (2003, p. 20)
complementam nossa ideia registrando que “[...] a
experiência familiar, que também parece ser a mais
comum no tempo e no espaço, distingue, portanto, de
maneira mais ou menos profunda, as várias culturas
e grupos, sendo também cada um deles atingido de
forma diferente pelas transformações sociais”. As crianças permanecem, em média, cerca
de dez horas nas instituições de Educação Infantil e
vivenciam
situações
diversas,
em
espaços
específicos e com uma rotina estabelecida. Nesse
ambiente, elas comem, brincam, socializam-se, são
cuidadas e educadas. Mas nem sempre foi assim. A educação das crianças pequenas,
segundo Kuhlmann Jr. e Fernandes (2004), foi, por
muito tempo, considerada responsabilidade única da
família e do grupo social ao qual pertenciam. Era
com os adultos e com as demais crianças que se
aprendia a tornar-se membro de um grupo, participar
das tradições consideradas importantes e apropriar-
se de conhecimentos que lhes seriam necessários
para a vida adulta. Por um bom período da história da
humanidade, nenhuma instituição incumbiu-se de
compartilhar a responsabilidade pela criança com
seus pais e com a comunidade da qual esta fazia
parte. Fala-se,
então,
de
pluralidade,
de
diversidade, de formas diferentes de constituir-se a
família. Fala-se de perceber e reconhecer a família
como categoria plural nas instituições de Educação
Infantil. Dessa forma, fortalece-se o objetivo deste
trabalho: discutir a relação entre creche e famílias no
contexto da pós-modernidade. Para tanto, utilizou-se
da pesquisa bibliográfica, uma modalidade de estudo
e análise de documentos de caráter científico
(OLIVEIRA, 2007), que remete, neste estudo, para
as contribuições de diferentes autores sobre a relação
escola e família na contemporaneidade. p
Na atualidade, essa relação é mantida de
maneira diversificada. O que antes era de extrema
responsabilidade
da
família
passou
a
ser
compartilhada com as instituições de Educação
Infantil. Como define Rosemberg (1995), os
movimentos de liberação da mulher, nos anos de
1960 e de 1970, diminuíram a distância entre papéis
masculinos e femininos, incentivaram à participação
das mulheres no mercado de trabalho e questionaram
o exercício da maternidade como o único destino
feminino. Introdução Portugal (1998) também ressalta que as
atuais condições de nossa sociedade fizeram com que
houvesse uma alteração de valores relativos à
atuação da mulher e da criança. Para a autora, a
emancipação da mulher é um fato que “[...] para além
de induzir profundas alterações socioeconômicas na
coletividade em geral, do ponto de vista psicológico,
expressa modificações profundas na própria maneira
de estar, de ser e de querer da mulher nas sociedades
actuais” (PORTUGAL, 1998, p. 123). O artigo organiza-se em dois momentos: o
primeiro trazendo a relação creche e famílias no
contexto da mudança, da mulher no campo de
trabalho e do esvaziamento da família nuclear. O
segundo momento reflete sobre a relação creche e
famílias no contexto da pós-modernidade, retratando
a singularidade, a diversidade de cada família que
precisa ser conhecida pela creche. Horizontes, v. 34, n. 2, p. 41-48, ago./dez. 2016 anos. Com essa decisão, estabelece-se, em termos
teóricos, uma prioridade que revoluciona a
perspectiva educacional no Brasil e traz questões que
precisam ser respondidas: Qual seria a especificidade
da Educação Infantil? Quais conceitos e concepções
estariam presentes nas ações e nas atividades das
instituições? Qual o papel do profissional dessa faixa
etária? Qual o papel das famílias nesse contexto? Quais interações se estabeleceriam entre creche e
famílias? Observamos esse sentido de família moderna
e nuclear distanciando-se da realidade de famílias
compostas por pai, mãe e irmãos ou apenas pela mãe,
ou somente pelo pai, pela mãe e os avós, pelas uniões
monoparentais, pelos filhos convivendo com pais
separados e os filhos do segundo casamento. Algumas mudanças, após os anos de 1960, na França,
são reconhecidas por Singly (2007) e marcam esse
novo contexto da família na sociedade: a diminuição
do número de casamentos e o aumento de uniões
livres; aumento dos divórcios, das separações; o
crescimento das famílias monoparentais, das famílias
compostas ou recompostas, no qual um dos cônjuges
é separado e casa-se novamente; diminuição do
número de nascimentos; aumento do número de
nascimentos fora do casamento; aumento do trabalho
assalariado das mulheres; cônjuges com atividade
profissional. Observa-se que não é mais possível
concebermos a família como uma instituição com
características comuns a todas, pois, como afirma
Peixoto, [...] o que observamos não foi exatamente o
enfraquecimento da instituição família, mas o
surgimento de novos
modelos familiares,
derivados desses fenômenos sociais e, sobretudo,
das transformações nas relações de gênero, que se
exprimem através do controle de natalidade, da
inserção intensiva da mulher no mercado de
trabalho, das mudanças ocorridas na esfera da
sexualidade, entre outros fatores [...] o cenário
torna-se, assim, cada vez mais complexo
(PEIXOTO, 2007, p. 9). Houve, assim, uma reorganização das
relações entre família e sociedade, e, por
consequência, das relações entre as famílias e as
instituições complementares como as creches, pré-
escolas e escolas. Saraceno e Naldini (2003, p.23)
destacam que “[...] a família possui igualmente uma
história
interna
própria,
que
transforma
continuamente quer as regras e os modos das relações
entre os sexos e as gerações, quer as relações e os
intercâmbios entre família e sociedade”. Para Lahire (2006, p. 57), “[...] é impossível
agir como se estivéssemos ligados a um espaço
cultural homogêneo”. Horizontes, v. 34, n. 2, p. 41-48, ago./dez. 2016 A relação creche e famílias: o contexto da
mudança A creche faz parte de um contexto histórico
que, por muito tempo, significou um lugar para as
mães deixarem seus filhos enquanto trabalhavam. Nesse
contexto,
as
ações
limitavam-se
ao
assistencialismo e à pura conduta de cuidado. Observa-se,
tanto
no
campo
acadêmico
(BONDIOLI; MANTOVANI, 1998; PORTUGAL,
1998; OLIVEIRA, 2002; CERISARA, 2002;
DAHLBERG; MOSS; PENCE, 2003; POST;
HOHMANN, 2003; GOLDSCHMIED; JACKSON,
2006; FORTUNATI, 2009; BARBOSA; RICHTER,
2009) quanto nas legislações (BRASIL, 1996, 2009)
uma premissa: a creche não deve ser um simples
lugar somente para as crianças serem deixadas, mas Na contemporaneidade, observa-se mais um
fenômeno de mudança. Os novos arranjos familiares
distanciam-se dos padrões familiares considerados
típicos da sociedade moderna: a família nuclear. Bruschini (1989) ilustra, por meio dos estudos de
Parsons e Bales (1955), as características dessa
família nuclear: Parsons propõe o estudo da família nuclear como
um pequeno grupo tarefa, no qual os membros
adultos
desempenham
papéis
altamente
diferenciados, assimétricos e complementares, o
que possibilita a presença de modelos masculinos Horizontes, v. 34, n. 2, p. 41-48, ago./dez. 2016 Creche e família ou creche e famílias: o contexto dessa relação na contemporaneidade 43 e femininos claramente definidos [...] o adulto
masculino ou marido-pai é o líder ‘instrumental’
do
grupo,
enquanto
o
adulto
feminino
desempenha
papéis
sociais
de
natureza
‘expressiva’, voltados principalmente para os
assuntos internos da família (BRUSCHINI, 1989,
p. 2). anos. Os irmãos são os responsáveis por
levar e buscar o Pedro da creche, mas, pelo menos
uma vez por semana, a mãe gosta de ir para
conversar com as professoras. [...]. Enquanto a
entrevista acontecia na cozinha, Pedro estava
completamente à vontade brincando no quintal, e
a mãe pedia que a irmã cuidasse dele, pois ele já
estava mexendo na água e no cachorro. Para a
mãe: “Não tenho nem palavras pra dizer que a
creche me ajuda bastante, se não fosse a creche
eu não sei, porque trabalhar pra pagar alguém
pra cuidar deles não compensa. Imagina
trabalhar de doméstica, ganhar um salário e
pagar pra cuidar das outras crianças enquanto
eu saio de dentro de casa” (CASANOVA, 2011,
p. 42-45). funcionária pública e trabalha na Secretaria de
Obras como secretária de um departamento. A
renda mensal gira em torno de 9 salários mínimos
[...] A mãe, que participou da entrevista, é
bastante comunicativa e registra suas ideias com
clareza. Fez questão de mostrar as lembrancinhas
que a Laura fez e que estão penduradas em um
quadro na parede da cozinha. A casa possui uma
cozinha grande, uma sala e dois quartos. Observamos muitos brinquedos por todos os
cômodos da casa. Para ela, para trabalhar com
bebês “[...] o principal é gostar de criança,
porque quando gosta do que tu fazes, tu não vai
medir esforços pra... [pensando]... tu vai estudar,
tu vai se especializar. É igual a mim, o meu sonho
e do meu marido era ter a Laura, então como era
uma coisa que a gente queria, uma coisa que a
gente gostava, foi aí que a gente ia atrás, que a
gente leu, pesquisou, se aprofundou no assunto”
(Mãe de Laura) (CASANOVA, 2011, p. 39-55). A família da Ana, de 1 ano e 5 meses, é composta
pela mãe e a avó, que moram na mesma casa. A
mãe, de 21 anos, foi quem participou da
entrevista e respondeu sempre de forma convicta
às perguntas. Ela possui o ensino médio
completo, trabalha como auxiliar de escritório,
recebe mensalmente dois salários mínimos e
meio, incluindo a pensão alimentícia. A mãe
conta que nunca foi casada com o pai da Ana e
que essa situação a incomodava no início, pois o
pai já estava em um outro relacionamento. Contudo, repensou algumas coisas e percebeu
que a filha precisava do pai. anos. Percebe-se que as identidades
unificadas, que representavam um estilo, uma classe
e que por tanto tempo estabilizaram a sociedade,
estão em declínio, fazendo surgir novas identidades. Esse movimento pode ser percebido por
intermédio dos perfis de quatro famílias que moram
no mesmo bairro, utilizam-se da mesma creche e
dispõem de características diversificadas. Essas
famílias participaram da pesquisa intitulada O que as
crianças pequenas fazem na creche? As famílias
respondem (CASANOVA, 2011)1. Seus perfis
ilustram a pluralidade e a diversidade de cada
família. Assim, as instituições, bem como o processo
de educação das crianças pequenas, também foram se
modificando. O que antes era de extrema
responsabilidade
da
família
passou
a
ser
compartilhada com as instituições de Educação
Infantil. São duas referências, família e professor que
se relacionam diante de um ponto em comum: a
criança. É nessa interação que surge um mundo de
novidades, de descobertas, de sensações, de
sentimentos da família em relação à instituição e vice
e versa. A família da Laura, de 1 ano, é composta pela
mãe de 22 anos, o pai com 34 anos, os quais
moram junto com o avô. Um pouco antes da
entrevista permanecemos na cozinha enquanto a
mãe terminava o banho da Laura e o avô
conversava com ela na banheira. O banho é uma
coisa que a mãe não faz questão que as
professoras deem na creche porque ela sempre
quer fazê-lo quando chega à casa. O pai trabalha
como segurança, não estava em casa no momento
da entrevista e vai à creche raramente. A mãe é Toda essa condição também tomou forma
legal a partir da nova Constituição Brasileira (1988)
e em decorrência da Lei de Diretrizes e Bases da
Educação Nacional (Lei nº9394/96), a qual prevê que
a educação oficial pública, do Estado, conta com uma
nova modalidade: Educação Infantil – dever do
Estado e direito subjetivo da criança desde 0 até 5 Horizontes, v. 34, n. 2, p. 41-48, ago./dez. 2016 44 Letícia Veiga Casanova dispõe de um quarto, cozinha e uma sala. Moram
a mãe, o Pedro, um irmão de 8 anos e uma irmã
com 9 anos. O pai está preso e a família sobrevive
com um salário mínimo do trabalho de doméstica
da mãe, de 27 anos e que estudou até o ensino
fundamental. anos. Hoje, Ana passa os
finais de semana com a família do pai e todos
conversam frequentemente. [...]. Na creche, ela
conta que a filha já fez amizades e relata que
começou a observar, depois de uma conversa com
a mãe da Laura, que a filha quando chega vai
direto ao berço em que a Laura está. É a avó que
leva Ana à creche pela manhã e ela vai buscá-la
no final da tarde. Quando perguntada sobre o que
ela achava que a filha ficava fazendo o dia inteiro
na creche, ela respondeu: “Eu acredito que ela
fique fazendo bastante atividade educativa,
porque a Ana de uns dias pra cá eu reparei que
tu pergunta pra ela assim: ‘Ana cadê teu olho?’
Que antes ela só sabia onde era a barriga. E
agora tu pergunta: ‘onde é o olho?’ Ela já te
mostra. ‘Onde que é o nariz?’ Ela já bota a mão. ‘A boca?’. Eu acredito que seja mais ou menos
isso” (Mãe da Ana) (CASANOVA, 2011, p. 38-
66). A família do José e da Maria, 10 meses e 2 anos
e 7 meses, respectivamente, dispõe de dois
salários mínimos para as despesas mensais da
mãe, de 24 anos, da irmã, de 5 anos - que também
frequenta o centro de educação infantil no
período matutino -, e do José e da Maria. O pai
faleceu há mais ou menos sete meses. Desde
então, a mãe, que só estudou até a quarta série do
ensino fundamental, começou a trabalhar com a
confecção de bolsas em casa para aumentar a
renda e precisou colocar as crianças na creche. A
entrevista foi marcada no final de semana pela
manhã e, quando a pesquisadora chegou à casa da
família, a mãe relatou que tinha acabado de
acordar, pois, como o José estava doente, ela
tinha ido dormir muito tarde. O José e a irmã mais
velha ainda dormiam no único quarto e a
entrevista aconteceu na cozinha. A casa dispõe
desses dois cômodos e fica aos fundos da
residência de parentes. A mãe contou que “[...]
dava mamadeira pra ele antes de ir e a
professora falou que não precisa dar porque
chega lá ganha mamadeira todo mundo, aí ele
come. Mas como ele chora de manhã, eu dou sem
elas saberem mesmo” (Mãe de José e Maria)
(CASANOVA, 2011, p. 41-74). anos. Podemos dizer que as instituições de
Educação Infantil interagem apenas com um tipo de
família? Que os momentos de interação são
percebidos da mesma forma por cada família? Que as
relações devem se estabelecer do mesmo jeito com
todas as famílias? Assim, como no caso da família do Pedro, de 2
anos e 3 meses, que mora nos fundos do terreno
da casa de parentes. Uma residência pequena que Esse é um movimento de transformações, de
perceber que, em uma sala de crianças pequenas, Horizontes, v. 34, n. 2, p. 41-48, ago./dez. 2016 Creche e família ou creche e famílias: o contexto dessa relação na contemporaneidade 45 assim como há uma criança diferente da outra, há
também uma família diferente da outra. Para autores
como Hall (2014) e Lyotard (2013), esse movimento
denomina-se pós-modernidade, no qual há uma
ruptura com os pensamentos da modernidade. prática pedagógica. Contudo, só conhecer as crianças
não basta. Na educação compartilhada, a interação
entre pais e professores, o envolvimento entre
instituição educacional e familiar é fundamental. Assim, conhecer as famílias torna-se essencial, pois
cada uma possui suas especificidades, com modos
próprios de relacionar-se com o mundo, de entender
e estabelecer contato com as pessoas que fazem parte
do seu cotidiano. p
p
Assim, para Hall (2014, p. 10, grifo do
autor), o sujeito da modernidade era aquele sujeito
totalmente “[...] centrado, unificado, dotado de
capacidades de razão, de consciência de ação, cujo
‘centro’ consistia num núcleo interior”. Já o sujeito
pós-moderno não tem uma identidade fixa, essencial
ou permanente, “[...] a identidade torna-se uma
‘celebração
móvel’:
formada
e
transformada
continuamente em relação às formas pelas quais
somos representados ou interpelados nos sistemas
culturais que nos rodeiam” (HALL, 2014, p. 10, grifo
do autor), Pensar a relação entre creche e famílias é
considerar o nível de compartilhamento existente
entre essas duas instituições responsáveis pela
educação dessas crianças. Para Fortunati: A ideia de que os serviços educacionais para a
primeira infância tenham como interlocutores
não só as crianças, mas também os pais, procede
de uma ideia sobre o desenvolvimento com
empresa conjunta entre as crianças e as pessoas
que as atendem e põe em evidencia a importância
de que existe uma troca contínua e recíproca entre
os diferentes contextos em que as crianças
crescem (FORTUNATI, 2009, p. 52). anos. Dessa forma, é necessário compreendermos
a relação creche e famílias nesse contexto pós-
moderno, de mudanças, de heterogeneidades, de
pluralismo cultural, de verdades provisórias e de
complexidades. Descarta-se, assim, a ideia de que a
creche atua exclusivamente com um tipo de família e
fortalece-se o conceito de que a creche está
constantemente relacionando-se com as famílias no
plural. Portugal
(1998)
alerta-nos
para
a
necessidade de observação do impacto da creche para
e nos pais, pois considera que a creche faz parte de
um sistema mais global de apoio às famílias. Por isso,
conhecer o que as famílias são e como elas se
caracterizam
pode
nos
trazer
informações
importantes. Como afirma Lahire (1995, p. 72), “[...]
o fato de os diferentes membros das famílias
contextualizadas agirem como agem, de seus filhos
serem o que são e comportarem-se como tal nos
espaços escolares não é fruto de causas únicas que
agiriam poderosamente sobre eles” - as famílias estão
envolvidas em um conjunto de fatos que os tornam o
que são. Horizontes, v. 34, n. 2, p. 41-48, ago./dez. 2016 Como afirma Hall: Ao refletir sobre a última questão, observa-
se que os profissionais da educação juntam, em uma
mesma categoria, realidades consideradas diferentes. É comum o discurso de que a família não participa e
não se interessa pela vida escolar de seus filhos. Segundo Lopes e Guimarães (2008), para os
profissionais da Educação Infantil, constrói-se uma
imagem de uma família que vê a instituição como um
“depósito de crianças”. Contudo, será que todas as
famílias veem da mesma forma e agem do mesmo
jeito? Será que conseguiríamos identificar formas
diferenciadas de participação? A identidade plenamente unificada, completa,
segura e coerente é uma fantasia. Ao invés disso,
à medida que os sistemas de significação e
representação cultural se multiplicam, somos
confrontados
por
uma
multiplicidade
desconcertante e cambiante de identidades
possíveis [...] (HALL, 2014, p. 12). A identidade plenamente unificada, completa,
segura e coerente é uma fantasia. Ao invés disso,
à medida que os sistemas de significação e
representação cultural se multiplicam, somos
confrontados
por
uma
multiplicidade
desconcertante e cambiante de identidades
possíveis [...] (HALL, 2014, p. 12). As famílias são vistas menos como
instituição e mais como espaço de socialização, no
qual “[...] o indivíduo não se define mais pelos laços
familiares (filho de sicrano ou mulher de fulano)”
(SINGLY, 2007, p. 16), marca da modernidade. Na
pós-modernidade, como exemplifica Singly (2007),
os indivíduos da família têm pertencimentos
múltiplos. O autor reforça que, na modernidade, o elo
social seria composto por fios mais sólidos como o
pertencimento à determinada nação, classe social,
raça
e
gênero. Em
contrapartida,
na
contemporaneidade, os elos seriam menos sólidos,
mas teriam muitos mais fios do que tinham antes. Como afirma Lahire (1995, p. 12, grifo do
autor), “[...] quando os professores falam de uma
forma muito genérica, as ‘grandes causas sociais’
tornam-se predominantes” - como as afirmações que
os pais não conversam mais com os filhos ou que eles
não têm mais tempo ou vontade. Dessa forma, a
homogeneidade do ambiente familiar é fortalecida no
ambiente escolar, assim como as ações generalistas
de interação com essas famílias. Observam-se, na prática educativa da creche,
ações universalistas de relação com as famílias que
definem uma mesma ação para todas as famílias. No
entanto, se é percebido que a creche se relaciona com
famílias na sua pluralidade, como é possível
assegurar um discurso totalizante, generalista e que
pretende dar respostas e soluções a todo contingente,
que é múltiplo e complexo? A relação creche e famílias: o contexto da pós-
modernidade Partimos do pressuposto de que as famílias
possuem diversas fontes de influência, no sentido de
tornarem-se pais e mães, de educarem seus filhos, de
aprenderem a lidar com as crianças. Os demais
membros das famílias como avós, tios, primos, bem
como o seu ambiente de trabalho, suas leituras, suas
conversas com vizinhos acabam influenciando as
atitudes dessa mãe e desse pai. A creche, o espaço
educativo institucionalizado, também é uma fonte de
influência para as famílias e deveria ser um espaço
em que todos os envolvidos, crianças, profissionais e
famílias, estivessem em constante interação. Para
tanto, é necessário que se fortaleçam mecanismos
conscientes e consistentes de interação, de forma que
a própria instituição mostre o que produz, fale sobre
o que faz, evidencie o desenvolvimento de cada
criança, conduza os pais e as crianças em um
processo de aprender. Dessa maneira, a creche
superará o conceito de ser um simples lugar para
deixar as crianças enquanto a família trabalha. Conjunto de fatos que se estabelecem nas
configurações das famílias e percorrem o grau de
escolarização, seu trabalho, a renda familiar, sua
relação com os outros membros da família e da
comunidade, suas vivências e experiências. Dessa
forma, podemos afirmar que a creche não se
relaciona com um único tipo de família: a família
relapsa ou a família interessada, a família pobre ou a
família rica, a família boa ou a família ruim. A creche
se relaciona com uma multiplicidade de famílias,
cada uma com suas configurações, suas vivências,
seus conhecimentos, suas experiências culturais. Essa relação com a multiplicidade, com a
família formada por indivíduos que estão imersos em
diferentes e inúmeras representações culturais, é
caracterizada pelo contexto da pós-modernidade. Nas instituições de educação dos pequenos,
conhecer cada criança torna-se imprescindível para o
sucesso do processo de desenvolvimento e para a Horizontes, v. 34, n. 2, p. 41-48, ago./dez. 2016 Letícia Veiga Casanova 46 Como afirma Hall: Para tanto, seria
necessário que a creche reconhecesse o contexto das
famílias na sua pluralidade e garantisse ações que
valorizassem
as
disposições
e
as
variações
individuais de cada família (LAHIRE, 2004). Dessa forma, as famílias não podem mais ser
caracterizadas, por exemplo, apenas pela sua
condição socioeconômica, de famílias pobres ou
ricas. Para Lahire (2006, p. 403), os elos da
contemporaneidade
são
caracterizados
pelas
vivências em condições materiais e culturais de
existências diferentes, pelas “[...] mobilidades
sociais, escolares ou profissionais, pequenas ou
grandes, que imprimem suas marcas nos perfis
culturais”. Percebe-se que a família não desaparece na
pós-modernidade, mas ela muda de sentido. Ela tem
como característica a abertura de um maior espaço
para o indivíduo e suas interações sociais. Assim, é
necessário interpretarmos as famílias como um
conjunto de atores. Para Lahire (2002, p. 198), “[...]
os atores são o que as suas múltiplas experiências
sociais
fazem
deles. São
chamados
a
ter
comportamentos, atitudes variadas segundo os
contextos em que são levados a evoluir”. Essa prática de entender as famílias de forma
generalizada transforma a relação dessas famílias
com a creche em uma relação superficial,
configurada pelas conversas rápidas e pouco
significativas. Fortunati (2009, p. 53, grifo do autor)
ressalta que essas “[...] formas de encontro entre
família e o serviço se apresenta mais como ‘rituais’
vazios que como o curso de um processo de relação
dinâmico”. A consciência de qualquer ser social só se
forma e adquire existência por meio das múltiplas
relações que ele estabelece, no mundo, com o outro
(LAHIRE, 1995). A creche, como contexto social
dessas famílias, também é fonte de construção dos
esquemas de ação, no qual as famílias aprendem
modos de agir, de apreciar, de orientar e de
relacionar-se. Diante das relações estabelecidas entre
creche e família, do que os pais estão se apropriando? O que as famílias estão aprendendo? Como a creche
vê as famílias? Para uma relação dinâmica, a creche
precisa reconhecer a pluralidade das famílias e não
condicionar as interações com os pais a formatos
universais. É necessário compreender que, no
contexto da pós-modernidade, não cabe mais
entender as famílias e planejar ações para elas como
se elas ainda representassem a família nuclear da
modernidade. Considerações Considerações Horizontes, v. 34, n. 2, p. 41-48, ago./dez. 2016 47 Creche e família ou creche e famílias: o contexto dessa relação na contemporaneidade CONGRESSO DE LEITURA DO BRASIL, COLE,
17., 2009, Campinas. Anais... Campinas, SP:
UNICAMP, 2009. No ambiente da creche, as relações entre as
famílias e a instituição dão-se praticamente em dois
momentos bem específicos: na entrada e na saída das
crianças; e; esporadicamente; nas reuniões de pais. As mães, os pais, os irmãos mais velhos, os avôs e as
avós interagem de forma diversificada, de acordo
com a disponibilidade de tempo de cada um. BONDIOLI,
Anna;
MANTOVANI,
Susanna. Manual de educação infantil: de 0 a 3 anos. Porto
Alegre: Artmed, 1998. Os momentos de interação entre creche e
famílias são marcados pela complexidade do contexto
pós-moderno. As famílias são configuradas em sua
multiplicidade,
por
indivíduos
que
vivenciam
experiências socializadoras diversas. Porém, para a
creche, a visão da família moderna e nuclear continua
marcada nos discursos generalistas de que a família
não participa, não tem conhecimento e nem interesse. BRASIL. Resolução CEB 5/2009, de 17 de
dezembro de 2009. Fixa as Diretrizes Curriculares
Nacionais para a Educação Infantil. Diário Oficial
[da] República Federativa do Brasil, Brasília, DF,
18 dez. 2009. Seção 1, p. 18. ______. Lei nº 9.394, de 20 de dezembro de 1996. Estabelece as Diretrizes e Bases da Educação
Nacional. Diário Oficial [da] República Federativa
do Brasil, Brasília, DF, v. 134, n. 248, 23 dez. 1996. Seção 1, p. 27834-27841. Encontra-se, aqui, a dualidade na relação
creche e famílias. Por um lado, as famílias são
caracterizadas no contexto da pós-modernidade como
não tendo uma identidade fixa, transformando-se
continuamente à medida que interagem com os
sistemas culturais que as rodeiam. Por outro lado, a
creche entende as famílias de forma generalista, como
uma instituição em que todos os indivíduos agem da
mesma maneira. Pode-se dizer que a creche planeja
ações de interação com um tipo de família e descarta-
se a ideia de que a creche está constantemente
relacionando-se com as famílias no plural. BRUSCHINI, Cristina. Uma abordagem sociológica
de família. Revista Brasileira de Estudos de
População, São Paulo, v. 6, n. 1, p. 1-23, jan./jun. 1989. CASANOVA, Letícia. O que as crianças pequenas
fazem na creche? As famílias respondem. 2011. 114
f. Dissertação
(Mestrado
em
Educação)
-
Universidade do Vale do Itajaí, Itajaí, 2011. Considerações Dessa
forma,
observa-se,
na
prática
educativa da creche, ações generalistas de relação
com as famílias que definem uma ação única para
todas elas. Porém, no contexto da pós-modernidade,
no qual a instituição de Educação Infantil relaciona-
se com famílias na sua pluralidade, torna-se inviável
assegurar um discurso totalizante e que pretende dar
respostas e soluções a todo contingente de famílias,
pois estas são traduzidas como múltiplas e
complexas. CERISARA, Ana Beatriz. Referencial Curricular
nacional para educação infantil no contexto das
reformas. Educação & Sociedade, Campinas, v. 23,
n. 80, p. 329-348, 2002. DAHLBERG, Gunilla; MOSS, Peter; PENCE, Alan. Qualidade na educação da primeira infância:
perspectivas pós-modernas. Porto Alegre: Artmed,
2003. Nota FORTUNATI, Aldo. A educação infantil como
projeto da comunidade. Crianças, educadores e pais
nos novos serviços para a infância e a família. Porto
Alegre: Artmed, 2009. 1 Os exemplos citados fazem parte da pesquisa de
Mestrado em Educação desenvolvida pela autora e
foi submetida ao processo de autorização solicitado
pelo Conselho de Ética para divulgação das
entrevistas. GOLDSCHMIED,
Elinor;
JACKSON,
Sonia. Educação de 0 a 3 anos: o atendimento em creche. 2. ed. Porto Alegre: Artmed, 2006. ______. Sucesso escolar nos meios populares: as
razões do improvável. São Paulo: Ática, 1995. ______. Sucesso escolar nos meios populares: as
razões do improvável. São Paulo: Ática, 1995. LYOTARD,
Jean-François. A
condição
pós-
moderna. Tradução Ricardo Corrêa Barbosa. Rio de
Janeiro: José Olympio, 2013. SARACENO,
Chiara;
NALDINI,
Manuela. Sociologia da família. Lisboa: Estampa, 2003. OLIVEIRA, Maria Marly. Como fazer pesquisa
qualitativa. Petrópolis, Vozes, 2007. SINGLY, François de. Sociologia da Família
Contemporânea. Rio de Janeiro: FGV, 2007. OLIVEIRA, Zilma Ramos de. Educação Infantil:
fundamentos e métodos. São Paulo: Cortez, 2002. SZYMANSKI, Heloisa. A relação família/escola:
desafios e perspectivas. Brasília: Liber Livro, 2010. PARSONS, Talcott; BALES, Robert Freed. Family,
socialization and interaction process. Glencoc: Free Press, 1995. Press, 1995. PORTUGAL, Gabriela. Crianças, famílias e creche
– uma abordagem ecológica da adaptação do bebé à
creche. Porto: Porto, 1998. POST, Jacalyn; HOHMANN, Mary. Educação de
bebés em infantários – cuidados e primeiras
aprendizagens. Lisboa:
Fundação
Calouste
Gulbenkian, 2003. LAHIRE, Bernard. A cultura dos indivíduos. Porto
Alegre: Artmed, 2006. LAHIRE, Bernard. A cultura dos indivíduos. Porto
Alegre: Artmed, 2006. ______. Homem plural: os determinantes da ação. Petrópolis: Vozes, 2002. Letícia Veiga Casanova: Professora da educação infantil no município de Itajaí (SC) e Mestre em Educação pela
Universidade do Vale do Itajaí – UNIVALI. Membro do Grupo de Pesquisa Contextos da Educação da Criança,
como doutoranda em Educação pela mesma instituição. ______. Homem plural: os determinantes da ação.
Petrópolis: Vozes, 2002. ROSEMBERG, Fúlvia. A criação de filhos
pequenos:
tendências
e
ambiguidades
contemporâneas. In: RIBEIRO, Ivete; RIBEIRO,
Ana
Clara. (Orgs.). Família
em
processos
contemporâneos: inovações culturais na sociedade
brasileira. São Paulo, Loyola, 1995, p.167-190. Horizontes, v. 34, n. 2, p. 41-48, ago./dez. 2016 Referências HALL, Stuart. A identidade cultural na pós-
modernidade. Tradução Tomaz Tadeu da Silva &
Guacira Lopes Louro. Rio de Janeiro: Lamparina,
2014. BARBOSA, Maria Carmen Silveira; RICHTER,
Sandra Regina Simonis. Os bebês interrogam o
currículo: as múltiplas linguagens na creche. In: BARBOSA, Maria Carmen Silveira; RICHTER,
Sandra Regina Simonis. Os bebês interrogam o
currículo: as múltiplas linguagens na creche. In: Horizontes, v. 34, n. 2, p. 41-48, ago./dez. 2016 Letícia Veiga Casanova 48 KUHLMANN
JR.,
Moysés;
FERNANDES,
Rogério. Sentidos da infância. In: FARIA FILHO,
Luciano Mendes de. (Org.). A Infância e sua
Educação. Belo Horizonte: Autêntica, 2004, p. 22-
37. Sobre a autora Letícia Veiga Casanova: Professora da educação infantil no município de Itajaí (SC) e Mestre em Educação pela
Universidade do Vale do Itajaí – UNIVALI. Membro do Grupo de Pesquisa Contextos da Educação da Criança,
como doutoranda em Educação pela mesma instituição. Recebido em janeiro de 2016. Recebido em janeiro de 2016. Aprovado em março de 2016. Aprovado em março de 2016. Horizontes, v. 34, n. 2, p. 41-48, ago./dez. 2016
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Prediction of Outcome in Acute Lower Gastrointestinal Bleeding Using Gradient Boosting
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PloS one
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Prediction of Outcome in Acute Lower
Gastrointestinal Bleeding Using Gradient
Boosting Lakshmana Ayaru1‡¶*, Petros-Pavlos Ypsilantis2‡, Abigail Nanapragasam1,
Ryan Chang-Ho Choi1, Anish Thillanathan1, Lee Min-Ho1, Giovanni Montana2¶* 1 Department of Gastroenterology, Charing Cross and Hammersmith Hospitals, Imperial College Healthcare
NHS Trust, London, United Kingdom, 2 Department of Biomedical Engineering, Kings College London,
London, United Kingdom ‡ These two authors are joint first authors. ¶ These two authors are joint senior authors. * Lakshmana.ayaru@imperial.nhs.uk (LA); giovanni.montana@kcl.ac.uk (GM) a1111 RESEARCH ARTICLE Methods Non-endoscopic variables from patients with ALGIB attending the emergency departments
of two teaching hospitals were analysed retrospectively for training/internal validation
(n=170) and external validation (n=130) of the GB model. The performance of the GB algo-
rithm in predicting recurrent bleeding, clinical intervention and severe bleeding was com-
pared to a multiple logic regression (MLR) model and two published MLR-based prediction
algorithms (BLEED and Strate prediction rule). Copyright: © 2015 Ayaru 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. OPEN ACCESS OPEN ACCESS
Citation: Ayaru L, Ypsilantis P-P, Nanapragasam A,
Choi RC-H, Thillanathan A, Min-Ho L, et al. (2015)
Prediction of Outcome in Acute Lower
Gastrointestinal Bleeding Using Gradient Boosting. PLoS ONE 10(7): e0132485. doi:10.1371/journal. pone.0132485
Editor: John Green, University Hospital Llandough,
UNITED KINGDOM
Received: March 24, 2015
Accepted: June 15, 2015
Published: July 14, 2015
Copyright: © 2015 Ayaru 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 paper. Funding: The authors received no specific funding
for this work. Competing Interests: The authors have declared
that no competing interests exist. OPEN ACCESS
Citation: Ayaru L, Ypsilantis P-P, Nanapragasam A,
Choi RC-H, Thillanathan A, Min-Ho L, et al. (2015)
Prediction of Outcome in Acute Lower
Gastrointestinal Bleeding Using Gradient Boosting. PLoS ONE 10(7): e0132485. doi:10.1371/journal. pone.0132485
Editor: John Green, University Hospital Llandough,
UNITED KINGDOM
Received: March 24, 2015
Accepted: June 15, 2015
Published: July 14, 2015
Copyright: © 2015 Ayaru 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 paper. Funding: The authors received no specific funding
for this work. Competing Interests: The authors have declared
that no competing interests exist. Citation: Ayaru L, Ypsilantis P-P, Nanapragasam A,
Choi RC-H, Thillanathan A, Min-Ho L, et al. (2015)
Prediction of Outcome in Acute Lower
Gastrointestinal Bleeding Using Gradient Boosting. PLoS ONE 10(7): e0132485. doi:10.1371/journal. pone.0132485 Citation: Ayaru L, Ypsilantis P-P, Nanapragasam A,
Choi RC-H, Thillanathan A, Min-Ho L, et al. (2015)
Prediction of Outcome in Acute Lower
Gastrointestinal Bleeding Using Gradient Boosting. PLoS ONE 10(7): e0132485. doi:10.1371/journal. pone.0132485 Background There are no widely used models in clinical care to predict outcome in acute lower gastro-
intestinal bleeding (ALGIB). If available these could help triage patients at presentation to
appropriate levels of care/intervention and improve medical resource utilisation. We aimed
to apply a state-of-the-art machine learning classifier, gradient boosting (GB), to predict out-
come in ALGIB using non-endoscopic measurements as predictors. Results The GB algorithm had the best negative predictive values for the chosen outcomes (>88%). On internal validation the accuracy of the GB algorithm for predicting recurrent bleeding,
therapeutic intervention and severe bleeding were (88%, 88% and 78% respectively) and
superior to the BLEED classification (64%, 68% and 63%), Strate prediction rule (78%,
78%, 67%) and conventional MLR (74%, 74% 62%). On external validation the accuracy
was similar to conventional MLR for recurrent bleeding (88% vs. 83%) and therapeutic inter-
vention (91% vs. 87%) but superior for severe bleeding (83% vs. 71%). Introduction Acute lower gastro-intestinal bleeding (ALGIB) is a common emergency increasing in inci-
dence with age [1], and may be more common than acute upper gastrointestinal bleeding in
the elderly [2]. The causes and severity are heterogeneous, e.g. large volume bleeding from
diverticulosis or minor bleeding from colitis with the aetiology and outcome often obscure to
the clinician at presentation. The majority of ALGIB presentations (80–85%) resolve spontaneously with no adverse out-
come and death is uncommon (2–4%) [3,4]. However a significant proportion (17–30%)
undergo therapeutic intervention (angiographic embolisation, colonoscopic-based therapies or
surgery) to control severe/recurrent bleeding [5,6]. Despite intervention re-bleeding occurs at
rate of approximately 20% in the first year [7]. Due to concern about severe/recurrent bleeding or need for intervention routine clinical
practice for the vast majority of patients with ALGIB who present to emergency department is
admission to hospital for in-patient observation for variable number of days with a proportion
undergoing endoscopy or radiological investigation. This strategy has the disadvantage of
being invasive, expensive [8] and exposes patients to hospital acquired complications. If a reli-
able predictive model was available at the point of presentation to hospital low risk patients
could be identified and triaged to outpatient management/a shorter inpatient stay. Resources
could be then freed up for high-risk patients to be appropriately transferred to higher levels of
care and undergo more aggressive investigation. Although multiple logistic regression (MLR)
based scores have been developed for ALGIB [9,10] none have been recommended for routine
clinical practice unlike the Glasgow Blatchford and Rockall scores for acute upper gastrointesti-
nal bleeding [11]. Possible reasons include the lack of validation in diverse settings and modest
accuracies in comparison with these scores. MLR based models may be limited in predicting
outcome in ALGIB as they are based on the assumption that a linear combination of the
observed features can be used to determine the probability of each particular outcome ignoring
any variable interaction which may be the key for the accurate prediction. In order to mitigate this potential limitation of MLR based scores and to accurately predict
ALGIB outcome, we chose to implement and assess the performance of a non-parametric algo-
rithm for classification, Gradient Boosting (GB) [12]. GB is a supervised machine learning
algorithm, and is able to approximate the unknown functional mapping between the inputs,
i.e. the non-endoscopic measurements, and the outputs, i.e. the ALGIB outcomes. Conclusion The gradient boosting algorithm accurately predicts outcome in patients with acute lower
gastrointestinal bleeding and outperforms multiple logistic regression based models. These
may be useful for risk stratification of patients on presentation to the emergency
department. Data Availability Statement: All relevant data are
within the paper. Competing Interests: The authors have declared
that no competing interests exist. 1 / 14 PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 Gradient Boosting for Acute Lower Gastrointestinal Bleeding PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 Study design The database had been generated retrospectively by identifying consecutive patients from elec-
tronic records who were aged 18 or over and presented to the emergency department with a
primary diagnosis of ALGIB defined as PR bleeding (bright red or maroon coloured blood
passed per rectum) within the previous three days. For this study we excluded patients with: 1)
presentation most indicative/final diagnosis of an upper GI bleed (haematemesis, melaena,
upper GI bleeding source detected at endoscopy or angiography), 2) inpatient ALGIB bleed, 3)
ALGIB as a secondary admission symptom, 4) patients transferred from other hospitals and 5)
incomplete patient data. Patients with haemodynamic instability and PR bleeding were
required to have an upper GI endoscopy before inclusion into the study unless a definitive
colonic source was identified. Patients admitted to Charing Cross Hospital with ALGIB were analysed for training and
internal validation of the GB algorithm. The external validation cohort consisted of patients
admitted to Hammersmith Hospital over the same time period. Charing Cross and Hammer-
smith hospitals are large, busy general teaching hospitals with separate emergency departments
and surgical teams. Ethics statement This study was performed by analysing an existing anonomysed database of patients presenting
with ALGIB collated for the purpose of audit to the emergency departments of Charing Cross
and Hammersmith hospitals, London, UK (1st January 2007 to 31st December 2011). The
study was approved by the Joint Research Compliance office at Imperial College Healthcare
NHS Trust (ref 125HH25060). The office confirmed that no formal ethical review or informed
consent was required as the study involved existing anonomysed routinely collected data, no
new data was being collected and there was no clinical intervention. Gradient Boosting for Acute Lower Gastrointestinal Bleeding In particular, the GB algorithm relies on decision trees as constituent or “base” predictive
algorithms. Decision tree are statistical models that recursively partition the input space in
order to find rules that are predictive of the output. The classical CART (Classification and
Regression Tree) algorithm was popularized in the 1980s by Breiman et al. [17]. Compared to
other machine learning methods, GB possesses several strengths: 1) it is less prone to over-
fitting [13] 2) it is robust to noise [18] 3) it has an internal mechanism to estimate error rates,
4) it provides indices of variable importance and 5) it can be used when the predictors are both
continuous and categorical. The aim of this study was to test whether the GB algorithm was able to accurately predict
clinical outcomes in patients presenting to emergency departments with ALGIB using non-
endoscopic variables available to clinicians at that time. We also set out to compare the perfor-
mance of the GB approach with conventional MLR and two previously published multivariate
logistic regression models [9,10]. We show in this study that the gradient boosting algorithm
accurately predicts rebleeding, severe bleeding and clinical intervention in patients with acute
lower gastrointestinal bleeding and outperforms multiple logistic regression based models. Introduction Supervised
learning algorithms are commonly trained on historical data consisting of examples of input-
output pairs. The GB algorithm embraces the notion of “ensemble learning”, whereby multiple simple
learning algorithms are used jointly in order to obtain better predictive performance than
could be achieved from any of the constituent learning algorithms [13]. Of clinical relevance
are several reports demonstrating that ensemble learning classification models are accurate in
predicting outcome in a variety of clinical settings [14–16]. 2 / 14 PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 Gradient boosting We deployed the GB algorithm, as originally proposed by Friedman [13], which has been suc-
cessfully applied in a number of clinical applications [19–23]. GB is a non-parametric algo-
rithm for supervised machine learning. It approximates the unknown functional mapping
from input explanatory variables to corresponding output variables. The non-parametric
nature of the GB algorithm enables the estimation of a functional mapping from non-
endoscopic measurements to ALGIB outcomes without the need to decide a priori the
parametric form of this function. By contrast, parametric models like logistic regression assume
that the log odds depend linearly on the covariates, and this linearity may be insufficient to cap-
ture the complexity of relationship between inputs and outputs [24]. For the training of the GB algorithm we consider m patients with non-endoscopic input
measurements and their corresponding ALGIB outcome in the form of (x1,y1),. . ., (xm,ym)
pairs, where each fxig
m
i ¼ 1 is a vector containing the non-endoscopic measurements for patient
i. We seek to approximate the unknown function y = F(x)so that predictions can be made on a
new patient for which we have observed the non-endoscopic input measurements x. The GB algorithm relies on an iterative model fitting procedure making use of many simple
predictive algorithms or “base” learners [13], and combine them to form more complex deci-
sion rules. The unknown function F is estimated by minimizing a loss function L defined over
the training set: ð1Þ F ¼ argminFSm
i¼1Lðyi ; FðxiÞÞ
ð1Þ GB constructs an approximation FN of F as a sum of N+1 “base” learners constructed
through N boosting iterations, FN ¼ P
N
n ¼ 0
fn. In our implementation, the “base” learners are
regression trees 12, which are particularly useful in clinical applications as they provide easy to
interpret decision rules. GB starts with an initial base learner F0 and then applies a steepest
descent step for the minimization of the loss function with respect to F0. These two steps are
repeated sequentially and each time a new learner is constructed to follow the direction along
which the loss of the previous learner is minimized. The steepest descent method takes steps
proportional to the negative gradient of the loss function in order to find the local minimum. Definitions of variables and final diagnoses Data on 39 previously published variables associated with need for intervention or adverse out-
come in ALGIB (Table 1) were identified from the literature and collected from prospectively
generated databases of presenting diagnoses, laboratory results, endoscopic, discharge and 3 / 14 PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 Gradient Boosting for Acute Lower Gastrointestinal Bleeding Gradient Boosting for Acute Lower Gastrointestinal Bleeding Table 1. Baseline Variables at initial ALGIB presentation. Demographic
Age
Gender
History
Use of omeprazole/lansoprazole
Use of NSAID drugs/antocoagulants
Alcoholism
Smoking
Nursing home resident
Colorectal polyp
Haemorrhoids
Diverticular disease history
Colonic AVM
Syncope
Comorbidities
Cardiovascular disease
Hypertension
Stroke history
COPD
Chronic renal failure
Diabetes mellitus
Dementia
Cancer
Chronic liver diease
Previous GI bleed history
Unstable comorbidities
Initial assessment
Heart rate
Systolic BP
Diastolic BP
Erratic mental status
Abdominal pain
Tender abdominal exam
Ongoing bleed in ED
Gross blood on DRE
Baseline bloods
Haemaglobin
Haematocrit
White blood cell count
Platelet
APTT
Prothrombin time
Urea
Creatinine
APTT, activated partial prothrombin time; AVM, arteriovenous malformation; DRE, digital rectal
examination; ED emergency department
doi:10.1371/journal.pone.0132485.t001 Table 1. Baseline Variables at initial ALGIB presentation. APTT, activated partial prothrombin time; AVM, arteriovenous malformation; DRE, digital rectal
examination; ED emergency department PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 4 / 14 4 / 14 Gradient Boosting for Acute Lower Gastrointestinal Bleeding coding databases. The variables were defined as follows: Unstable co-morbidity-any organ sys-
tem abnormality that usually requires ICU admission, erratic mental status-clouding of con-
sciousness due to any cause or presence of syncope confusion or coma, cardiovascular disease-
history of angina, myocardial infarction, cardiomyopathy or heart failure, respiratory disease-
current or past history of copd, liver disease-history or presence of jaundice, cirrhosis or portal
hypertension and renal failure-creatinine >125mircomol/l. Definite colonic bleeding was defined as any signs of active or recent bleeding on endoscopy
or angiography (stigmata of recent haemorrhage: active bleeding, a non bleeding visible vessel,
or an adherent clot). Presumptive was defined haematochezia or blood per rectum and no sus-
picion of upper GI bleeding, with one or more potential bleeding sources below the ligament of
Treitz. 8 end Algorithm After training, the parameters of the learned regression trees enclose rules capturing the
(possibly non-linear) relationship between non-endoscopic variables and the ALGIB outcome. A new patient with non-endoscopic measurements x is then assigned to a specific outcome
class y simply by following the decision rules associated to that class. To enable an optimal training of the GB algorithm, first we randomly divided our cohort of
170 patients collected at Charing Cross Hospital into training and validation datasets. Specifi-
cally, 70% of patients were assigned to the training set and the remaining 30% were utilized for
internal validation. Internal validation was carried out with the objective of optimally tuning
the GB hyperparameters (i.e. the number of boosting iterations, the depth of the regression
trees and the learning rate) before assessing the performance of the algorithm externally on a
cohort of patients admitted to Hammersmith Hospital (completely independent dataset). For each one of the clinical outcomes in our studies we ranked the covariates using the inter-
nal GB mechanism for variable ranking, and selected the 10 best predictive variables for each
outcome. We then investigated whether re-fitting the GB algorithm using only this reduced set
of covariates would yield comparable performance. Gradient boosting 4
an ¼ argmina;b
P
m
i ¼ 1
½gnðxiÞ bhðxi; aÞ
2, fit a regression tree hn(x,am)
5
Fn(x) = Fn-1(x)+γnh(x,an), for
6
gn ¼ argming
Pm
i ¼ 1 Lðyi; Fn1ðxiÞ þ ghðxi; anÞÞ
d f Gradient Boosting Algorithm
1 F0ðxÞ ¼ argminp
Pm
i ¼ 1 Lðyi; pÞ, where p is the response of the regression t
the training data. 2 for n = 1 ! N do
3
gn ¼ rFn1ðxÞLðy; Fn1ðxÞÞ gradient at the training data x. 4
an ¼ argmina;b
P
m
i ¼ 1
½gnðxiÞ bhðxi; aÞ
2, fit a regression tree hn(x,am)
5
Fn(x) = Fn-1(x)+γnh(x,an), for
6
gn ¼ argming
Pm
i ¼ 1 Lðyi; Fn1ðxiÞ þ ghðxi; anÞÞ g
g
1 F0ðxÞ ¼ argminp
Pm
i ¼ 1 Lðyi; pÞ, where p is the response of the regression tree to
the training data. 2 for n
1 ! N do
3
gn ¼ rFn1ðxÞLðy; Fn1ðxÞÞ gradient at the training data x. 3
gn ¼ rFn1ðxÞLðy; Fn1ðxÞÞ gradient at the training data x. 4
an ¼ argmina;b
P
m
i ¼ 1
½gnðxiÞ bhðxi; aÞ
2, fit a regression tree hn(x,am)
5
Fn(x) = Fn-1(x)+γnh(x,an), for
6
gn ¼ argming
Pm
i ¼ 1 Lðyi; Fn1ðxiÞ þ ghðxi; anÞÞ 7 end for Gradient boosting More explicitly, the gradient of the loss function L for each training point xi at the iteration
step n is given by ð2Þ gi;nðxiÞ ¼ rFn1ðxiÞLðyi; Fn1ðxiÞÞ;
1 i m:
ð2Þ The gradient is defined only at the data points fxig
m
i ¼ 1 and cannot be generalized to other
x values. One way to enable generalization is to choose a regression tree h(x,an) that produces The gradient is defined only at the data points fxig
m
i ¼ 1 and cannot be generalized to other
x values. One way to enable generalization is to choose a regression tree h(x,an) that produces PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 5 / 14 ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 Gradient Boosting for Acute Lower Gastrointestinal Bleeding hn ¼ fhðxi; anÞg
m
1 most parallel to-gn Rm. This regression tree can be obtained from the
solution an ¼ argmina;bSm
i¼1½gnðxiÞ bhðxi; aÞ
2;
ð3Þ ð3Þ where an are the parameters of the regression tree h(x,an) and β is the learning rate, which
determines the contribution of each tree to the approximation. Having estimated the regression
tree that is most highly correlated with-gn (x) over the data distribution, the next update of the
approximation FN is given by where an are the parameters of the regression tree h(x,an) and β is the learning rate, which
determines the contribution of each tree to the approximation. Having estimated the regression
tree that is most highly correlated with-gn (x) over the data distribution, the next update of the
approximation FN is given by FnðxÞ ¼ Fn1ðxÞ þ gnhðx; anÞ;
ð4Þ ð4Þ which uses the optimal length, gn ¼ argmingSm
i¼1Lðyi; Fn1ðxiÞ þ ghðxi; anÞÞ:
ð5Þ ð5Þ The GB algorithm is summarized in the following pseudo-code: The GB algorithm is summarized in the following pseudo-code: The GB algorithm is summarized in the following pseudo-code:
Gradient Boosting Algorithm
1 F0ðxÞ ¼ argminp
Pm
i ¼ 1 Lðyi; pÞ, where p is the response of the regression tree to
the training data. 2 for n = 1 ! N do
3
gn ¼ rFn1ðxÞLðy; Fn1ðxÞÞ gradient at the training data x. Characteristics and clinical outcomes of cohorts For the Charing Cross cohort (CXC) following the initial search through emergency and
endoscopy databases at Charing Cross hospital 174 patients were identified as having had a his-
tory of ALGIB and presentation to the emergency department. Four patients were excluded
due to incomplete information for risk scoring. The 170 remaining patients made up the train-
ing/internal validation cohort. The same search process was used to compile the Hammersmith
cohort (HC). 133 patients were attended the emergency department of Hammersmith hospital
with a primary diagnosis of ALGIB. Of these patients three were excluded due incomplete data
for risk scoring. The remaining 130 patients made up the external validation cohort. The accu-
racy of data collection was by random re-assessment of 10% of notes by another author (LA). The demographic characteristic, clinical features and final diagnoses of the internal and
external validation cohorts are shown in Tables 2 and 3. The patients in the internal Charing
Cross cohort were similar to the Hammersmith cohort with regard to sex ratio, median age
and length of stay. Nearly all patients (98–100%) in both cohorts were admitted to hospital for
management of ALGIB which lasted a median of 4 days. Upper GI endoscopy to rule out an
upper GI bleed was carried out in 20% of cases in the internal cohort and 14% of cases in the
external cohort. Patients in both cohorts were similar in terms of undergoing a lower endos-
copy procedure (74% vs. 81%) but in-patient colonoscopy was more common in the Charing
Cross cohort (76% vs. 46%). This consisted of colonoscopy (56% Charing cross cohort, 45%
Hammersmith cohort) with the remainder having flexible sigmoidoscopy, rigid sigmoidoscopy For the Charing Cross cohort (CXC) following the initial search through emergency and
endoscopy databases at Charing Cross hospital 174 patients were identified as having had a his-
tory of ALGIB and presentation to the emergency department. Four patients were excluded
due to incomplete information for risk scoring. The 170 remaining patients made up the train-
ing/internal validation cohort. The same search process was used to compile the Hammersmith
cohort (HC). 133 patients were attended the emergency department of Hammersmith hospital
with a primary diagnosis of ALGIB. Of these patients three were excluded due incomplete data
for risk scoring. The remaining 130 patients made up the external validation cohort. Outcomes The outcomes measured were therapeutic intervention (endoscopic, angiographic, surgical),
severe bleeding (defined as ongoing or recurrent bleeding) and recurrent bleeding. These out-
comes were chosen as they indicate the need for inpatient care. Therapeutic intervention to
stop the source of a bleed was included as this suggested the presence of an ongoing bleed that
was not resolving spontaneously. Definitions for the three outcomes were taken the published
literature [5,9,25,26]. Severe bleeding was defined as the following: continued bleeding in the
first 24 hours of hospitalisation (defined as a RBC transfusion of 2 units, and/or a haemato-
crit decrease of 20%), or recurrent bleeding after 24 hours of stability (defined as more than
one transfusion of RBCs, a further haematocrit decrease of 20%, or readmission for ALGIB
within 1 week of discharge). Recurrent bleeding was defined as recurrent haematochezia after
24 hours of stabilisation during which no active bleeding was observed, associated with any of
the following as a new finding: decrease in haemoglobin of 2g/dl, decrease in haematocrit of
5%, haemodynamic instability, or having an additional RBC transfusion (2 units received
in total). MLR and published MLR based models for ALGIB Conventional multiple logistic regression was applied to the Charing Cross and Hammmer-
smith cohorts using the same 39 non-endoscopic as in GB. Moreover, two published MLR
based models for ALGIB, the BLEED score [10] (persistent bleeding, low systolic blood pres-
sure, elevated prothrombin time, erratic mental status and unstable comorbid disease) and
Strate prediction rule [9] (heart rate 100, systolic blood pressure 115, syncope, non tender
abdominal examination, rectal bleeding within first 4hrs of evaluation, aspirin use and > 2 co-
morbid conditions) were calculated for the both cohorts. 6 / 14 PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 Gradient Boosting for Acute Lower Gastrointestinal Bleeding Statistical Analysis The following statistical figures to predict severe bleeding, recurrent bleeding and therapeutic
intervention were derived for all models in the internal and external validation cohorts: Sensi-
tivity, Specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and Accu-
racy (sum of correct predictions over total predictions) using HDS Epimax, 2004 and Graph
Pad Prism software. Comparison of continuous and categorical data (between the internal and
external validation cohorts) was carried out using Mann-Whitney U and Fisher exact tests
respectively. A two-tailed significance of 5% was used in all comparisons. PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 Characteristics and clinical outcomes of cohorts The accu-
racy of data collection was by random re-assessment of 10% of notes by another author (LA). The demographic characteristic, clinical features and final diagnoses of the internal and
external validation cohorts are shown in Tables 2 and 3. The patients in the internal Charing
Cross cohort were similar to the Hammersmith cohort with regard to sex ratio, median age
and length of stay. Nearly all patients (98–100%) in both cohorts were admitted to hospital for
management of ALGIB which lasted a median of 4 days. Upper GI endoscopy to rule out an
upper GI bleed was carried out in 20% of cases in the internal cohort and 14% of cases in the
external cohort. Patients in both cohorts were similar in terms of undergoing a lower endos-
copy procedure (74% vs. 81%) but in-patient colonoscopy was more common in the Charing
Cross cohort (76% vs. 46%). This consisted of colonoscopy (56% Charing cross cohort, 45%
Hammersmith cohort) with the remainder having flexible sigmoidoscopy, rigid sigmoidoscopy 7 / 14 PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 Gradient Boosting for Acute Lower Gastrointestinal Bleeding or proctoscopy. A CT or mesenteric angiogram was carried out in 8% of patients in the Char-
ing Cross cohort and 5% in the Hammersmith cohort. The three most common diagnoses were diverticulosis, colitis and anorectal disorders such
as haemorrhoids. Final diagnoses were similar in both cohorts apart from a presumptive
Table 2. Characteristics and outcomes of cohorts. Characteristic
Charing Cross (n = 170)
Hammersmith (n = 130)
P value
Male
90 (53%)
69 (53%)
ns
Median Age (range)
70 (16–99)
70 (17–101)
ns
Discharge within 24 hrs
4 (2%)
0 (0%)
ns
Median, mean hospital stay (range)
4, 7 (0–102)
4, 7 (0–182)
ns
Blood cell transfusion
58 (34%)
23 (18%)
0.001
Had lower GI Endoscopy
125 (74%)
105 (81%)
ns
Outcomes
Severe bleeding
60 (35%)
28 (22%)
0.01
Recurrent bleeding
34 (20%)
19 (14%)
ns
Therapeutic intervention
26 (15%)
9 (7%)
0.02
endoscopic
9 (5%)
4 (3%)
ns
angiographic
9 (5%)
4 (3%)
ns
surgery
8 (4.7%)
1 (0.7%)
ns
Death
4 (2.3%)
3 (2.3%)
ns
ns-not significant
doi:10.1371/journal.pone.0132485.t002
Table 3. Final Diagnoses in Cohorts. Charing Cross (n = 170)
Hammersmith (n = 130)
Definite
Diverticulosis and its complications
13
16
Colitis
14
5
Anorectal (including varices)
6
12
Neoplasia and post-neoplasia therapy
14
5
Angiodysplasia
3
4
Isolated large bowel ulcers
3
1
Coagulation disorders
1
0
Small bowel bleeding
1
1
Presumptive
Diverticulosis and its complications*
44
13
Colitis
11
11
Anorectal (including varices)
11
9
Neoplasia and post-neoplasia therapy
2
4
Angiodysplasia
3
2
Coagulation disorders
0
1
solated large bowel ulcers
4
0
Small bowel bleed
1
0
No diagnosis made*
39
46
* significant difference between cohorts p<0.05
doi:10.1371/journal.pone.0132485.t003 Table 2. Characteristics and outcomes of cohorts. Table 2. Characteristics and outcomes of cohorts. Gradient Boosting for Acute Lower Gastrointestinal Bleeding diagnosis of a diverticular bleed which was more common in the Charing Cross cohort and no
diagnosis made which was more common in the Hammersmith cohort. Therapeutic intervention for bleeding (endoscopic therapy, angiographic embolisation or
surgery) was more common in the Charing Cross cohort. Endoscopic intervention in both
cohorts consisted of clipping, APC and banding. Angiographic embolisation of colonic vessels
was carried out in nine patients in the internal cohort and four in the external cohort. Blood
transfusion was significantly more common in the Charing Cross cohort (mean 1.6 units vs. 0.9 units) as was severe bleeding. There was no significant difference between the cohorts in re-
bleeding or death. All patients who died were 65 years of age and the causes of death were
cancer (n = 2) cardiac failure (n = 1), pneumonia (n = 1), colonic ischemia (n = 1), pulmonary
hypertension (n = 1) and unknown (n = 1). No patient died because of uncontrolled bleeding. Characteristic
Charing Cross (n = 170)
Hammersmith (n = 130)
P value
Male
90 (53%)
69 (53%)
ns
Median Age (range)
70 (16–99)
70 (17–101)
ns
Discharge within 24 hrs
4 (2%)
0 (0%)
ns
Median, mean hospital stay (range)
4, 7 (0–102)
4, 7 (0–182)
ns
Blood cell transfusion
58 (34%)
23 (18%)
0.001
Had lower GI Endoscopy
125 (74%)
105 (81%)
ns
Outcomes
Severe bleeding
60 (35%)
28 (22%)
0.01
Recurrent bleeding
34 (20%)
19 (14%)
ns
Therapeutic intervention
26 (15%)
9 (7%)
0.02
endoscopic
9 (5%)
4 (3%)
ns
angiographic
9 (5%)
4 (3%)
ns
surgery
8 (4.7%)
1 (0.7%)
ns
Death
4 (2.3%)
3 (2.3%)
ns
ns-not significant
doi:10.1371/journal.pone.0132485.t002 or proctoscopy. A CT or mesenteric angiogram was carried out in 8% of patients in the Char-
ing Cross cohort and 5% in the Hammersmith cohort. The three most common diagnoses were diverticulosis, colitis and anorectal disorders such
as haemorrhoids. Final diagnoses were similar in both cohorts apart from a presumptive Table 3. Final Diagnoses in Cohorts. Charing Cross (n = 170)
Hammersmith (n = 130)
Definite
Diverticulosis and its complications
13
16
Colitis
14
5
Anorectal (including varices)
6
12
Neoplasia and post-neoplasia therapy
14
5
Angiodysplasia
3
4
Isolated large bowel ulcers
3
1
Coagulation disorders
1
0
Small bowel bleeding
1
1
Presumptive
Diverticulosis and its complications*
44
13
Colitis
11
11
Anorectal (including varices)
11
9
Neoplasia and post-neoplasia therapy
2
4
Angiodysplasia
3
2
Coagulation disorders
0
1
solated large bowel ulcers
4
0
Small bowel bleed
1
0
No diagnosis made*
39
46
* significant difference between cohorts p<0.05
doi:10.1371/journal.pone.0132485.t003
PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015
8 / 14 Table 3. Final Diagnoses in Cohorts. Table 3. Final Diagnoses in Cohorts. 8 / 14 Predictive performance of gradient boosting and multiple logistic
regression The best GB algorithms using the 39 variables had predictive accuracies of 88%, 91% and 83%
(Table 4) for recurrent bleeding, therapeutic intervention and severe bleeding respectively. The
accuracies were similar in the Charing Cross and Hammersmith cohorts. The positive predic-
tive value of all GB algorithms were not high in either cohort although importantly for clinical
decision making the negative predictive values were high (88–98%) for the three outcomes. Table 4. Predictive performance of models in Charing Cross (CXC) and Hammersmith (HC) cohorts. Outcome variable
Accuracy
Sensitivity
Specificity
PPV
NPV
GB model: Recurrent bleeding
CXC
88
67
91
50
95
HC
88
57
91
50
94
GB model: Therapeutic intervention
CXH
88
80
89
44
98
HC
91
60
92
27
98
GB model: Severe bleeding
CXH
78
73
80
61
88
HC
83
57
89
58
90
MLR model: Recurrent bleeding
CXC
74
22
85
25
83
HC
83
20
85
6
95
MLR model: Therapeutic intervention
CXC
74
16
82
11
97
HC
87
20
90
9
96
MLR model: Severe Bleeding
CXC
62
46
69
39
75
HC
71
35
83
42
80
BLEED classification (CXC only)
Recurrent bleeding
64
24
75
21
77
Therapeutic intervention
68
27.5
76
19
84
Severe bleeding
63
33
79
44
69
Strate prediction rule (CXC only)cut off >3
Recurrent bleeding
78
16
94
33
81
Therapeutic intervention
78
4
92
8
84
Severe bleeding
67
13
96
66
66
doi:10.1371/journal.pone.0132485.t004 redictive performance of models in Charing Cross (CXC) and Hammersmith (HC) cohorts. PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 9 / 14 Gradient Boosting for Acute Lower Gastrointestinal Bleeding Table 5. Top ten variable importance using gradient boosting models. Contribution %
Severe bleeding
Platelet count
13.4
APTT
13.0
Haematocrit
12.4
Urea
10.9
Creatinine
9.7
Prothrombin time
8.9
Diastolic blood pressure
6.8
Heart rate
4.1
Systolic blood pressure
3.9
Alcohol abuse
3.9
Therapeutic Intervention
Haemoglobin
15.7
Diastolic blood pressure
13.9
haematocrit
9.5
APTT
9.0
Creatinine
8.2
Fresh blood on PR
7.1
Prothrombin time
6.7
Heart rate
5.0
Past medical history of colorectal polyp
3.4
Use of NSAIDs or anticoagulants
3.4
Recurrent bleeding
Creatinine
19.1
Haemoglobin
18.8
Age
17.9
Diabetes
13.2
APTT
11.5
Diastolic blood pressure
6.8
Heart Rate
4.6
Urea
4.4
Alcoholism
2.4
Total number of co-morbidities
1.3
doi:10.1371/journal.pone.0132485.t005 Table 5. Top ten variable importance using gradient boosting models. PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 Discussion There is a need for non-endoscopic risk scores to help risk stratify patients with ALGIB for
early discharge/outpatient management or higher levels of care and thereby utilise resources
efficiently. Current clinical practice includes colonoscopic-based triage which is invasive, ade-
quate preparation difficult to achieve and treatable stigmata of haemorrhage infrequent [1]. This study has shown that a GB algorithm based on clinical and laboratory variables was accu-
rate (>80%) in predicting the clinical outcomes of recurrent bleeding, therapeutic intervention
and severe bleeding. GB had high negative predictive values (88–98%) in both Charing Cross and Hammersmith
Cohorts. This suggests that these models may be useful to triage patients into a low-risk group
who could be managed with an abbreviated stay in hospital avoiding high levels of care or as
outpatients. The median and mean inpatient stay in the Charing Cross and Hammersmith
cohorts was four and seven days respectively and therefore a reduction in this would allow for
significant cost-savings and decrease exposure of patient to hospital associated hazards such as
infections. A particular strength of this study is the validation and good performance in an external
cohort with a lower incidence of severe bleeding indicating the algorithm can maintain accuracy
in a different setting. Our GB algorithm used only non-endoscopic variables available to the cli-
nician in the emergency department and therefore has clinical applicability for decision-making. We would however emphasise that such a model is not aimed at replacing experienced decision
making but rather aiding the process. This is the case with recommended risk scores for acute
upper gastrointestinal bleeding such as the Rockall and Glasgow Blatchford scores [11]. One study to our knowledge has examined an ensemble machine learning model in acute
gastrointestinal bleeding and was developed to identify the bleeding source, need for resuscita-
tion and those who require urgent endoscopy [27]. This study differs substantially from ours in
that a random forests algorithm was used by building classification trees ignoring the error of
the previous tree in the sequence [28]. Also a mixed population of patients with both acute
upper, middle and lower gastrointestinal bleeding was studied (no numbers given for ALGIB),
there was no comparison with previously published scores and the model was not tested in an
external cohort. Nevertheless accuracies of >75–80% were found for the studied outcomes pro-
viding evidence of the utility of the ensemble machine learning techniques. Predictive performance of gradient boosting and multiple logistic
regression Contribution % The top ten contributing predictors used in the GB algorithms for each outcome are listed on
Table 5 in descending order. Four variables (heart rate, diastolic blood pressure, creatinine and
APTT) were in the top ten most frequently used variables for all outcomes. On internal valida-
tion the accuracy of the GB models for predicting recurrent bleeding, therapeutic intervention
and severe bleeding was (88%, 88% and 78% respectively) and superior to the BLEED classifi-
cation (64%, 68% and 63%) (Table 4), Strate prediction rule (78, 78, 67%) and conventional
MLR (74%, 74% 62%). On external validation the accuracy of the GB algorithm was similar to
conventional MLR for recurrent bleeding (88% vs. 83%), and therapeutic intervention (91% vs. 87%) but superior for severe bleeding (83 vs 71%). GB models using just the top ten predictive
variables were less accurate in predicting rebleeding, severe bleeding and therapeutic interven-
tion than those with the full set of 39 variables by 8–10% on average. The top ten contributing predictors used in the GB algorithms for each outcome are listed on
Table 5 in descending order. Four variables (heart rate, diastolic blood pressure, creatinine and
APTT) were in the top ten most frequently used variables for all outcomes. On internal valida-
tion the accuracy of the GB models for predicting recurrent bleeding, therapeutic intervention
and severe bleeding was (88%, 88% and 78% respectively) and superior to the BLEED classifi-
cation (64%, 68% and 63%) (Table 4), Strate prediction rule (78, 78, 67%) and conventional
MLR (74%, 74% 62%). On external validation the accuracy of the GB algorithm was similar to
conventional MLR for recurrent bleeding (88% vs. 83%), and therapeutic intervention (91% vs. 87%) but superior for severe bleeding (83 vs 71%). GB models using just the top ten predictive
variables were less accurate in predicting rebleeding, severe bleeding and therapeutic interven-
tion than those with the full set of 39 variables by 8–10% on average. 10 / 14 PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 Gradient Boosting for Acute Lower Gastrointestinal Bleeding PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 Gradient Boosting for Acute Lower Gastrointestinal Bleeding and hospital mortality) rather than the end-points we examined. The Strate prediction rule [9]
was designed to predict severe bleeding which we studied but in our cohort performed least
well for this outcome and better for recurrent bleeding and need for clinical intervention. Arti-
ficial neural networks, another machine learning classifier, have also been shown to be accurate
in predicting re-bleeding and clinical intervention in ALGIB [5]. However for our cohort, the
classification performance of neural networks was found inferior to GB in ALGIB (data not
shown). Our study has a number of potential limitations: First the database of patients was collated
retrospectively and relied on the inherent accuracy of patient records. The majority of data col-
lected was however quantitative and also available from prospectively generated electronic lab-
oratory, endoscopic and patient records. Second the GB requires the input of many more
variables than BLEED or Strate prediction rule (39 vs. 5 vs. 8) which increases complexity. Reduction in the number of variables used in the GB model to 10 led to decreased accuracy of
8–10% for the studied outcomes which would compromise clinical utility of the GB algorithm. Importantly however in our experience input of data for the 39 variables into the programme
takes less than 5 minutes and therefore would be suitable for use in an emergency department/
ward particularly given the explosion of smart phone apps which allow for quick data entry
with drop down menus where only positive inputs are required. This would be similar to
endoscopy reports which are generated electronically, typically require >50 pieces of data and
are used in routine clinical practice. Third the decision to give blood transfusion and apply
endoscopic therapy was not protocol based which could have led to bias. In mitigation our
analysis showed that endoscopic therapy was consistently applied in this study according to
consensus guidelines (data not shown) and therefore limits this as a potential source of bias. Finally death was not examined as an outcome due its infrequent nature in our cohort. Death
is rare in ALGIB occurring in <4% in large series and generally occurs in those with co-morbid
conditions [6] and after an in-patient bleed [32] that latter which was an exclusion criteria for
our study. Author Contributions Conceived and designed the experiments: LA PY GM. Performed the experiments: LA PY RC
AT LM GM. Analyzed the data: LA PY AN RC GM. Contributed reagents/materials/analysis
tools: LA PY GM. Wrote the paper: LA PY GM. Future work to examine the GB model in a larger cohort could be undertaken to
examine its utility in predicting death. To determine the impact of the model in predicting out-
comes in ALGIB a randomised study of the GB model plus routine clinical decision-making
versus routine clinical decision-making could be performed. In summary, gradient boosting accurately predicts outcome in patients with acute lower
gastrointestinal bleeding. This machine learning approach has the potential to aid in the risk
stratification of patients with ALGIB on presentation to the emergency department. PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 Discussion The performance of the GB algorithm in our study was superior to MLR and two published
MLR based models. Ensemble machine learning models have been shown to be more accurate
than conventional logistic regression to classify disease or predict outcome in a variety of clini-
cal settings [29–31]. Theoretical reasons for this are that logistic regression predicts outcomes
based on linear combinations of independent variables by fitting a single model that best
explain the relationship between observed values and outcome. On the other hand, the ratio-
nale of the GB algorithm to fit many simple models whose predictions are then combined can
produce a good fit of the predicted outcome values to the observed values, even if the specific
nature of the relationship between the predictor variables and the corresponding outcome is
complex (e.g. nonlinear, interacted or noisy with outliers). Also, unlike multiple logistic regres-
sion, GB method can handle a large number of input variables and generate an internal unbi-
ased estimate of the generalization error as the simple classification tree estimation progresses. Finally, the stage-wise model fitting procedure of the GB algorithm allows to automatically
assess the influence of each non-endoscopic variable in the construction of a robust classifica-
tion rule [12]. Other explanations are that the BLEED score [10] was designed to predict a composite end-
point of in-hospital complication (recurrent haemorrhage, surgery to control haemorrhage 11 / 14 PLOS ONE | DOI:10.1371/journal.pone.0132485
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acute lower-gastrointestinal haemorrhage based on an artificial neural network: internal and external
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able and accurate prediction of receipt of transfusion in patients undergoing percutaneous coronary
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neous intracerebral hemorrhage with remarkable discrimination. Eur J Neurol Off J Eur Fed Neurol
Soc. 2010; 17: 945–950. doi: 10.1111/j.1468-1331.2010.02955.x 17. Breiman L, Friedman J, Stone CJ, Olshen RA. Classification and regression trees. CRC press; 1984. 18. Kalai A, Servedio RA. Boosting in the Presence of Noise. Proceedings of the Thirty-fifth Annual ACM
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780542.780573 19. Dodd S, Berk M, Kelin K, Zhang Q, Eriksson E, Deberdt W, et al. Application of the Gradient Boosted
method in randomised clinical trials: Participant variables that contribute to depression treatment effi-
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July 14, 2015 PLOS ONE | DOI:10.1371/journal.pone.0132485
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nomics. 2006; 7: 1003–1016. doi: 10.2217/14622416.7.7.1003 PMID: 17054411 24. Breiman L. Statistical Modeling: The Two Cultures (with comments and a rejoinder by the author). Stat
Sci. 2001; 199–231. doi: 10.1214/ss/1009213726 25. Strate LL, Orav EJ, Syngal S. Early predictors of severity in acute lower intestinal tract bleeding. Arch
Intern Med. 2003; 163: 838–843. doi: 10.1001/archinte.163.7.838 PMID: 12695275 13 / 14 PLOS ONE | DOI:10.1371/journal.pone.0132485
July 14, 2015 Gradient Boosting for Acute Lower Gastrointestinal Bleeding 26. Velayos FS, Williamson A, Sousa KH, Lung E, Bostrom A, Weber EJ, et al. Early predictors of severe
lower gastrointestinal bleeding and adverse outcomes: a prospective study. Clin Gastroenterol Hepatol
Off Clin Pract J Am Gastroenterol Assoc. 2004; 2: 485–490. 27. Chu A, Ahn H, Halwan B, Kalmin B, Artifon ELA, Barkun A, et al. A decision support system to facilitate
management of patients with acute gastrointestinal bleeding. Artif Intell Med. 2008; 42: 247–259. doi:
10.1016/j.artmed.2007.10.003 PMID: 18063351 28. Breiman L. Random Forests. Mach Learn. 2001; 45: 5–32. 28. Breiman L. Random Forests. Mach Learn. 2001; 45: 5–32. 29. Casanova R, Saldana S, Chew EY, Danis RP, Greven CM, Ambrosius WT. Application of random for-
ests methods to diabetic retinopathy classification analyses. PloS One. 2014; 9: e98587. doi: 10.1371/
journal.pone.0098587 PMID: 24940623 30. Maroco J, Silva D, Rodrigues A, Guerreiro M, Santana I, de Mendonça A. Data mining methods in the
prediction of Dementia: A real-data comparison of the accuracy, sensitivity and specificity of linear dis-
criminant analysis, logistic regression, neural networks, support vector machines, classification trees
and random forests. BMC Res Notes. 2011; 4: 299. doi: 10.1186/1756-0500-4-299 PMID: 21849043 31. Austin PC, Lee DS, Steyerberg EW, Tu JV. Regression trees for predicting mortality in patients with car-
diovascular disease: what improvement is achieved by using ensemble-based methods? Biom J Biom
Z. 2012; 54: 657–673. doi: 10.1002/bimj.201100251 32. Strate LL, Ayanian JZ, Kotler G, Syngal S. Risk factors for mortality in lower intestinal bleeding. Clin
Gastroenterol Hepatol. 2008; 6: 1004–1010. doi: 10.1016/j.cgh.2008.03.021 PMID: 18558513 14 / 14
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Consumer Perceptions of Cigarette Design in France: A Comparison of Regular, Slim, Pink and Plain Cigarettes
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This is a pre-copyedited, author-produced PDF of an article accepted for publication in Nicotine & Tobacco Research following peer
review. The version of record Karine Gallopel-Morvan, Crawford Moodie, Romain Guignard, Figen Eker, Emmanuelle Béguinot,
Consumer Perceptions of Cigarette Design in France: A Comparison of Regular, Slim, Pink and Plain Cigarettes, Nicotine & Tobacco
Research, Volume 21, Issue 7, July 2019, Pages 911–917 is available online at: https://doi.org/10.1093/ntr/nty105 4 Comité National Contre le Tabagisme (French National Committee Against Tobacco), Paris,
France. anuscript
Eker MSc4, Emmanuelle Béguinot MSc4
Affiliations:
1 EHESP School of Public Health, EA 7348 MOS, Rennes, France. 2 Centre for Tobacco Control Research, University of Stirling, Stirling, Scotland, UK. Ti l
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This is a pre-copyedited, author-produced PDF of an article accepted for publication in Nicotine & Tobacco Research following peer
review. The version of record Karine Gallopel-Morvan, Crawford Moodie, Romain Guignard, Figen Eker, Emmanuelle Béguinot,
Consumer Perceptions of Cigarette Design in France: A Comparison of Regular, Slim, Pink and Plain Cigarettes, Nicotine & Tobacco
Research, Volume 21, Issue 7, July 2019, Pages 911–917 is available online at: https://doi.org/10.1093/ntr/nty105 Ti l
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This is a pre-copyedited, author-produced PDF of an article accepted for publication in Nicotine & Tobacco Research following peer
review. The version of record Karine Gallopel-Morvan, Crawford Moodie, Romain Guignard, Figen Eker, Emmanuelle Béguinot,
Consumer Perceptions of Cigarette Design in France: A Comparison of Regular, Slim, Pink and Plain Cigarettes, Nicotine & Tobacco
Research, Volume 21, Issue 7, July 2019, Pages 911–917 is available online at: https://doi.org/10.1093/ntr/nty105 This is a pre-copyedited, author-produced PDF of an article accepted for publication in Nicotine & Tobacco Research following peer
review. The version of record Karine Gallopel-Morvan, Crawford Moodie, Romain Guignard, Figen Eker, Emmanuelle Béguinot,
Consumer Perceptions of Cigarette Design in France: A Comparison of Regular, Slim, Pink and Plain Cigarettes, Nicotine & Tobacco
Research, Volume 21, Issue 7, July 2019, Pages 911–917 is available online at: https://doi.org/10.1093/ntr/nty105 This is a pre-copyedited, author-produced PDF of an article accepted for publication in Nicotine & Tobacco Research following peer
review. The version of record Karine Gallopel-Morvan, Crawford Moodie, Romain Guignard, Figen Eker, Emmanuelle Béguinot,
Consumer Perceptions of Cigarette Design in France: A Comparison of Regular, Slim, Pink and Plain Cigarettes, Nicotine & Tobacco
Research, Volume 21, Issue 7, July 2019, Pages 911–917 is available online at: https://doi.org/10.1093/ntr/nty105 Title: Consumer perceptions of cigarette design in France: A comparison of regular, slim, pink Title: Consumer perceptions of cigarette design in France: A comparison of regular, slim, pink and plain cigarettes Accepted Manuscript
Authors: Karine Gallopel-Morvan PhD1, Crawford Moodie PhD2, Romain Guignard MSc3, Figen
Eker MSc4, Emmanuelle Béguinot MSc4
Affiliations:
1 EHESP School of Public Health, EA 7348 MOS, Rennes, France. 2 Centre for Tobacco Control Research, University of Stirling, Stirling, Scotland, UK. 3 Santé publique France (the national public health agency), Saint-Maurice, France. 4 Comité National Contre le Tabagisme (French National Committee Against Tobacco), Paris,
France. Corresponding author: Karine Gallopel-Morvan, EHESP School of Public Health, Rennes,
France. Email: karine.gallopel-morvan@ehesp.fr Tel: (+33) 6 75 36 91 62. pt
Authors: Karine Gallopel-Morvan PhD1, Crawford Moodie PhD2, Romain Guignard MSc3, Figen
Eker MSc4, Emmanuelle Béguinot MSc4 ed Manuscript
Eker MSc4, Emmanuelle Béguinot MSc4
Affiliations:
1 EHESP School of Public Health, EA 7348 MOS, Rennes, France. 2 Centre for Tobacco Control Research, University of Stirling, Stirling, Scotland, UK. 3 Santé publique France (the national public health agency), Saint-Maurice, France. y
y
on 31 May 2018 Affiliations: d Ma
Conclusions: The study suggests that altering the appearance of the cigarette may reduce
cigarette appeal, increase harm perceptions, and deter both young people and smokers. d Ma
Conclusions: The study suggests that altering the appearance of the cigarette may reduce
cigarette appeal, increase harm perceptions, and deter both young people and smokers. Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 Affiliations: anu
2 Centre for Tobacco Control Research, University of Stirling, Stirling, Scotland, UK. Ma
3 Santé publique France (the national public health agency), Saint-Maurice, France. epted M
h
ll
l epted M cept
Corresponding author: Karine Gallopel-Morvan, EHESP School of Public Health, Rennes,
France. Email: karine.gallopel-morvan@ehesp.fr Tel: (+33) 6 75 36 91 62. ept
orresponding author: Karine Gallopel-Morvan, EHESP School of Public Health, Rennes, ep
Corresponding author: Karine Gallopel-Morvan, EHESP School of Public Health, Rennes, cep
Corresponding author: Karine Gallopel-Morvan, EHESP School of Public Health, Rennes,
France Email: karine gallopel-morvan@ehesp fr Tel: (+33) 6 75 36 91 62 cep
Corresponding author: Karine Gallopel-Morvan, EHESP School of Public Health, Rennes, ccep
rance. Email: karine.gallopel-morvan@ehesp.fr Tel: (+33) 6 75 36 91 62. Acce
Abstract A
Abstract Introduction: The cigarette, like the cigarette pack, is used by tobacco companies as a romotional tool. We explore how the cigarette could potentially be used as a dissuasive too promotional tool. We explore how the cigarette could potentially be used as a dissuasive tool. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for
Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail:
journals.permissions@oup.com. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for
Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail:
journals.permissions@oup.com. by University o
on 31 May 2018 cript
Methods: An online survey was conducted with 15-30 year old smokers and non-smokers
(N=998) in France to explore their perceptions of a plain cigarette (grey with no brand name)
and three branded cigarettes (regular, slim, pink). Participants were randomly assigned to view
the plain cigarette and either the regular, slim or pink cigarette. They were asked to rate the
cigarettes by Appeal (tastiest, highest quality, most expensive), Harm (most dangerous, most
effective for motivating people to talk about tobacco dangers), and Perceived behavioral impact
(most effective to convince teenagers not to start, to motivate smokers to reduce consumption
and quit). anusc
Results: In comparison to the grey cigarette, each of the branded cigarettes were considered
more appealing, less harmful, and more likely to motivate teenagers to start and less likely to
motivate smokers to reduce consumption or quit. ed Ma
Conclusions: The study suggests that altering the appearance of the cigarette may reduce
cigarette appeal, increase harm perceptions, and deter both young people and smokers. by University o
on 31 May 2018 Implications Accept
Very little research has focused on dissuasive cigarettes whereas the cigarette stick has become
very important for tobacco companies for communication purposes. This is the first study to
compare the effect of various branded cigarettes (regular, slim, pink) with a plain grey cigarette
on young adult smokers and non-smokers. The findings suggest that a plain grey cigarette can
reduce cigarette appeal, increase perceptions of harm, and may deter use among both smokers
and non-smokers. by University of
on 31 May 2018 by U
e s ty o
on 31 May 2018 Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 INTRODUCTION script
Article 13 of the Framework Convention for Tobacco Control (FCTC) bans all tobacco marketing
and recommends plain (or standardized) tobacco packaging.[1] Many countries have adopted
comprehensive tobacco advertising bans and some have fully implemented plain packaging. Following Australia in December 2012, France became the second country to require plain
packaging in January 2017; the United Kingdom has since followed suit in May 2017.[2]
Consequently, the cigarette stick has become more important for tobacco companies for brand
communication,[3] with promotional elements often displayed on cigarettes (e.g. brand names,
logos, brand descriptors, capsule symbols) and filter innovation (e.g. different shapes and
colors) increasingly common.[4] Accepted Manus
Tobacco companies’ use of branded cigarettes to influence audience preferences has
been revealed in past research and in internal documents.[5-8] For instance, slim cigarettes
have been marketed at women and associated with advertising campaigns promoting the belief
that smoking is an effective way of controlling weight: Philip Morris launched Virginia Slims
with its advertising stressing themes of thinness, glamour and independence.[9] Industry
research has found that among women smokers, irrespective of age, certain ‘superslims’ brands
and cigarettes were viewed as stylish or projected a feminine elegance.[10,11] Studies have also
found that certain slim brands were perceived as less harmful than other
cigarettes,[12] although blood cadmium levels have not been found to be lower among females
who smoke slim rather than non-slim cigarettes.[13] Ac
Colored and flavored cigarettes have also been developed by tobacco companies to
create appeal and communicate favorable brand impressions.[14,15] In the 1950s for instance,
pink colored ‘Vanity Fair’ cigarettes were advertised alongside the slogan ‘Be glamorous all day
long’.[16] Internal tobacco documents reported that colored tipping or cigarette paper could
make smoking more pleasurable for women,[17] with these products perceived as novel by
young people.[18] In addition, colored cigarettes are used to communicate flavor and taste,[14] Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 by University o
on 31 May 2018 e.g. black indicates chocolate flavor for the ‘Black devil’ brand and pink indicates vanilla flavor
and sweet taste for the ‘Pink elephant’ brand. Accepted Manuscript
Rather surprisingly, only a few studies have explored consumer response to cigarettes. Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
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on 31 May 2018 by U
e s ty o
on 31 May 2018 INTRODUCTION Two focus groups studies were conducted in Scotland, one with 15 year old smokers and non-
smokers and the other with 12-24 year old female never smokers and occasional smokers, to
explore perceptions of a range of cigarettes.[19,20] In both studies, slimmer cigarettes were
considered more appealing and less harmful than regular cigarettes with a cork filter and white
cigarette paper. Color also influenced product perceptions, with a pink cigarette generating
considerable interest among 12-24 female never smokers and occasional smokers, being
considered appealing, pleasant tasting and less harmful. A qualitative study with young female
smokers in New Zealand found that colored cigarettes (lilac, red, silver-white or gold) were
judged to be attractive and improved smokers’ social image, with these cigarettes thought to
help them look better, be seen as classy, and avoid social stigma.[21] When regular cigarettes
with cork or white filters and slim cigarettes were compared, there was a preference for slim
and white cigarettes as participants thought that these helped distance themselves from
negative associations with smoking by portraying a glamorous, slim, delicate and feminine
image. Using a face-to-face survey with a large European sample of current and former adult
smokers, Agaku et al. found that colored cigarettes were thought to be sweet tasting and that
flavored cigarettes were associated with first smoking experiences and reduced harm
perceptions.[22] Finally, an online survey in Australia compared young adults smokers’
reactions to different cigarettes, with regular cigarettes rated as more attractive and higher
quality than extra-long, short, slim or extra slim sticks.[23] Most participants indicated that they
would prefer to smoke the regular stick. To reduce the appeal of the types of cigarettes on sale in most markets, researchers have
begun to explore consumer response to cigarettes designed to be dissuasive. Few studies have
explored the impact of plain, unattractively colored cigarettes. A qualitative study and an online by University o
on 31 May 2018 t
survey in New Zealand both found that green or yellow-brown cigarettes were considered
aversive and rated as less appealing than regular cigarettes.[21,24] Focus groups in Scotland
also found that young people perceived brown cigarettes as particularly strong and
harmful.[19,20] These findings suggest that altering the color of cigarettes may be able to
influence how they are perceived, the image they convey and smoking intentions. INTRODUCTION nuscript
Research has yet to compare the effect of a plain, unattractively colored cigarette, with a
number of different cigarette styles which are available in most countries. In this study, we
explore how young adult smokers and non-smokers perceive a plain grey colored cigarette
(designed for this study) and three cigarettes (regular, slim and brightly colored) that were on
sale in France at the time of the study, in terms of appeal, harm and behavioral impact. d Manu
METHODS
Design and sample Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 by University o
on 31 May 2018 Design and sample Design and sample Accepted
An online survey was conducted in France from 4th to 16th December 2013 (before the
implementation of tobacco plain packaging in 2017), with 998 smokers and non-smokers aged
15-30 years. The sample was chosen because prevalence of daily smoking is high in France for
this population (33.4%).[25] A quota sample was sought, balanced by gender, age group, socio-
economic status, geographic area (nine broad areas that cover all of France) and size of urban
unit (<2000 inhabitants; 2000-20,000; 20,000-100,000; >100,000; and the Ile de France region,
which includes Paris, with 12 million inhabitants), following the national percentages of the
general population indicated by the National Institute of Statistics and Economics Studies (see
Table 1 for sample characteristics). Almost two-fifths (38%) were smokers (of which 67% were
daily smokers) and 62% were non-smokers (of which 39% were former smokers). by University of
on 31 May 2018 Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 by University o
on 31 May 2018 Procedure ript
Participants were recruited by an established market research company (LH2), in collaboration
with its partner ‘Survey Sampling International’. Members of their online panel were sent an
email invitation explaining that the survey was part of a national public health study on tobacco
prevention messages; the email invitation and survey were both in French. We do not have the
response rate as recording contact, participation and refusal rates is impractical when using this
sampling methodology. Participants received a modest incentive, in the form of ‘points’ which
can be redeemed for vouchers, as is common with online panels. Accepted Manuscrip
For those eligible for inclusion, they were randomly assigned to view one of three
images: 333 were shown an image of a regular cigarette with the brand name ‘Marlboro’ written
on the stick and a grey plain cigarette (PC); 332 were shown an image of a slim cigarette with
the brand name ‘Vogue’ displayed on the tip and a grey cigarette; 333 were shown an image of a
pink cigarette with the symbol of the brand (an elephant) displayed on the tip and a grey PC (see
Figure 1: cigarettes shown to participants). These three branded cigarettes (regular, slim and
pink) were chosen because they were sold on the French market when the study was conducted:
regular sticks were the most commonly sold cigarettes, with slim and pink cigarettes, targeted at
women and young people, also available.[26, pages 57-60] Grey was selected for the PC because
previous research in France found that grey tobacco packaging was perceived as unattractive
compared to white and brown packs,[27] with grey reported to be the color that French
consumers disliked the most in a study that compared 190 color pairs.[28] Participants were
asked about Appeal, Harm and Perceived behavioral impact, with the ordering of the questions
randomised. The study was ethically approved by the Institut National de Prévention et
d’Education pour la Santé (INPES). by University of
on 31 May 2018 Demographic information Demographic information t
Smoking status, gender and socio-economic status (based on occupation of the head of the
household) was assessed. To assess smoking status, we used the same items employed in the
European Commission's Eurobarometer survey. Participants were asked: ‘Are you: 1/ a non-
smoker; 2/ an occasional smoker (you do not smoke daily); 3/ a regular smoker (you smoke at
least one cigarette per day)’. script script
Appeal Appeal by U
e s ty o
on 31 May 2018 Appeal anusc
Participants were asked, in three separate questions: ‘of these two cigarettes, which one do
you consider: the most expensive, tastiest, the highest quality’. These three items were taken
from past research on plain packaging.[29,30] ed Man
from past research on plain packaging.[29,30]
Harm Harm epted
Participants were asked in two questions: ‘of these two cigarettes, which one do you consider:
the most dangerous, the most effective to make people talk about tobacco dangers’. Accep
Perceived behavioral impact A
Participants were asked in three questions: ‘of these two cigarettes, which one do you
consider: the most effective for convincing teenagers not to start, the most effective for
motivating smokers to quit and the most effective for motivating smokers to reduce
consumption’. For all questions, participants had the option to select either image, ‘Don’t know’, or ‘None’. For all questions, participants had the option to select either image, ‘Don’t know’, or ‘None’. Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
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on 31 May 2018 Analysis pted Manuscript
Using Stata/SE 13.1 software, all analyses were conducted on weighted data that reflected the
demographic structure of the national population. Adjusted Wald tests for weighted data were
used to examine differences in the proportion of participants selecting each cigarette. Logistic
regression models were run to examine differences in perceptions of the different cigarettes. For
each cigarette, the dependent variables for Appeal were ‘most expensive’, ‘tastiest’, ‘highest
quality’ (where 0 = selecting the PC and 1 = selecting the branded cigarette). The dependent
variables for Harm were ‘most dangerous’ and ‘most effective to make people talk about tobacco
dangers’ (where 0 = selecting the branded cigarette and 1 = selecting the PC). For Perceived
behavioral impact, the dependent variables were ‘most effective in convincing teenagers not to
start’, ‘most effective for motivating smokers to quit’, and ‘most effective for motivating smokers
to reduce consumption’ (where 0 = selecting the branded cigarette and 1 = selecting the PC). Gender, age (15-20; 21-24; 25-30), smoking status (non-smoker vs. occasional smokers vs. daily
smoker) and type of cigarette (regular vs. slim vs. pink) were entered as predictor variables in
each of the models. Accept
RESULTS
Perceptions of the plain cigarette (in comparison to the regular, slim or pink cigarettes) Ac
RESULTS Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
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e s ty o
on 31 May 2018 Ac
Perceptions of the plain cigarette (in comparison to the regular, slim or pink cigarettes) A
The branded regular, slim, and pink cigarettes were viewed as more appealing (the most
expensive, the tastiest and the highest quality) than the PC, and less likely to have a greater
impact on smoking initiation and cessation (most effective to convince teenagers not to start,
and motivate smokers to reduce consumption and quit) (see Table 2: perceptions of regular,
slim, pink and PC). While the regular and the pink cigarettes were viewed as less dangerous than Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 by University of
on 31 May 2018 the PC, there were no significant differences with respect to making people talk about the
dangers of tobacco. As for the slim cigarette, it was perceived as less harmful than the PC (least
dangerous, least effective in making people talk about the dangers of tobacco). Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
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Perceptions of cigarettes according to participants’ profiles and type of cigarette anuscript
Logistic regression models were conducted to examine the effect of gender (reference: male),
age (reference: 15-20), smoking status (reference: non-smoker) and type of cigarette (reference:
regular cigarette) on selecting the PC or branded cigarette (see Table 3). Wald tests were
computed to test the differences in odds-ratios between 21-24 and 25-30 year olds, and
between occasional and daily smokers. Accepted Man
Females were significantly more likely than males to select the branded cigarette
(regular, slim or pink) as highest quality and the PC as the most effective for convincing
teenagers not to start, for motivating smokers to quit and to reduce tobacco consumption. Those
aged 25-30 years were significantly more likely than 15-20 year olds to report the PC as most
effective for motivating smokers to quit. There was no significant difference between 21-24 and
25-30 year olds. As for smoking status, occasional smokers were more likely than non-smokers
to choose the branded cigarettes as highest quality and to select the PC as most effective for
motivating teenagers not to start. Daily smokers were less likely than non-smokers to consider
the PC as most effective for motivating smokers to quit. Occasional smokers were more likely
than daily smokers to select the branded cigarettes as tastiest and highest quality, and they were
more likely to select the PC as most effective for convincing teenagers not to start and most
effective for motivating smokers to quit. With respect to type of cigarette, participants exposed to the ‘pink cigarette / PC’ option
(compared to those exposed to ‘regular cigarette / PC’) were more likely to select the branded
cigarette as the most expensive and less likely to choose it as the highest quality. For perceived by University o
on 31 May 2018 script
behavioral impact, participants exposed to the ‘pink cigarette / PC’ option were less likely to
select the PC as the most effective for motivating smokers to quit and to reduce their
consumption compared to those exposed to ‘regular cigarette / PC’ option. Participants exposed
to the ‘slim cigarette / PC’ image were less likely to select the branded cigarette as tastiest
compared to those exposed to the ‘regular cigarette / PC’ option. pt
Perceptions of cigarettes according to participants’ profiles and type of cigarette They were also more likely to
select the PC as the most dangerous and the most effective to make people talk about tobacco
dangers, and they were less likely to choose the PC as the most effective for motivating smokers
to quit and to reduce consumption (compared to those exposed to the ‘regular cigarette / PC’
option). Manuscrip
p
g
g Manuscr
option). DISCUSSION Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
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on 31 May 2018 by University o
on 31 May 2018 Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 DISCUSSION Accepted Ma
Almost three decades ago, an advertising agency suggested that Philip Morris “brand the
cigarette”, proposing that “colors and designs could be carried through to the cigarette itself – a
visible extension of the personality of the brand (and the user)”.[31] We found that cigarettes,
whether brightly colored or with a slim or regular design, and with a brand name or symbol
displayed on the stick, were more likely than a plain grey cigarette to influence perceptions of
appeal, harm and behavioral impact. The regular, slim and pink cigarettes were considered the
most expensive, highest quality, tastiest and less dangerous than the PC. The PC, in contrast, was
perceived as a more effective means of preventing initiation among teenagers and motivating
smokers to reduce consumption and quit. These results suggest that the appearance of a
cigarette, in terms of its color (bright or dull), size (slim or regular), and presence of a brand
name, can influence product perceptions and perceived behavioral impact. This is also true for
regular cigarettes; the white cigarette paper may help consumers distance themselves from the
health risks associated with smoking.[21] These findings are similar to research on plain by University o
on 31 May 2018 packaging, where the use of a darker pack color can reduce the attractiveness of the pack,
increase harm beliefs and motivation to quit.[29,30] ed Manuscript
We found no significant differences with respect to the cigarettes being more likely to
make people talk about the dangers of tobacco, except between the PC and the slim cigarette. As
such, an unattractive color alone may be insufficient to provoke such reactions. Some research
has revealed that displaying a warning message on cigarettes was considered a reminder of
health risks. For instance, cigarettes displaying a ‘minutes of life lost’ message have been found
to increase smokers’ intentions to quit,[32] and sticks displaying ‘minutes of life lost’ or
‘smoking kills’ warnings were rated as less appealing than regular cigarettes among
smokers.[24] Other studies have also found that on-cigarette message (‘smoking kills‘) may help
to put teenagers off starting[33] and would be less likely to encourage product trial among
young adult smokers and non-smokers.[34] Future research could explore cigarettes combining
unattractive colors and health warnings, as recommended in previous research with marketing
experts.[35] ccepted
We identified differences in how the cigarettes were perceived by gender, age, smoking
status and cigarette type. DISCUSSION Branded cigarettes had a stronger influence on females compared to
males in conveying quality, with females more likely than males to view the PC as most effective
for convincing teens not to start and for motivating smokers to reduce consumption or quit. Accepted
We identified differences in how the cigarettes were perceived by gender, age, smoking
status and cigarette type. Branded cigarettes had a stronger influence on females compared to
males in conveying quality, with females more likely than males to view the PC as most effective
for convincing teens not to start and for motivating smokers to reduce consumption or quit. These results are in line with research that showed that women are more likely than men to
view fully branded cigarette packs as attractive compared to plain packs.[36] Cigarette design
also had a stronger impact on occasional smokers than non-smokers and daily smokers, with
branded cigarettes more likely to be selected as the highest quality stick (and the tastiest
compared to daily smokers) than the PC. In addition, the PC was more often chosen as the most
effective cigarette for convincing teens not to start among occasional smokers than among non-
smokers and as the most effective for convincing smokers to quit than among daily smokers. It
may be that occasional smokers are more vulnerable to the impact of tobacco marketing (and Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 by University o
on 31 May 2018 thus the design of cigarettes) as they are more concerned with their image when they
smoke.[37] Manuscript
Participants’ reactions differed according to the type of branded cigarette they were
exposed to. In the regular/PC option, the regular cigarette was more often selected as the
tastiest and highest quality compared to the slim/PC and the pink/PC options, but less often
chosen than the slim/PC or pink/PC options as the most effective cigarette for convincing
smokers to reduce consumption and to quit. This may be explained by the fact that people are
more familiar with the regular cigarette style and may consider it is reassuring regarding taste
and quality, whereas slim and pink sticks may be perceived as newer products of which the
taste/quality they are less able to discern. Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 by University o
on 31 May 2018 DISCUSSION In the slim/PC option, participants less often selected
the slim stick as the most dangerous, consistent with previous research which has found that
slim cigarettes are perceived as less harmful, cleaner and safer than regular cigarettes.[19-21] Accepted M
Several conclusions can be drawn from these results. First, our study suggests that
dissuasive sticks may be worth considering in countries, such as France, that have implemented
plain packaging. In such markets, while smokers can hide their packs because they are
unattractive, they cannot hide the cigarettes when they smoke. Second, if a pack a day smoker
(20 cigarettes) is exposed to cigarettes approximately 70,000 times per year (with around 10
puffs per cigarette),[4] then removing branding from cigarettes and standardizing the color may
help reduce the attractiveness of cigarettes. Third, branded cigarettes, whether regular, slim or
colored, appear to be more misleading than PC and, as such, regulation of the cigarette itself may
be warranted. These regulations may be particularly important in developing countries where
cigarettes are often sold by the stick.[38] There are certain limitations that need to be considered. First, as the branded cigarettes
we tested differed from the plain cigarette in a number of respects (color, shape and brand name
or symbol displayed on the cigarette), our research does not allow us to isolate the specific effect by University o
on 31 May 2018 cript
of each attribute on participants’ reactions. Nevertheless, should policy makers legislate for
unattractively colored cigarettes without any markings, consumers will be confronted with a
similar scenario to the one presented in this study, in that cigarettes on sale, which currently
have brand names and/or symbols, will no longer have any of these features. Second, we
assessed behavioral intentions rather than actual behavior. Third, forced exposure of our
experimental design may have had an impact on responses. Fourth, while we intentionally
explored young adults’ perceptions of cigarette design, given that this is a key demographic for
public health, we are unable to provide any insight into the response of older smokers. Manusc
In conclusion, our paper highlights that cigarettes, like cigarette packs, can be a powerful
communication tool. Research could meaningfully extend this study by exploring other types of
branded cigarettes, including cigarettes with flavored capsules in the filter that have been found
to be particularly appealing to young people.[39,40] In addition, PC colors other than grey could
be examined in future research. DISCUSSION epted
Funding: This work was supported by a grant from the INPES (Institut National de Prévention
et d’Education pour la Santé) in France. Crawford Moodie is funded by Cancer Research UK. cep
Competing interests: None declared Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 by University o
on 31 May 2018 by U
e s ty o
on 31 May 2018 Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 10 Unknown. Operating plan Asia. 1995. RJ Reynolds records. Bates No. by University o
on 31 May 2018 cep
Competing interests: None declared cep
Competing interests: None declared Accep
Competing interests: None declared
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on 31 May 2018 cep
Competing interests: None declared Sample characteristics (weighted data)
Smokers
(n=375)
Non-
smokers
(n=623)
Total
(n=998)
p-value
Age group
0.185
15-20 years
33%
39%
37%
21-24 years
26%
24%
25%
25-30 years
41%
36%
38%
Gender
0.437
Male
52%
50%
51%
Female
48%
50%
49%
Cigarette consumption per day
(daily smokers)
Less than 11
41%
11-20
43%
21-30
8%
31 or more
1%
It varies
8%
Tobacco most often smoked
Manufactured cigarettes
85%
Roll-Your-Own cigarettes
14%
Cigars or pipe
1%
Intention to quit
No
18% Accepted Man
Cigarette consumption per day
(daily smokers)
Less than 11
41%
11-20
43%
21-30
8%
31 or more
1%
It varies
8%
Tobacco most often smoked Accepted Man
Cigarette consumption per day
(daily smokers)
Less than 11
41%
11-20
43%
21-30
8%
31 or more
1%
It varies
8%
Tobacco most often smoked
Manufactured cigarettes
85%
Roll-Your-Own cigarettes
14%
Cigars or pipe
1%
Intention to quit
No
18% Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 cep
Competing interests: None declared Regular and occasional smoking by college students:
personality attributions of smokers and nonsmokers. Psych Reports 1998;83:1299–306. anuscrip
36 Moodie C, Ford A. Young adult smokers’ perceptions of cigarette pack innovation, pack colour
and plain packaging. Australasian Marketing J 2011;19:174–80. 37 Hines D, Fretz AC, Nollen NL. Regular and occasional smoking by college students:
personality attributions of smokers and nonsmokers. Psych Reports 1998;83:1299–306. Man
38 de Ojeda A, Barnoya J, Thrasher JF. Availability and costs of single cigarettes in Guatemala. Nicot Tob Res 2013;15:83–7. doi: 10.1093/ntr/nts087. Man
38 de Ojeda A, Barnoya J, Thrasher JF. Availability and costs of single cigarettes in Guatemala. Nicot Tob Res 2013;15:83–7. doi: 10.1093/ntr/nts087. pted M
39 Abad-Vivero EN, Thrasher JF, Arillo-Santillán E, et al. Recall, appeal and willingness to
try cigarettes with flavour capsules: assessing the impact of a tobacco product pted M
39 Abad-Vivero EN, Thrasher JF, Arillo-Santillán E, et al. Recall, appeal and willingness to
try cigarettes with flavour capsules: assessing the impact of a tobacco product
innovation among early adolescents. Tob Control 2016;25:113–9. doi: epted
try cigarettes with flavour capsules: assessing the impact of a tobacco product
innovation among early adolescents. Tob Control 2016;25:113–9. doi: epte
innovation among early adolescents. Tob Control 2016;25:113–9. doi: cep
10.1136/tobaccocontrol-2015-052805. Acce
40 Thrasher JF, Abad-Vivero EN, Moodie C, et al. Cigarette brands with flavour capsules in the
filter: trends in use and brand perceptions among smokers in the USA, Mexico and Australia,
2012–2014. Tob Control 2016;25:275–83. doi: 10.1136/tobaccocontrol-2015-052805. by University of
on 31 May 2018 Table 1. Sample characteristics (weighted data) Accepted Manuscript
Table 1. Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 Accepted Harm Accepted Ma
Most effective to make people
talk about tobacco dangers
38
29
17
16
ns
Perceived behavioral impact
Most effective for convincing
teenagers not to start
13
49
29
10
***
Most effective for motivating
smokers to quit
6
52
34
8
***
Most effective for motivating
smokers to reduce
consumption
7
54
30
9
***
Slim cigarette
(Vogue)
Plain
Cigarette
None a
Don’t
know a
p-
value b Intention to quit No by University of
on 31 May 2018 Yes, in the next 30 days
22%
Yes, in the next 6 months
27%
Yes, but not in the next 6 months
15%
Don’t know
18% Accepted Manuscript
d from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
sity of Stirling user
2018 by University of
on 31 May 2018 Table 2. Perceptions of regular, slim, pink and plain cigarettes (PC) (%) Accepted Manuscript
Regular cigarette
(Marlboro)
Plain
Cigarette
None a
Don’t
know a
p-
value b
Appeal
Most expensive
61
14
13
12
***
Tastiest
58
6
21
15
***
Highest quality
61
6
24
9
***
Harm
Most dangerous
25
44
19
12
***
Most effective to make people
talk about tobacco dangers
38
29
17
16
ns
Perceived behavioral impact
Most effective for convincing
teenagers not to start
13
49
29
10
***
Most effective for motivating
smokers to quit
6
52
34
8
***
Most effective for motivating
smokers to reduce
consumption
7
54
30
9
***
Slim cigarette
(Vogue)
Plain
Cigarette
None a
Don’t
know a
p-
value b
Appeal
Most expensive
67
14
7
12
***
Tastiest
48
19
17
17
*** Harm Accepted
Appeal
Most expensive
69
4
12
14
***
Tastiest
65
4
16
15
***
Highest quality
47
13
26
14
***
Harm
Most dangerous
23
40
23
14
*** Most effective to make people
talk about tobacco dangers Perceived behavioral impact Appeal Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 d Manuscript
Highest quality
61
11
18
11
***
Harm
Most dangerous
14
55
17
14
***
Most effective to make people
talk about tobacco dangers
20
38
19
24
***
Perceived behavioral impact
Most effective in convincing
teenagers not to start
16
46
27
11
***
Most effective in motivating
smokers to quit
13
48
30
8
***
Most effective in motivating
smokers to reduce
consumption
16
44
28
12
***
Pink cigarette
(Pink Elephant)
Plain
Cigarette
None a
Don’t
know a
p-
value b Accepted Manuscript
Highest quality
61
11
18
11
***
Harm
Most dangerous
14
55
17
14
***
Most effective to make people
talk about tobacco dangers
20
38
19
24
***
Perceived behavioral impact
Most effective in convincing
teenagers not to start
16
46
27
11
***
Most effective in motivating
smokers to quit
13
48
30
8
***
Most effective in motivating
smokers to reduce
consumption
16
44
28
12
***
Pink cigarette
(Pink Elephant)
Plain
Cigarette
None a
Don’t
know a
p-
value b
Appeal
Most expensive
69
4
12
14
***
Tastiest
65
4
16
15
***
Highest quality
47
13
26
14
***
Harm
Most dangerous
23
40
23
14
***
Most effective to make people
talk about tobacco dangers
30
27
22
22
ns
d b h
l Highest quality Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 Perceived behavioral impact Accepted Manuscript
Most effective in convincing
teenagers not to start
18
45
26
11
***
Most effective in motivating
smokers to quit
10
44
35
11
***
Most effective in motivating
smokers to reduce
consumption
10
46
34
10
***
a These responses were removed from the chi-square analysis
b ns: not significant ; *** p<0.001 pt
Most effective in convincing
teenagers not to start
18
45
26
11
***
Most effective in motivating
smokers to quit
10
44
35
11
***
Most effective in motivating
smokers to reduce
consumption
10
46
34
10
***
a These responses were removed from the chi square analysis a These responses were removed from the chi-square analysis Accepted Manuscript
a These responses were removed from the chi-square analysis
b ns: not significant ; *** p<0.001
aded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
versity of Stirling user
May 2018 a These responses were removed from the chi-square analysis by University of
on 31 May 2018 ipt
Table 3. Binary logistic regression: odds-ratios for the selection of branded / plain rather than plain / branded cigarettes by gender (reference =
male), age (reference = 15-20), smoking status (reference = non-smoker) and type of branded cigarette displayed (reference = regular cigarette) Accepted Manuscript
© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For
permissions, please e-mail: journals.permissions@oup.com. Table 3. Binary logistic regression: odds-ratios for the selection of branded / plain rather than plain / branded cigarettes by gender (reference =
male), age (reference = 15-20), smoking status (reference = non-smoker) and type of branded cigarette displayed (reference = regular cigarette)
Gender
(Female)
Age
(21-
24)
Age
(25-
30)
p-value
21-24 /
25-30
Occasional
smoker
Daily
smoker
p-value
occasional
/ daily
Pink
cigarette
Slim
cigarette
Likelihood of selecting the branded cigarettes (1)
Most expensive
1.29
1.03
0.88
ns
1.31
0.80
ns
4.03***
1.21
Tastiest
1.36
1.14
1.16
ns
1.65
0.67
*
2.01
0.29***
Highest quality
2.24***
0.97
1.48
ns
2.65*
1.07
*
0.39**
0.64
Likelihood of selecting the plain cigarette (PC) (2)
Most dangerous
1.22
0.76
0.80
ns
1.31
1.47
ns
1.03
2.31***
Most effective to make people
talk about tobacco dangers
1.15
0.87
1.18
ns
1.29
1.17
ns
1.09
2.44***
Most effective for convincing
teenagers not to start
1.62*
0.62
0.73
ns
2.26*
0.72
**
0.69
0.78 Accepted Manuscript
© The Author(s) 2018. Perceived behavioral impact Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For
permissions, please e-mail: journals.permissions@oup.com. Table 3. Binary logistic regression: odds-ratios for the selection of branded / plain rather than plain / branded cigarettes by gender (reference =
male), age (reference = 15-20), smoking status (reference = non-smoker) and type of branded cigarette displayed (reference = regular cigarette)
Gender
(Female)
Age
(21-
24)
Age
(25-
30)
p-value
21-24 /
25-30
Occasional
smoker
Daily
smoker
p-value
occasional
/ daily
Pink
cigarette
Slim
cigarette
Likelihood of selecting the branded cigarettes (1)
Most expensive
1.29
1.03
0.88
ns
1.31
0.80
ns
4.03***
1.21
Tastiest
1.36
1.14
1.16
ns
1.65
0.67
*
2.01
0.29***
Highest quality
2.24***
0.97
1.48
ns
2.65*
1.07
*
0.39**
0.64
Likelihood of selecting the plain cigarette (PC) (2)
Most dangerous
1.22
0.76
0.80
ns
1.31
1.47
ns
1.03
2.31***
Most effective to make people
talk about tobacco dangers
1.15
0.87
1.18
ns
1.29
1.17
ns
1.09
2.44***
Most effective for convincing
teenagers not to start
1.62*
0.62
0.73
ns
2.26*
0.72
**
0.69
0.78 Likelihood of selecting the branded cigarettes (1) Likelihood of selecting the plain cigarette (PC) (2) © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For
permissions, please e-mail: journals.permissions@oup.com. ttps://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
Stirling user Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 Manuscript
Most effective for motivating
smokers to quit
1.92**
1.32
2**
ns
1.14
0.53*
*
0.50*
0.44**
Most effective for motivating
smokers to reduce
consumption
1.95**
0.75
1.11
ns
0.57
0.69
ns
0.52*
0.32***
*p<0.05; **p<0.01; *** p<0.001; ns: not significant
(1) It was predicted that positive attributes (‘most expensive, tastiest and highest quality’) would be associated with branded cigarettes, which is why participants
were asked about the likelihood of selecting branded cigarettes. d Ma
(2) It was predicted that negative attributes (’most dangerous, most effective to make people talk about tobacco dangers, most effective for convincing teenagers not
to start, most effective for motivating smokers to quit/to reduce’) would be associated with the plain cigarette, which is why participants were asked about the
likelihood of selecting plain cigarettes. Accepted Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 Figure 1. Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 Perceived behavioral impact Cigarettes shown to participants by University o
on 31 May 2018 Downloaded from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
by University of Stirling user
on 31 May 2018 Figure 1. Cigarettes shown to participants Figure 1. Cigarettes shown to participants Accepted Manuscript
g
g
p
p
Image 1: Regular cigarette (‘Marlboro’) and Grey cigarette (n=333)
Image 2: Slim cigarette (‘Vogue’) and Grey cigarette (n=332) nuscript
Image 1: Regular cigarette (‘Marlboro’) and Grey cigarette (n=333) usc
Image 1: Regular cigarette (‘Marlboro’) and Grey cigarette (n=333) Accepted Manu
Image 2: Slim cigarette (‘Vogue’) and Grey cigarette (n=332) by University of
on 31 May 2018 Accepted Manuscript
Image 3: Pink cigarette (‘Pink Elephant’) and Grey cigarette (n=333) script
Image 3: Pink cigarette (‘Pink Elephant’) and Grey cigarette (n=333) cepted Manuscript
Image 3: Pink cigarette (‘Pink Elephant’) and Grey cigarette (n=333) Accepted Manuscript
Image 3: Pink cigarette (‘Pink Elephant’) and Grey cigarette (n=333)
from https://academic.oup.com/ntr/advance-article-abstract/doi/10.1093/ntr/nty105/5003364
ty of Stirling user
018 crip
Image 3: Pink cigarette (‘Pink Elephant’) and Grey cigarette (n=333) by University of
on 31 May 2018
|
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